Chromosomes;

Its Nature-Before, During and After Gene Activation:

 

Basic chromosomal structure:

Chromosomal DNA of eukaryotes is linear, double stranded and very long ranging from 25Mb to 258Mb+ per chromosome.  Human haploid DNA is 3-3.2x10^9 bp long, but compacted into 22 + X or Y chromosomes; 2n = 46.  Any such long DNA free from any structural support gets broken during replication, recombination, gene Expression and transcription. That is the raison d’etre eukaryotic systems have designed to compact such long DNA in to compact threads called chromosomes. Chromosomal DNA is associated with histone and nonhistones proteins, where histones participate as structural components and provide strength and stability and protect DNA from shearing and breakage; nonhistones act as functional or regulatory components either in activation or repression of genes.

                       

Chromosomes n Human Evolution;

Chromosomes are our Genetic materials; In course of time many-MANY thousands of years, chromosomes changed their numbers and genes, so monkey family with 2n= 48 developed from four legged animals- Chimpanzee, Orangutans, Apes etc. to Bipeds 2n=46; one of the chromosomes “Y” is going to be lost, what will happen to the present Homo sapiens in future? It is predicted that the ‘Y’ chromosome may/would disappear.  If so what?

                                                           

Chromosomes-human; Ape to Hom sapiens (Hominus)

                                                Modern humans developed in time

                                                           

Modern Sapiens evolved from Ape-Chimpanzee/ via chromosomal fusion of chromosome 2dimer.

                                                                       

http: Wikipedia.com

 

 

Scientists are still figuring out when all this inter-group mating took place. Modern humans may have mated with Neanderthals after migrating out of Africa and into Europe and Asia around 70,000 years ago. Apparently, this was no one-night stand—research suggests there were multiple encounters between Neanderthals and modern humans. http: Wikipedia.com.

 

Less is known about the Denisovans and their movements, but research suggests modern humans mated with them in Asia and Australia between 50,000 and 15,000 years ago.

Until recently, some researchers assumed people of African descent didn’t have Neanderthal ancestry because their predecessors didn’t leave Africa to meet the Neanderthals in Europe and Asia. But in January 2020, a paper in Cell upended that narrative by reporting that modern populations across Africa also carry a significant amount of Neanderthal DNA. Researchers suggest this could be the result of modern humans migrating back into Africa over the past 20,000 years or more after mating with Neanderthals in Europe and Asia.

“It seems like the more DNA evidence that we get—every question that gets answered, five more pop up,” he says. “So, it’s a bit of an evolutionary whack-a-mole.”

Early Human Ancestors Shared Skills; Human groups that encountered each other probably swapped more than just genes, too. Neanderthals living in modern-day France roughly 50,000 years ago knew how to start a fire, according to a 2018 Nature paper on which Sorensen was the lead author. Fire-starting is a key skill that different human groups could have passed along to each other—possibly even one that Neanderthals taught to some modern humans.

Scientists are still figuring out when all this inter-group mating took place. Modern humans may have mated with Neanderthals after migrating out of Africa and into Europe and Asia around 70,000 years ago. Apparently, this was no one-night stand—research suggests there were multiple encounters between Neanderthals and modern humans.

Given these types of discoveries, it may be better to think about human evolution as a “braided stream,” rather than a “classical tree of evolution,” says Andrew C. Sorensen, a postdoctoral researcher in archaeology at Leiden University in the Netherlands. Although the majority of modern humans’ DNA still comes from a group that developed in Africa (Neanderthal and Denisovan DNA accounts for only a small percentage of our genes), new discoveries about inter-group mating have complicated our view of human evolution.

“It seems like the more DNA evidence that we get—every question that gets answered, five more pop up,” he says. “So, it’s a bit of an evolutionary wacky-a-mole.

What Prehistoric Cave Paintings Reveal About Early Humans Lived;

 

                                             Homo erectus,

 

Did Neanderthals Die Out Much Earlier Than We Thought?

 

The human lineage of Australopithecus afarensis, Homo habilis, Homo erectus, Neanderthals and Homo sapiens. Encyclopedia Britannica/Universal Images Group/Getty Images Britannica/Universal Images Group/Getty Images have no prediction of the present day Homo sapiens.

Scientists are still figuring out when all this inter-group mating took place. Modern humans may have mated with Neanderthals after migrating out of Africa and into Europe and Asia around 70,000 years ago. Apparently, this was no one-night stand—research suggests there were multiple encounters between Neanderthals and modern humans.

Less is known about the Denisovans and their movements, but research suggests modern humans mated with them in Asia and Australia between 50,000 and 15,000 years ago.

Until recently, some researchers assumed people of African descent didn’t have Neanderthal ancestry because their predecessors didn’t leave Africa to meet the Neanderthals in Europe and Asia. But in January 2020, a paper in Cell upended that narrative by reporting that modern populations across Africa also carry a significant amount of Neanderthal DNA. Researchers suggest this could be the result of modern humans migrating back into Africa over the past 20,000 years after mating with Neanderthals in Europe and Asia.

Given these types of discoveries, it may be better to think about human evolution as a “braided stream,” rather than a “classical tree of evolution,” says Andrew C. Sorensen, a postdoctoral researcher in archaeology at Leiden University in the Netherlands. Although the majority of modern humans’ DNA still comes from a group that developed in Africa (Neanderthal and Denisovan DNA accounts for only a small percentage of our genes), new discoveries about inter-group mating have complicated our view of human evolution.

“It seems like the more DNA evidence that we get—every question that gets answered, five more pop up,” he says. “So, it’s a bit of an evolutionary wacky-a-mole.”

Early Human Ancestors Shared Skills; Human groups that encountered each other probably swapped more than just genes, too. Neanderthals living in modern-day France roughly 50,000 years ago knew how to start a fire, according to a 2018 Nature paper on which Sorensen was the lead author. Fire-starting is a key skill that different human groups could have passed along to each other—possibly even one that Neanderthals taught to some modern humans.

Paintings to show how and where they lived What Prehistoric Cave Paintings Reveal About Early Human Life and how they ived;

 

Early Humans May Have Scavenged More than They Hunted

 

 

Early Humans Slept Around with More than Just Neanderthals

 

 

What Prehistoric Cave Paintings Reveal About Early Human Life

 Scientists are still figuring out when all this inter-group mating took place. Modern humans may have mated with Neanderthals after migrating out of Africa and into Europe and Asia around 70,000 years ago. Apparently, this was no one-night stand—research suggests there were multiple encounters between Neanderthals and modern humans.

Less is known about the Denisovans and their movements, but research suggests modern humans mated with them in Asia and Australia between 50,000 and 15,000 years ago.

Until recently, some researchers assumed people of African descent didn’t have Neanderthal ancestry because their predecessors didn’t leave Africa to meet the Neanderthals in Europe and Asia. But in January 2020, a paper in Cell upended that narrative by reporting that modern populations across Africa also carry a significant amount of Neanderthal DNA. Researchers suggest this could be the result of modern humans migrating back into Africa over the past 20,000 years after mating with Neanderthals in Europe and Asia.

Given these types of discoveries, it may be better to think about human evolution as a “braided stream,” rather than a “classical tree of evolution,” says Andrew C. Sorensen, a postdoctoral researcher in archaeology at Leiden University in the Netherlands. Although the majority of modern humans’ DNA still comes from a group that developed in Africa (Neanderthal and Denisovan DNA accounts for only a small percentage of our genes), new discoveries about inter-group mating have complicated our view of human evolution.

“It seems like the more DNA evidence that we get—every question that gets answered, five more pop up,” he says. “So, it’s a bit of an evolutionary wacky-a-mole.”

Early Human Ancestors Shared Skills; Human groups that encountered each other probably swapped more than just genes, too. Neanderthals living in modern-day France roughly 50,000 years ago knew how to start a fire, according to a 2018 Nature paper on which Sorensen was the lead author. Fire-starting is a key skill that different human groups could have passed along to each other—possibly even one that Neanderthals taught to some modern humans.

 

 

 

Neanderthal and Homo sapiens reconstructions by the Kennis brothers

“It seems like the more DNA evidence that we get—every question that gets answered, five more pop up,” he says. “So, it’s a bit of an evolutionary wacky-a-mole.”

Human groups that encountered each other probably swapped more than just genes, too. Neanderthals living in modern-day France roughly 50,000 years ago knew how to start a fire, according to a 2018 Nature paper on which Sorensen was the lead author. Fire-starting is a key skill that different human groups could have passed along to each other—possibly even one that Neanderthals taught to some modern humans.

Scientists are still figuring out when all this inter-group mating took place. Modern humans may have mated with Neanderthals after migrating out of Africa and into Europe and Asia around 70,000 years ago. Apparently, this was no one-night stand—research suggests there were multiple encounters between Neanderthals and modern humans.

Less is known about the Denisovans and their movements, but research suggests modern humans mated with them in Asia and Australia between 50,000 and 15,000 years ago.

Until recently, some researchers assumed people of African descent didn’t have Neanderthal ancestry because their predecessors didn’t leave Africa to meet the Neanderthals in Europe and Asia. But in January 2020, a paper in Cell upended that narrative by reporting that modern populations across Africa also carry a significant amount of Neanderthal DNA. Researchers suggest this could be the result of modern humans migrating back into Africa over the past 20,000 years after mating with Neanderthals in Europe and Asia.

Precursor- DNA/RNA/PROTEINS compacted into Chromosomes:

                                         Very small protein-RNA or DNA Particles which in living-beings living, but outside they are just crystalline particles.

    

 

 

 

Bacterial Genome-contracted

 

| Models of bacterial chromosome organization. Models of nucleoid organization with Ori and Ter represented by red and purple circles. (a) Model of the E. coli genome with the four macro-domains Ori, Ter, left, right, represented by circles in red, purple, pink and blue, respectively. (b) Model of the B. subtilis genome adapted from ref. 52. (c) 3D models of the M. pneumoniae genome conformation.

Human chromosomal DNA

Structural organization om DNA to chromosome

                    

                                    The Nucleus; fig.cox.miami.edu/

.

"Histone-depleted chromosomes (were studied) in the electron microscope. Our results show that: the histone-depleted chromosomes consist of a scaffold or core, which has the shape characteristic of a metaphase chromosome, surrounded by a halo of DNA; the halo consists of many loops of DNA, each anchored in the scaffold at its base; most of the DNA exists in loops at least 10-30 µm long (30-90 kilobases).'' Paulson, J.R. and Laemmli, U.K. Cell 12 (1977) 817-828

 

Composed of DNA and protein (histones) all tightly wrapped up in one package; duplicated chromosomes are connected by a centromere. Y chromosome contain SRY and X chromosome contain SOX 3/

Acerogenin as a marker for sex identification in forensics and describe four additional Y chromosome markers, sex-determining region Y (SRY), Y-encoded testis-specific protein (TSPY), locus DXYS156, and steroid sulfatase (STS). The SRY, TSPY, DXYS156, and STS markers each have properties that could be used for developing more rigorous methods of testing forensic DNA samples for a Y chromosome or the presence of specific reproductive or secondary sex characteristics. http://www.avensonline.org/ http://www.uic.edu/; Applied Genetics-Chromosomes; http://on-line.ucol.ac.nz/genetics; http://www.apsubiology.org

X chromosomes contain 1000 genes with Sox 3/9 (female sex genes?), but Y chromosome contain ~60 genes, the SRY determine male sex.

Two juvenile central chimpanzees, the nominate subspecies

Diagrammatic representation of the location of the fusion site of chromosomes 2A and 2B and the genes inserted at this location.

 

Chimpanzee-human chromosome differences. A major structural difference is that human chromosome 2 (green color code) was derived from two smaller chromosomes that are found in other great apes (now called 2A and 2B ^[8]). Parts of human chromosome 2 are scattered among parts of several cat and rat chromosomes in these species that are more distantly related to humans (more ancient common ancestors; about 85 million years since the human/rodent common ancestor ^{https://en.wikipedia.org/}

 

http://www.biologydiscussion.com

Salivary gland Polytene Chromosomes; (Balbiani); http://www.biologyexams4u.com/;http://www.biyanigirlscollege.com/; www.slideplayer.es/slide

Lampbrush chromatid, central nucleoprotein produces chromatin loops which are transcriptional active; https://clinicalcenter.nih.gov

 

www.Keywordsking.com

Chromosomes sequenced at the Sanger Institute. [Genome Research Limited]; http://webcache.googleusercontent.com/

 

Chromosome number, DNA content in BP and gene number per chromosome; SC = Secondary Constriction (NOR) in chromosomes 13, 14, 15, 21 and 22:

I

If you were getting excited about having a company like 23andme sequence your genome for you, it's time to put a lid on it. Apparently the State of California has decided that people should not be allowed to sequence their own genomes without supervision from a medical professional (despite the fact that many medical professionals are not trained to understand genomic data). It is weird remark. each of the chromosomes has two arms on either side of centromere.  The smaller arm is called P-arm and the longer arm is called Q-arm.  Based on linkage studies each of these arms are subdivided in P1, P2, p3 etc; similarly, Q arms. Chromosomes showed in the diagrams are metaphasic most condensed chromatins (chromosomes); NCBI, Genetic Review; http://www.ncbi.nlm.nih.gov/

In human beings, XX generates Female sex, but XY generates Males, why and how? The Y with X chromosomes dominates the X chromosomal genetic expression. In living humans, males dominate women? Note: even plants have XY genes for sex organ determination; S. latifolia. Dioecious papaya.  It revealed a short male specific region on the Y chromosome (MSY) 

Note: Even plants have XY genes for sex organ determination; Ex. S. latifolia and Dioecious papaya.  It revealed a short male specific region on the Y chromosome (MSY) determines stamen formation of male flowers where ovary formation is deleted.

 

 

Human Chromosomal Base pairs and Genes: 

 

N0.	Base pair x10^6	No. of genes
1	263	2769
2	255	1776
3	214	1445
5	203	1023
6	194	1261
7	183	1401
8	171	1410
9	155	952
10	145	1086
11	144	1042
12	143	1626
13	114	1347	sc
14	109	477	sc
15	106	821	sc
16	98	915
17	92	1139
18	85	1471
19	67	408
20	72	1715
21	50	357	sc
22	56	657	sc
x	164	1090
Y	144	59

 

 

 

 

 

 

 

 

 

                                           Human gene analysis -with chromosomes.

                                                                       

Nucleolar organizing centers (fibrillar centers): Pale staining regions containing DNA encoding rRNA,ii. Pars fibrosa: Electron dense fibrillar region composed of ribosomal RNA transcripts. iii. Pars granulosa: Granular-appearing region-composed of maturing ribosome particles.; http://dc304.4shared.com/

Nuclear structures- heterochromatin is bound to inner nuclear membrane matrix; the nucleolus shows different structures such as pars fibrosa and pars granulosa.

 

The Y chromosome likely contains ~ 300 to 410 or more genes that provide instructions for making proteins. Because only males have the Y chromosome with X, the genes on this Y chromosome tend to be involved in male sex determination and development. Sep 28, 2022 in each cell. The Y chromosome spans more than 59 million building blocks of DNA (base pairs) and represents almost 2 percent of the total DNA in cells. The Y chromosome do contain genes that provide instructions for making male related proteins and few others. Because only males have the Y chromosome, the genes on this chromosome tend to be involved in male sex determination and development.  The SRY genes responsible for male organs development and suppress female genes?. The Y chromosome spans more than 59 million building blocks of DNA (base pairs) and represents almost 2 percent of the total DNA in cells.

1.    The Y chromosome contains a "male-determining gene," the SRY/SOX9 gene(s)? that causes testes to form in the embryo and results in development of external and internal male genitalia. If there is a mutation in the SRY gene, the embryo will develop female genitalia despite having XY chromosomes. The Y chromosome is one-third the size of the X chromosome and contains about 55 genes while the X chromosome has about 900 (+) genes.

2.     The X chromosome spans about 155 million DNA building blocks (base pairs) and represents approximately 5 percent of the total DNA in cells.

What X chromosomal genes determine Female Sex-DAX1-WANT4a?

3.     Females contain-total number of Neuronal cells including Glial cells is 171 billion cells, the number in male brains is less than the female brains.

 

 

 

 

https://www.slideshare.net

 

Metaphasic type of condensation is due to Structure (ral) Maintenance of Chromosome (SMC) proteins and non-SMC proteins such as Condensins (contract the length) and Cohesins (glue two parallel chromatid strands). Differential staining with DAPI (4, 5-Diamino phenyl Indole) shows some darker bands and some lighter bands called G and R bands respectively; such bands can be discerned in compacted metaphasic chromosomes.  Such dark bands are called heterochromatin and the lighter regions are called euchromatin; which exists in various states.  Heterochromatin is classified into constitutive and facultative types; the first is found to be condensed always and the facultative locus varies from one tissue to the other.  Constitutive heterochromatin is found at Pericentric and telomeric regions and Sub- telomeric regions.  The one of the two X-chromosome in mammals and many vertebrate’ cells is always heterochromatic. The metaphasic chromosomes are condensed 1400-fold in contrast to nucleosomal threads of 11nm thick.  The metaphase dark bands contain more DNA per unit than euchromatin.

DAPI-4', 6-diamidino-2-phenylindole, dihydrochloride (DAPI) is a blue fluorescent DNA dye that targets double-stranded AT clusters in the DNA minor groove; The level of DAPI-DNA fluorescence is proportional to DNA content

Synteny maps (bottom map) for each Tetraodon (it is puttering fish contains just 350MBps smallest among vertebrates) chromosome, colored segments represent conserved synteny with a particular human chromosome. Synteny is defined as groups of two or more Tetraodon genes that possess an orthologue on the same human chromosome, irrespective of orientation or order. Tetraodon chromosomes are not in descending order by size because of unequal sequence coverage. The entire map includes 5,518 orthologues in 900 syntenic segments.  You can also do this in the other direction, and take each Tetraodon chromosome, color code them, break them apart, and reassemble them into the order they would be in the human genome, as in the second diagram.

1.     Female mammals have two X chromosomes in every cell. However, one of the X chromosomes gets inactivated. Such inactivation stops transcription, hence making sure a potentially toxic double dose of X-linked genes does not occur but only one chromosomal genes works.

2.     An inactivated X chromosome gets condensed into a small, dense structure in the nucleus, and is called a Barr body. Barr bodies are commonly used to determine sex.

3.     X chromosomes contain 900-1000 genes and the genes Dax1, Want-4a act as female determining genes and Y chromosome contains about 500-600 genes and SRY/Sox9 are male determining genes and they dominate its partner X chromosomal genes.

4.     Is it true that in Human population all over the world, socially, Males dominate females, is it true?

5.     Human brain contains 171 billion cells (80 billion neuronal cells and 85 million Glial cells).  Do these genes with male sex genes dominate females?

6.     Do female and males have the same number of brain cell?

 

Figure above: The ontogeny of X-chromosome inactivation in the mouse: two current views.

 

a) The de novo inactivation model requires many rounds of inactivation and reactivation: the paternal germline initiates meiotic sex-chromosome inactivation, but the X chromosome is completely reactivated after meiosis. The zygote inherits two fully active X chromosomes and begins re-inactivation of the paternal X chromosome (XP) at the 4- to 8-cell stage. In the trophectoderm (extra-embryonic cells, shown in blue), XP silencing is maintained, therefore accounting for the imprinted form of X-chromosome inactivation. By contrast, in the epiblast (green cells), yet another round of reactivation takes place in preparation for a final round of inactivation in the form of random X-chromosome inactivation. b) In the pre-inactivation model, the female zygote inherits a partially silent XP and maintains the silent state throughout pre-implantation development. Silencing becomes globalized and complete in extra-embryonic tissues. This accounts for the imprinted form of X-chromosome inactivation. By contrast, the epiblast cells of the inner cell mass (ICM) undergo a single round of reactivation followed by a random form of X-chromosome inactivation.

© 2005 Nature Publishing Group Huynh, K. D. et al. X-chromosome inactivation: a hypothesis linking ontogeny and phylogeny. Nature Reviews Genetics 6, 10 (2005).

 

 

Genes sit on a loop right near the large regulator molecules needed to start and stop their production (promoters, enhancers and repressors). Loops can be flexible and the contact of the sites can be intermittent. This loop region makes it much easier to use the DNA. Often these loops create the environment for the activity, but a further stimulus is, also, needed. Chromatin loops; http://jonlieffmd.com/

Each type of chromatin structure works to form regions with TADs. Some factors keep the different types apart, making them more localized. TAD-topologically Associated Domains; http://jonlieffmd.com

Human mitotic chromosomes stained to reveal a scaffold-like structure along the chromosome axis

In these confocal fluorescence micrographs, the DNA has been stained with a blue dye, and the axis has been stained red with a fluorescent antibody against a protein in the Condensin complex. Only part of the scaffolding is visible in these optical sections. (A) A typical mitotic chromosome, which has a gently coiled scaffold along each of the two chromatids. (B) A metaphase chromosome from a cell artificially blocked in metaphase; in the chromosomes of these cells, the scaffold has condensed by further helical folding. A 2M NaCl extracted scaffold protein is fibrous with molecular weight of 37kDa and 83 (85) kDa. The scaffold protein is also associated with Topoisomerase II (140kDa).  Perhaps the most abundant chromosomal nonhistone protein may be TopoisomeraseII.  It can expand and contract.

(Courtesy of Ulrich Laemmle and Kazuhiro Maeshima).

 

Sister chromatid cohesion is mediated by entrapment of sister DNAs by a tripartite ring composed of cohesin’s Smc1, Smc3, and α-kleisin subunits. Condensins consists of SMC2/CAP-E and SMC4/cap-c, plus they also contain few associated proteins, cohesins glue chromosomal threads and Condensins contract the chromosomal thread; http://mcb.asm.org/http://openi.nlm.nih.gov/

Condensin- I associates with structural and gene regulatory regions in vertebrate chromosomes; The Condensin complex is essential for correct packaging and segregation of chromosomes during mitosis and meiosis in all eukaryotes.  We find that Condensin I binds predominantly to promoter sequences in mitotic cells.  Ji Hun Kim, etal; http://www.nature.com/

 

http://www.valdosta.edu/~jfelder/molecular1.ppt ;http://edu.docdat.com/

 

The central scaffold protein is visible in Lamp brush chromosomes of oocyte cells and one can observe the opened loops of various lengths, which are all active in transcription; each loop can be considered as one chromomere; from the extension one can measure the length of DNA of each chromomere. http://earthkart2011.blogspot.in/

Nucleosomal thread of 11nm thickness; it is the basic chromosomal form; this form exists in certain regions of the chromatin at interphase.  The third level organization of chromatin is that the 30 nm fiber associated with various nonhistones proteins, among them Topoisomerases and HMG proteins are found in large numbers.  Histone depleted chromatin shows a long structural protein runs the entire length of chromatin called scaffold protein and the chromosomal DNA is found looped out of such structure; the base of the loops is attached to the scaffold; the loops are of various sizes 20 to 90kb (15 to 30um).  A cross sectional view of a typical chromosome consists of a central scaffold protein from which histone bond DNA threads (coiled) appears to loop out; thus the thickness of the structure is 1um, it agrees with the thickness of the intact but relaxed chromosome. 

Chromosomes with 140 -250million base pairs could easily produce about 2000 -3000 such ~70kb long loops.  The base of DNA loops containing AT rich sequences are bound to scaffold protein complex; such regions of chromonema are called Matrix Attachment sites (MARs or SARs scaffold attachment).  MARs/SARs are AT-rich DNA sequences, often containing topoisomerase II at the base that mediate the anchoring of the chromatin fiber to the chromosome scaffold or nuclear matrix and might delimit the boundaries of discrete and topologically independent higher order domains. The scaffold proteins can be associated with ScII 85KDa; they have ATPase domains.  Chromatin SMC proteins also play important role chromatin organization.

Interestingly interphase chromatin is also associated with several TFs bound to specific regions of the DNA coils. Interestingly the base can also associate with histone deacetylases or Acetylases. DNA associated activators (or TFs) or repressors at specific positions throughout the cell cycle is a fact; if one finds such proteins associated with chromatin, provide the specificity of the gene loci. The chromatin compaction leads to 300nm thickness. This further compacts to 700nm to 1400nm thick at metaphase. The 700nm compaction is due to coiling of 300nm loops.  Such spiral coiling facilitates the opening of chromatin easily.  At the same time these threads are strengthened by scaffold and non-histone proteins.

 

Nucleosomes folded and compacted into a bundle of fibers; http://www.studyblue.com/

 

 

 

 

 

New structural model for the metaphase chromosome based on thin plates; Experiments performed using several different microscopy techniques have allowed researchers at the UAB Chromatin Laboratory to discover that, during cell division, chromosome DNA is packaged within planar structures formed by many extremely thin layers. These planar structures are stacked, occupy the entire volume of the chromosomes, and are probably oriented perpendicular to the central chromatid axis. The planar geometry of these structures is very well defined, but the nucleosomes inside the successive layers are irregularly oriented. Pablo Castro-Hartmann, et al; http://www.uab.es/

 

“We (above authors) have discovered that in condensed chromosomes, chromatin is densely packaged forming plate-like structures instead of the typical fibers considered in the current models of metaphase chromosomes. Our electron microscopy images have shown that chromosome plates can form multilayered structures, having a thickness of approximately 6 nm each layer”.

 

Chromo DNA to metaphasic Chromatin; http://en.wikipedia.org/wiki/Chromatin.

 

When chromatin duplicates at S-phase, the replication fork drives through ds DNA separating parental strands into leading strand and the other as lagging strand.  As the strands separate, the histone octamer randomly associates with single strands distributed equally among them. During replication, as new dsDNA produced gets associated with histone octamers.   Histones synthesized at this point are more or less acetylated and the same organize into H3x2 and H4x2 tetramers and bind to dsDNA and then H2Ax2 and H2Bx2 join. Most of the histone tails are acetylated at specific amino acids. The assembly is facilitated not only by acetylation of H3 histones but also by Nucleoplasmin; and probably Topoisomerases assist in the formation of new chromatin threads; nucleoplasmins are considered as Chaperonins. 

 

Model of the chaperone function of Nucleoplasmin: (a) Face views. (b) Side views. The nucleoplasmins pentamers (blue and yellow) dimerize to form decamers, a process that might be favored by the binding of H2A–H2B dimers (red). The formation of decamers, either alone or in the presence of H2A–H2B dimers, might trigger a conformational change in the pentamers such that the β hairpins (blue and yellow) become extended outwards. The dimers bind to the β hairpins and A1 tracts of the nucleoplasmins decamer. An H3–H4 tetramer (green) then binds to each dimer to form an octamer. This binding is repeated five times on the lateral surface on the decamer to form a decamer–octamer complex. www.cell.com

 

https://www.slideshare.net

Chromosome (continued): loop domains-

“Each extended loops as shown in the upper diagram looks like a knob like projection at the surface and it is believed to represent the tip of a separate looped domain. Note that the two identical paired chromatids in the diagram can be clearly distinguished. (From M.P. Marsden and U.K. Laemmli, Cell 17:849-858, 1979. © Cell Press.); “organization of these loops looks like Zea mays grains on its central axis in the cob”.  The binding of DNA to central scaffold provides strength to 30nm fibers it does not interfere with replication of DNA, recombination or Repair”.  The composition of scaffold protein is still to be understood.

 Yet one can observe metaphase chromosome perse certain regions where the chromatin is more compact, in Pericentric and telocentric regions.  These compactions are due heterochromatization which can be distinguished by staining method. Telomeric HC is due to RAP1 and Sir Proteins, where Sir2 is histone deacetylases. Deacetylation can lead to binding of more deacetylase’s activity and more binding of proteins such as RAP1 and SiR.  But the pericentromeric heterochromatization is due to small micro RNAs such as siRNAs as well as histone deacetylation, methylation of histone H3K9 and K27; and HP1binding to them. Heterochromatization spreading in euchromatin and other region is blocked by specific DNA sequence-based protein subunits called insulators.

 

Chromatin Insulators and Enhancer-blockers

 “The ability of chromatin to organize into functional autonomous units characterized by specific levels and patterns of expression is ensured through the establishment of boundaries that delimit these domains. In some cases, transcriptionally active genes may be embedded in an environment containing extensive regions of condensed chromatin capable of inappropriately silencing their expression. In other cases, signals from extraneous enhancers could cause incorrect pattern of expression of silent genes located nearby. Boundaries are responsible for ensuring the maintenance of the appropriate level of expression of each gene or gene cluster by marking the barrier between chromatin domains of distinct states.

These boundaries might occur at various positions, as a result of a balance between countervailing processes (such as chromatin condensation and decondensation), or be fixed at specific DNA loci. Chromatin insulators are DNA elements that mark the boundaries of chromatin domains by limiting the range of action of enhancers and silencers and by preventing incursions of neighboring chromatin domains. Although they have a wide variation in DNA sequences and proteins that bind to them, they are characterized by at least one of the following abilities; enhancer-blocking or barrier activity”.

A chromatin insulator with enhancer-blocking properties is able to block the enhancer-promoter communication when positioned between them. Enhancer-blockers restrict the long-range activation potential of eukaryotic enhancers in order to strictly limit their influence to one or few specific target promoters.

“The processivity model, envisions the relay of the enhancer signal to the promoter as a tracking action along the chromatin fiber. That model corresponds to a protein-tracking model in which the RNA-polymerase II-complex assembled at the enhancer tracks from the enhancer along the DNA to reach the promoter and activate mRNA synthesis. The transmission of this type of signal could be disrupted by the interposition of the insulator nucleoprotein complex interposed between the enhancer and the promoter”.

 

 “In the topological model, instead of sending a signal from afar, a distant enhancer has to be brought close to the promoter through mechanisms that allow direct interaction between the enhancer and the promoter (e.g. by loop formation). Insulators may recruit chromatin-modifying enzymes to locally change the chromatin state disabling loop formation and thus preventing the direct enhancer-promoter interaction”.

 

“Barriers Elements: Barrier elements are none other than insulators and they have been shown to be able to recruit histone-modifying enzymes, locally competing with the spreading of silent chromatin markers often found between active genes prevent the spreading of gene activation to the neighboring gene improperly”.

 

Specifically, an insulator may have an enhancer-blocking activity or a barrier activity.  Here is an illustration of these two functions of insulators: ;http://www.anti-agingfirewalls.com/

Eukaryotic Genome Organization

Legend: Eukaryotic genomes are composed of chromatin harboring in different states: Heterochromatin corresponds to closed chromatin, non-permissive for gene expression, highly dense in nucleosomes bearing markers of silent chromatin (red circles). In this domain, silencers (red triangles) are responsible for limiting genes’ transcription level (thin blue arrows). On the other hand, euchromatin domains are composed of nucleosomes bearing marks of permissive chromatin (green circles) and contain highly expressed genes (thick blue arrows). Enhancers (green squares) are driving the expression of these genes and are capable of activating genes over large distances (green arrows). Chromatin insulators secure the delimitation of chromatin domains by limiting the spread of silent chromatin (barrier element, yellow circles) and also restrict the long-range influence of enhancers (truncated green arrow) when interposed between them and promoters (enhancer-blocker element, orange circle). Viral vectors (grey triangle) flanked by effective chromatin insulators, combining both enhancer blocking and barrier properties, raise the prospect of safer and more efficient gene therapy vectors.

 

Alan Cohen etal; http://www.nyas.org/

 

http://emboj.embopress.org

Insulator activity can be regulated by ubiquitination and sumoylation of insulator proteins. A. Two active insulators coming together at an insulator body. dTopors is present at the insulator sites, Mod(mdg4)2.2 and CP190 are not sumoylated and dTopors serves as a bridge to the lamina. B. Two inactive insulators that cannot be part of an insulator body. dTopors is absent and Su (Hw) is not ubiquitinated, whereas Mod(mdg4)2.2 and CP190 are sumoylated. The two insulator sites cannot interact with the lamina or each other and form insulator bodies

Chromatin domains and insulators; http://wwwuser.cnb.csic.es/

 

Insulators also block the effect of repressor spreading.  A transgene (represented by gold DNA) integrated in the chromosome in a region of condensed chromatin is not properly expressed; the repressive chromatin structure of the surrounding region presumably spreads into transgene sequences, inhibiting enhancer-promoter interactions. B. If the transgene is flanked by barrier insulators (red DNA with two proteins represented as dark blue and green spheres), these sequences inhibit the spreading of the repressive chromatin, allowing an open chromatin and normal transcription of the gene.

 

Nucleating Centers in the Chromatin:

Interphase Nucleus full of active components

 

Gene expression starts with assembly of Basal Transcription Associate factors or apparatus called BTA factors in sequence, it need not be, but discerned by experiments; TFIID binds to TATAA site, how it is identified?  It is identified by the sequence and nucleosomal acetylation region; this provides the site for nucleation.  It is not a chance but it is determined by TBP to their specific DNA sequence TATAA this acts as nucleating center for the assembly of other Transcriptional complexes. The assembly of the BTA is complex but once in its place they can interact with upstream elements, whatever they are, whichever they may be, and by conformational changes in the protein complex induce the opening of dsDNA into transcriptional bubble, which is required for ensuing transcription.  In another way transcriptional initiation can be possible by the binding of upstream factors in sequence specific manner and recruiting other components so as to modify histone tails by acetylation and loosening the nucleosomal structure to facilitate the binding of the rest of Transcriptional complex downstream.  Loosening of the chromatin structure makes the promoter region to be freed from nucleosomes for the assembly of transcriptional factors, finally the RNA pol complex.

 

It is important to realize the promoter and regulatory elements in DNA should be made available free from proteins in the chromosomes. Before initiation of transcription; chromatin has to be remodeled in such a way at least some prime gene activating factors identify the said gene site (s) in sequence specific manner in the chromatin and bind and induce changes in chromatin structure.  The same thing holds good for gene silencing or repression.  There are remodeling protein complexes which are multisubunit structures; they are responsible for remodeling the chromatin so as to make the specific DNA sequence made available for the transcriptional apparatus assemble or not made available for the binding of transcriptional complexes.  Perpetuation of chromatin status, active or inactive is an intrinsic process and it is executed precisely and exactly, until other factors are innovated to change the status.

 

Chromosomal State During Gene Expression:

Regulation of gene expression in eukaryotes is more complex and intrinsic.  The genome is organized into a nucleoprotein complex of different orders.  Chromosomes bear genes of different types such as protein coding mRNAs; NC RNAs such as rRNA, tRNA, RNAi’s (Si RNAs and MiRNAs), ScRNAs, SnRNAs, SnoRNAs, activator RNAs, TmRNA, antisense RNAs, Riboswitch RNAs, Lnc RNA, Linc RNAs, X-inactivating RNAs, TasiRNA, RasiRNA, PiRNA, telomerase RNA, RNaseP RNA, 7sK RNA, 7sLRNA and many more which are expressed differentially during development and also in tissue specific manner during and after development.  Even after development genes are expressed in tissues in response a variety of signals.  

 

Preferential association between co-regulated genes at Transcriptional factories:

Chromosomes undergo structural changes, has been observed, during cell cycle, from relaxed state at interphase to highly condensed state at metaphase.  At interphase substantial numbers of genes are expressed in tissue specific manner.  At metaphase the chromosomes are condensed to such an extent, all genes in them are shut off.  Completion of cell division leads to chromatin to relax and transcribe the required transcripts for the cell.

 

 

It is during this process chromatin relaxes and positions in nuclear milieu and get attached to the inner surface of the nuclear membrane associated nuclear matrix proteins through their heterochromatin loci.  Even in response to signals, for cell division, cells acquire inputs for cell determination and differentiation. In the interphase whatever the cell types; the relaxed chromatin, certain regions or loci loop out into nucleoplasm.  This region of the chromatin of 11-30nm thick, now it is accessible to transcription complex; it does not mean that all those genes present in euchromatic region are expressed, it is not so.  The looped euchromatin DNA that is engaged in transcription exhibit what is called ‘transcriptional factories’ where active loops from different domains of chromatins of different chromosomes are clustered together where one finds active transcription of different set of genes located.

Colocalization of genes in the nucleus for expression or coregulation.  Fraser & Wendy Bickmore;http://www.nature.com/

Active genes on decondensed chromatin loops that extend outside chromosome territories can colocalize both in cis and in trans at sites in the nucleus with local concentrations of Pol II (namely transcription factories; dark pink) and adjacent to splicing-factor-enriched speckles (pale pink). Interactions can also occur between regulatory elements and/or gene loci and lead to coregulation in trans (blue circle).

 

 

Long range chromatin interactions

 

Genome organization in mammalian nuclei: Chromosome conformation varies between cell types and this inevitably places whole groups of genes in particular nuclear environments, such as regions in the nuclear interior that are rich in splicing factors; or next to transcription factories, where many RNA polymerases simultaneously transcribe different transcription units. Genome architecture within the interphase nucleus is inextricably linked with gene regulation.

 

                        Active genes (red) with transcription factories (green) in T cells.

 

 Lamin Associated Domains LADs may consist of relatively condensed chromatin (thick lines) and aggregate at the nuclear lamina. Other repressed regions may interact with each other in the nuclear interior, as do active regions. Complexes formed by components of the transcription machinery (transcription factories) and CTCF may tether active regions together. Parts of only two chromosomes are depicted, each in a different color for clarity. Most interactions occur within chromosomes, and relatively few occur between chromosomes; Bas van Steensel & Job Dekker; http://www.nature.com/

 

It is logical to expect that chromosomal loci where gene to be expressed requires unwinding and the nucleosomal structures are relaxed or at least some part of the DNA of the said gene is to be free from histones for the binding of nucleating pioneer proteins, which recruits histone acetylases and transcriptional complex and its related factors. 

Whether the regions that are active in gene expression or not can be tested by DNase1 or micrococcal nuclease treatment, which on partial digestion of the chromatin DNA wherever it is free from proteins is digested and the same can be analyzed on gels as several bands of uniform sizes.  Nucleosome bound regions show ladders and nucleosome free regions as blanks.

 

Analysis of DNA fragments by Gel.

 

DNase-1footprint analysis of ER binding to probe DNA is different from binding to mono-nucleosomes. EPAGE; (A) DNase I footprint with labeled probe DNA (ERE) in the absence or presence of 2 and 4 μg recombinant human ERα. After DNase treatment reaction products were run on an 8% acrylamide-8M urea sequencing gel. The bracket indicates the position of the consensus ERE and protected sites. (B) DNase I footprint with labeled mono-nucleosomes in the absence or presence of 20 μg ER-containing nuclear extract plus or minus 10 μg HMGB-2. Arrows indicate sites of enhanced cleavage of DNA bases with addition of ER ± HMGB-2 vs. absence of ER. The symbol (*) indicates sites of decreased cleavage.

 

This can be observed by gel electrophoresis.  So, the DNA of a gene that is active state should be free from nucleosomes, and mostly it is of promoter regions; it is the promoter region where transcriptional apparatus and its associated factors bind upstream of InR and inclusive of InR.  The most fascinating aspect is what makes the DNA of gene or genes accessible or not accessible for transcriptional initiation and transcription?  The simple explanation is that when histones are free from promoter region of DNA, the promoter of the genes accessible but if it is bound it is not.  What makes the histone to be freed form such regions?  Is it due to the binding repressor to DNA that perpetuates that makes the promoter not accessible or DNA is tightly compacted by specific proteins so it is not accessible.  For the DNA to be free such chromatin compacting proteins should be freed.  The crux of gene activation and gene inactivation is in understanding of chromatin remodeling during the process.

 

State of chromatin when Gene Expression is on Grand scale:

1. Ribosomal RNA gene expression:

 

All eukaryotic cells irrespective of species, do exhibit rRNA synthesis all the time in the nucleolar region of the nucleus.  Nucleolar size changes, when pericyclic cells are activated, in the case of IBA induced root initiation in Phaseolus vulgaris, the size of the nucleolus is so large it occupies ¾ of the nucleus. This shows the requirement of rRNA for pericyclic cell not for others. Requirement of rRNA for any cell is very high and so requires the transcription of rRNA all the time and in large amounts.  To supply such high quantity, 100-200 rRNA genes in tandem repeats in the secondary constriction region, looped out as naked DNA, depending upon the species and the stage of development. In human’s chromosome 13, 14, 15, 21 and 22 contain rRNA genes in a region called NOR or nucleolar organizer region. After telophase as the nuclear membrane reconstitutes, chromosomes relax and open; at this point of time rRNA coding DNA open out in the form of loops of various sizes and organize into nucleolus, where the DNA is freed from all chromatin proteins and gets associated with transcription complex and transcription goes on. And one can visualize each of the rRNA genes that are in the process of transcription, on the open DNA, and one can observe Christmas tree like transcripts arranged-from the nascent to old transcript.  In this the chromosomal DNA is totally devoid of any histones, and the only proteins associated are RNAP I and its associated factors are found.  This goes on 24 hrs. a day and 365 days a year. This implies for massive transcription the DNA should be free for the transcription complex to operate.

 

Nucleus is green in color and nucleolus in blue color

 

 

 

 

 

 

 

 

 

 

 

It has been observed nucleolar loci exist in certain chromosomes and not in all chromosomes.  They are inherited by the same parent, i.e., but pollen-male and egg-female are contributor; it is virtually similar to biparental contribution.  This is true for biparental off springs. It so happens, the nucleolar DNA that opens up for transcription is either from one parent or the other.  So, one set of rRNA genes are kept silent and the other is expressed; which is similar to X chromosome inactivation.

http://www.indiana.edu

 

 

 

 

 

 

 

 

 

 

 

rRNA genes exist in tandem arrays. Each of the genes contains core sequences and upstream promoter-enhancer components.  The rDNA is transcribed in very high quantities and higher rates.  As a series of RNAP I assemble at the start point and initiate transcription and end; one can observe the transcripts from short to long ones, looks like Christmas tree pattern. Note not all rRNA gees are expressed; only 40 to 50% of them are expressed; In the nucleoli of the oocyte nucleus of Triturus virescens, an American newt species (as well as with other amphibians) occurs free DNA. The picture shows the transcription of genes that are carriers of the information about ribosomal RNA formation ("MILLER-trees"). More about the interpretation of the electron microscopic picture (O. L. MILLER, B. R. BEATTY, Biology Division, Oak Ridge National Laboratory, http://www1.biologie.uni-hamburg.de

 

2. Lamp brush chromosomes: 

Another system that shows such grand scale transcription at a particular stage of development is Xenopus oocyte stage.  During meiosis at pachytene-diplotene transitory stage, one finds meiotic chromosomes are maximally elongated and one can observe large number of granular structures all along the length of each synaptically paired chromonemal threads, at some points one can observe chiasmata also.

Top figure, Lampbrush chromosomes’ active in transcription. http, diagrammatic features of structural organization of Lampbrush chromatin. http://www.nptel.ac.in/

 

Chromosomes contain chromomeres (cytological observation), which are nothing but coiled-coiled compacted chromonemata containing clusters of genes.  Some chromomeres in such synapsed chromatin are opened out into naked DNA loops of 5kbp to 50kbp long. Such loops are free from chromatin associated histones. Nearly 5000 such loops have been counted. In between such opened or say active chromomeres there are chromomeres compacted into inactive chromatin. Such looped out DNA is free from histone complexes and found to be actively transcribing. Such kind of transcription is required for the future development of egg into an embryo after fertilization.  This is a preparatory stage where the developmental process requires proteins, rRNA, mRNAs and many others in massive quantity.  They are produced and stored. 

3. Salivary gland chromosomes:

 

Another grand scale expression is seen in insect larval development like drosophila and its related species. Such a scenario is also observed in plants’ haustoria during the development of plant embryo.  Developing plant embryo requires all such inputs for the embryo development and it is provided by the polytene chromosomal formation and gene expression on grand scale or say large scale.

 

When the fertilized egg develops into larva, in the case of Drosophila, on reaching 11th day it enters into pupa stage.  There is a dramatic transformation of the larval body into pupal structures and later into insect per se.  This huge transformation requires a large-scale expression all those genes involved in metamorphosis.  The larval cells have four pairs of chromosomes and the total number of genes in the insect is about ~18000 or so.  The homologous chromosomes are found paired, as if it is meiosis. The number of genes’ expression required for such transition has been analyzed by micro array chips and found to be in large numbers.  But most of the genes exist as a pair of genes on their respective homologous chromosomes.  But the requirement in such in that period is huge.  In order to supply to such demands, each of the required genes has to be expressed at very high rates in the transitional period; this looks like an impossible task.  But large-scale expression made possible by chromosomal DNA duplication into ~1080 chromonemal strands irrespective of what genes this is are expressed what genes remain silent.  Thus, each gene is represented ~1080 times. The gene expression at each locus amounts to thousand genes that suffices the demands.  So, insects have designed that their chromosomes in Salivary glands on 11th day undergo transformation into multistranded, visible under normal microscope, this is differential gene expression at cytological level. Starting from the early 11th day larva till the pupal initiation, sets of genes are expressed temporally, it means at the early stage one set of gene are expressed, as the development progresses, another set of genes start expressing and the early genes expression regresses.  By the end of this progression of thousands of individual genes are expressed and thousands become silent a cascade of gene expression.

 

Chromosomal puffs at 75EF and 74B are visible

http://bioinfosu.okstate.edu

http://www.yourarticlelibrary.com

 

The polytene chromosome in its glory with 1080 duplicated strands show many very prominent chromosomal bands.  Each of the bands represents a gene or group of genes.  In some of the bands DNA loops out, free from associated structural proteins, 10kb to 50kbp size associated with transcription machinery producing transcripts required to be translated and some to be stored. The loci at which thousands DNA loops opened and expressed, looks like puffs (chromosomal puffs) when stained; some of the long loops, identified by Balbiani are called Balbiani rings. In between many chromosomal bands remain unexpressed.  During transition from larva to pupa gene expression or puff formation and regression at different positions can be observed. Here again one observes chromosomal DNA at specific positions should be free from histone complexes to produce massive number of transcripts.

 

 

 

Balbiani Rings: Electron micrograph of a Balbiani ring (A) and a schematic presentation of a transcription loop with growing RNP particles (B). The loop consists of three portions: an upstream region (a), the transcribed template (b), and a downstream region (c). In the electron micrograph, two segments of transcription loops have been indicated by arrows. The bar equals 1 µm. From- Daneholt (1992).

 

 

 

Specific loci containing a set of genes related are expressed in high order, thus one can see the Puff like regions.  During formation stage, in salivary glands, chromosomes undergo repeated replication without separation to produce multistrands.  This provides a scope for a gene to be present in 1080 copies; this provides an opportunity for transcription is massive scale.

 

 

Gene Expression Requires DNA of the Promoter either to be Free or to be loosened from the Grip of Histones and Repressor Proteins:

From the above description, for gene expression, the DNA in the chromatin has to be dissociated or freed from compacting histones and other associated proteins; for they block specific sites for the binding of activators and transcriptional complex.  Chromosomes’ composite structure is made up of four histones. Histones constitute structural components of chromosomes as organized structural elements called nucleosomes.  But chromosomes are also associated with thousands of nonhistones, which can be differentiated from histones from their very nature of being little acidic; not all of them.  Most of them are involved in regulation of gene expression and DNA replication, DNA damage repair; and maximum number of nonhistones found on chromatin is found to be topoisomerases at the base of chromatin loops where the DNA is bound to scaffold proteins. Topoisomerase II has important function in removing super coiled DNA into relaxed forms either at replication stage or at transcriptional stage or whenever required; they can also induce super coiled structures and make it inactive. Can positive supercoiled DNA block transcriptional initiation?  Why one finds such huge number of topoisomerases? Along with Topoisomerases another protein that is found in large amounts is a DNA binding protein called High mobility group {HMG); they are a family of proteins involved in binding and bending DNA and transcription. Another protein found at scaffold region is an 85KDa protein; they are like SMC proteins with ATP binding sites.

 

One notices the presence of gene activation and gene repression proteins all along the chromosomal threads, their loci depend on the specific DNA sequences, cell type and stage at which they are found. The number of such proteins involved in gene expression and repression may run into thousands. Delineation of it requires the expertise of functional genomic professionals. In this description, the regulation of gene expression is dealt in two different functional aspects but with inter-related structures and functions.

 

Bacterial genomic DNA which is also associated with proteins at their promoter regions, not so compacted as found in eukaryotic systems. The compacted DNA into chromatin often undergoes tight compaction and relaxation.  The compacted regions can be observed by staining as dark bands.

 

The Role of Histones and Nonhistones in General:

 

It is very well documented that all cells in all tissues express certain set of genes for house-keeping functions, common to all cells in the body, they are called house keepings genes. Some are expressed in tissue specific manner; they are expressed using tissue specific factors.  There are genes, whatever tissue or cell types, are exposed to certain signal molecules, and they induce signal specific gene expression. All other genes remain unexpressed or silent.  The genes that are expressed have access to transcriptional apparatus and those not expressed or kept silent don’t have access to transcriptional proteins or the access is blocked.

 

In this context it is important to know which of the chromosomal proteins prevent transcription and which of them allow transcription in specific manner?  To answer this question scientists, have isolated DNA from an organism.  They also isolated histones and nonhistones from different tissues. Very simple experiments (even high school students can understand) are designed to show which of the prime chromosomal proteins are involved in transcription and that too in tissue specific manner and which proteins block transcription.

 

First experiment:

DNA isolated from a system is same; common DNA for the experiment. Histones and nonhistones are isolated from specific tissues i.e., from Liver (L) and Brain (B).

In one experiment non histones from liver and brain tissue are added separately to the common DNA and then histones were added and allowed to express; experiments showed nonhistones from liver expressed liver specific genes and brain specific nonhistones expressed brain specific genes.  Histones could not prevent nonhistones’ mediated expression. Tissue specific expression is due to specific nonhistones.

In the second set of experiments histones were added first to the DNA, and then non histones from brain and liver added separately, results showed gene expression failed, for the histones binding to DNA blocked access to non-histones.  Though this experiment is simple yet its result showed nonhistones find their promoter elements and initiated transcription, histone cannot block when nonhistones already bound, but when histones are bound nonhistones cannot act.  Identification and characterization of those nonhistones involved in gene expression is fascinating.

Second experiment:

Isolate a specific DNA segment, say 5s rDNA, add histone first then add required components such as IIIA, IIIC, IIIB and RNAP III; result no transcription.  Instead add IIIA first then add histones and then other components; transcription takes place.  This shows the binding of IIIA to specific sequence is a gene identity factor; it does not allow histones to inhibit.  Once IIIA binds in sequence specific manner; when it binds to its sequence, it recruits other components to initiate transcription even in the presence of histones; so histones cannot prevent transcription once sequence identity factor binds; i.e. ‘nucleating’ factor binds.  Can nucleating factors perpetuate through cell lineages? Yes, in bacteria they do and there no doubts that eukaryotes have this facility, though not completely elucidated.

 

Inference:

In eukaryotes the promoter elements have more complex combination of sequence boxes to be recognized by a combination of factors. The transcriptional activators bind to different elements located at different positions away from the promoter and START site.  So also transcriptional repressors, they do so by binding to specific sites identified by sequences. So activators activate specific gene transcription and repressors repress specific gene expression. Repressed genes can be induced to express and active genes can repressed when required. There are others called silencers and insulators, which have their own specific roles to play.

 

The regulator proteins of one kind are activators, co-activators, mediator complexes and transcriptional complexes.  The others are repressors, corepressor and their associated proteins. They bind to different structural elements and activate the gene expression or gene repression in specific.  Activators first seek specific promoter sequences and bind if the region is free from histones, then co activators, Mediator complexes join, the latter don’t bind to DNA. Repressors are specific to specific genes, so also specific silencers.  In some cases the repression is not just few loci but the whole chromosomal genes are totally inactivated, in an extreme case, but it is prevalent in higher systems for example inactivation of human X chromosome.   In many of the chromosomes certain regions of chromosomes are repressed such regions are called heterochromatin.

It is important to know repression can be due to specific repressor binding and preventing the access to transcriptional machinery; or the repressor remains in place until it is induced to change either to become activator or they get released from the DNA. There is another mode of repression that is heterochromatization.  One such state is telomeric region and pericentromeric region, where the chromosomal region has constitutive heterochromatin.  There are other heterochromatin blocks in euchromatin such and they found to change the loci from one tissue to the other; they are called facultative heterochromatin; it is the facultative heterochromatin that blocks some blocks of genes.  In some cases, certain gene expression is blocked by localized heterochromatization.  There are many such examples due such abnormal heterochromatization leads to epigenetic expression or repression of genes that are unrelated to normal gene expression and cause diseases.  Histones should not be considered as sole repressor proteins, but they are fundamental building blocks of chromatin where DNA is strengthened as the fiber and prevent DNA from shearing and breakdown. They provide remarkable strength for the DNA 10A^o thick to remain unsheared during cell cycle and gene expression. Another very important structural protein that reinforces and sustains the rigors of chromosomal changes during replication, recombination and DNA breakage and repair is that of scaffold protein; it also sustains several modes of chromatin changes such as relaxation and differential compaction.

 

Chromosome Remodeling Protein Complexes and their Function:

Important Gene Regulation Factors:

In eukaryotes, the DNA, for that matter any organism, such as bacteria or viruses (some viruses contain RNA as the genome), that has encoded message, contains promoter elements with more complex combination of sequence boxes to be recognized by specific factors.  DNA is associated with histones and nonhistones and organized into chromatin thread.  This, when it is in relaxed i.e 11-to 30nm state the chromatin is open and transcription complex can assemble; but if it is compacted, the transcriptional factors cannot access the promoter, so no transcription. Chromatin in relaxed state is basic 11-30nm chromonemal thread where nucleosomes are bound together by Histone1.  This thread in 30nm state, where the chromonemal thread loops out and its base is bound to scaffold region.  This is subjected to activation or repression of genes.  It is the same thread gets compacted differentially into heterochromatin and can be relaxed as euchromatin.  Repression is not always due to heterochromatization; but it is also due to the binding of specific repressor complexes associated with 30-110nm structure.  Binding of one factor induce and recruit other accessory factors either for activation or repression. In order to understand the complexity of activation and repression one has to look deep into complex components of proteins and their role.

 

Chromatin Activators and Repressors:

Activators bind to specific elements located at different sites upstream of the START site.  So also, transcriptional repressors bind to specific sites; they are identified by sequences.

The structural and functional features of both gene activators and repressors have specific motifs and domains, each have specific functions in gene regulation. In spite of the variety of components involved in regulating gene expression, the overall mechanism, from yeast to man to plants, is more or less similar. Why? For expression of genes chromatin in specific regions has to open and provide access to transcriptional factors and RNA polymerase for binding and initiate transcription.

Basic structural components of chromatin are histones and DNA; in addition a large number of non-histones are also found associated with basic histone DNA threads.  If one observes chromatin in vivo, its surface shows the chromatin is studded with whole lot proteins not just histones. Probably every gene or gene loci are already associated with specific proteins; identity of them and their location is important.

In general, what is now known today that it is histone components bound to DNA and their structural modifications that make the chromatin to be in inactive or active?  Though histone octamer enwrapped around by DNA and nucleosomes are compacted by H1, yet nucleosomal histone tails of ~42nm long in comparison to ~65bp long linker DNA interact with one another and their modifications perform most enticing functions.  Modification of histone tails and additional components added to such tails make the chromatin compact or loosen. Even DNA CpG modifications also contribute to its compactness. Understanding of such modification is very important in understanding chromosomal remodeling and regulation of gene activity.  At chromosomal level histones and nonhistones perform functions as to the cell requirements.  The crux of the problem is that chromatin DNA should be free from histones so that activator or repressor factors can bind.  If chromatin is compacted DNA won’t be free for the binding of these regulator proteins.  The 30nm chromonemal loops which are bound to scaffold protein (300nm structures) should provide access to factors for the binding.  But this chromatin is compacted differentially into tight compaction and loose compaction, the former is called heterochromatin and the latter is called euchromatin.  Even euchromatin is not free for the binding of factors for H1 is bound to linker DNA and compacted to some extent.  What is interesting is that the H1 binding and the positions of nu bodies are dynamic, in the sense they assemble and disassemble and the DNA in euchromatin is very often made free and close in short period of time.  Is this time period is enough for the assembly of factors to bind and execute their activity?  Another important aspect of the chromatin is that whether or not all those nonhistones bound before the replication of chromatin DNA remain associated when the daughter DNAs or after DNA replication, do they reassociate afresh? if so this has to happen even before the chromatin thread is formed. 

It is known that cells perpetuate their characters to their lineage of cells, but the pluripotent cells undergo differentiation with every cell division.  Maintenance of cells structure and functional features in their lineage of cells should contain all the regulatory factors bound to their respective positions.  There is a lot of bias among the scientist in interpreting this perpetuation.  Perpetuation of old histones and complementing them with new histones is known.  Perpetuation of DNA methylation is also established.  Similarly modified old Histones remain associated and evenly distributed among the newly formed DNA strands.  Perpetuation of repressors and activators in prokaryotes is established.  Similarly, perpetuation of nonhistones is not unthinkable.  Take for example, the GAL4 protein as repressor remains bound to chromatin for many generations of yeast cells.  After its activation the same is also perpetuated in dividing cells.

Histones and histone modifications and their effects on chromatin:

Histone fold- most of the histones contain a central helix domain with smaller helices on either side of the main helices, and also contain N terminal tails with specific amino acid sequences, but H2A in addition to NH2 tail contains C terminal tail too. Histone tails with specific a.a sequence get modified-this feature provides what is called as Histone code.

 

 

Chromatin II, histone modifications: http://bricker.tcnj.edu/

Epigenetic regulation of key vascular genes and growth factors- Histones are subject to post-transcriptional modifications, which occur in histone tails. The best-known post-transcriptional modifications (acetylation and methylation) are shown. The number under each amino acid represents its position. Greek ‘Epi' means ‘over, on-top or above', therefore Epigenetics refers to something above genetics itself. Epigentic marks are not in DNA sequence itself but on top of them, as chemical additions on DNA stretch (DNA methylation, Figure 2) or on those proteins in which the DNA is wrapped around (Histones, Figure 1).  These modifications act as switches turning gene expression on or off. Functional epigenome is necessary for health of a cell or organism. Epigenome is likely an easier target for therapeutic modifications when compared with genome itself; http://cardiovascres.oxfordjournals.org/

 

N terminal tails of H3 and H4, their modifications play very important role in chromatin condensation or relaxation; for that matter activation of genes and repression of genes.  Position of specific amino acids is indicated for specific kind of modifications; such as methylation, acetylation and phosphorylation.  Some amino acids are get modified either by acetylation or methylation.  If they acetylated, they can be removed by deacetylases and if they are methylated, they are demethylated by demethylase.

Arginine is preferred for methylation; but R can also be acetylated. But K in certain positions as shown in the figure is mostly acetylated and, in some positions, it can also be methylated.  Rarely it can be ubiquitylated. Serine is the most preferred site for phosphorylation.  It is important to know the site of amino acids in the tail and its modification provides what is called histone code.  Such modifications provide the information for the binding of other proteins.

DNA in eukaryotes is packaged into nucleosomes which consist of DNA wrapped around histone proteins. Covalent modification of histones plays a critical regulatory role in controlling transcription, replication and repair. Different histone modifications are recognized by different protein modules found in regulatory complexes with different, even antagonistic functions; acetylated sequences are recognized by Bromodomain proteins and Methylated sequences are recognized by Chromodomain proteins.  Positions of acetylation, methylation, phosphorylation and ubiquitination (SUMOylating) are shown below.

The chromodomain helicase DNA binding protein 1 (CHD1) is well known for its remodeling activity in the maintenance of stemness. It also has main function in recognizing a substrate of transcription regulatory histone acetylation complex SAGA. CHD1 has been suggested to act as a molecular adaptor, which bring several epigenetic complexes together [29]. In ESCs, this adaptor has been suggested to be indispensable for the maintenance of pluripotent chromatin state where it is highly expressed when compared to differentiated cells. After knockdown of the CHD1 with RNAi, the pattern of diffuse ESCs heterochromatin disappears showing a higher amount of heterochromatin. In turn, CHD1 knockdown fibroblasts reprogrammed less efficiently [30]. The nature of CHD1 in pluripotent cells specifies that it can prevent the formation of heterochromatin foci [30]. CHD1 has also been reported to be one of the genes that activate Oct4, Sox2 and Nanog; http://www.intechopen.com/

Histone modifications have been associated with either 'active' or 'inactive' chromatin states, as well as with particular cellular processes, including mitosis, spermatogenesis and DNA repair. Some modifications, such as histone lysine methylation, are known to recruit specific binding proteins (for example, HP1 to methylated histone H3 lysine 9 and PRC1 to methylated histone H3 lysine 27, whereas acetylation at various residues is believed to have a more structural role, making the nucleosome structure 'looser' and more accessible to transcription factors. Several synergistic and antagonistic interactions have been described between different histone modifications.  On the basis of these observations, it has been proposed that patterns of post-translational modification form a combinatorial 'histone code’. However, the degree of interdependence between different histone modifications, and the various distinct chromatin states they define (individually or in combination), the state of chromatin; this feature is still not entirely understood

 

 

H1 = 208 (222) aa, has central globular body with N and C terminal tails.  H1 composition varies among the species. It has a 5 R s and 53 Ks and 5 D & Es.

Hhh2A = 129 aa, it contains both C (39 aa) and N terminal tail- N S G R G K Q G G K A R A K A K T R S S R A G L, with 12 R, 14K and 7 D/ Es.

H2B = 126(125) aa, it has 34 N-terminal tail with 7R, 21K and 10 D and Es-

P E P S K  S A P A P K K G S K K A I T K A Q K K D G K K R K R S R K.

 

H3 = 136 (135) aa, its N tail 32 contains 18R, 13K and 11 D n Es-.

-A R T K Q T A R K S T G G K A P R K Q L A T K A A R K S A P A T G G V K K; where K9 and K14 are acetylated in euchromatin, even S9 can be phosphorylated.

H4 = 103 (102) aa, N-tail 32 contains 14R, 11K and 7 D n Es-n-S G R G K G G K G L G K G G A K R H R K V L R D where R3 can be acetylated in euchromatin. 

Structural features of Lysine and Arginine: http://www.dls.ym.edu.tw/

Chromatin-Modifying Enzymes- The diversity of chemical states obtained by selective and sequential methylation of lysine and arginine residues within proteins, as catalyzed by the histone methyltransferase class of enzymes. Copeland et al (2012) Targeting Genetic Alterations in Protein Methyltransferases for Personalized Cancer Therapeutics. http://www.epizyme.com/epigenetics.

Lysine acetylation and deacetylation by specific acetylases and deacetylases

 

Histone tail modifications and their functions:

 

 

 

Modification of H3 and H4 tails has important effect on chromatin compaction or relaxation. The K can be acetylated or it can be methylated, similarly R can be methylated and serine can be phosphorylated.  These modifications can be reversed i.e. deacetylation, demethylation and dephosphorylation. Specific modifications at specific sites have tremendous effects on chromatin activation (loosening) and chromatin inactivation (compaction).

acK = Acetylation to K (lysine,

meR = methylation to R (Arginine),

meK = methylation to K,

pS = phophorylation to Serine,

pT = phosphorylation to Threonine

uk = ubiquitination to K,,

 

The order of amino acids in the H3 and H4 tails play significant role in chromosomal activation or inactivation i.e activation of a gene or suppression of a gene.  David Allis proposed that histones N-terminal amino acid sequences provide encoded information, what is called Histone code, where certain modifications evoke certain chromatin based functions, a combination of modifications evoke specific biological functions.

 

It is not only Lysine and Arginine gets modified, even DNA at specific dimers of CpG cytosine is added with methyl group.

 

Role of histones:

Organization of Nucleosomal thread, provide strength and stability to DNA.

Packing nucleosomes with H1 lead to chromonemal thread formation.

Histones with non-histones produce chromatin.

 

Modified histones mostly with CH3 can lead to compaction and gene silencing,; modified Histones with acetylation relax chromatin and genes can be activated, Modifications of Histone tails in the region of promoter elements have a role in heterochromatization and relaxation of the same. Histones perse don’t bind to specific regulatory elements of genes; but nucleosomal threads provide stability and strength to DNA. However, association of histones with DNA is random and there is every possibility, they may cover regulatory sequence of genes.

 

Non-histones and their role:

There are more than 2000 nonhistones proteins, most of them are slightly acidic or neutral; Topoisomerase II is found in large quantity,

HMG family of proteins is another found in large amounts, another protein is 85 KD (?) whose function is not clear, a scaffold protein? Chromatin is associated with large number transcriptional complexes-transcriptional regulators-activators, co activators, repressors, and corepressor, silencers and insulators, which are all grouped as nonhistones; Their quantity and quality of protein varies from one tissue type to the other.

 

Histone modifying enzymes and enzyme complexes:

 

 

ATP-dependent chromatin modifying enzyme complexes:

Complex	Organism	function
RSC	Yeast	Restructure
SWI/SNF	Yeast, fly, Hu	loosening
NuRF	Fly,Hu	Nu restructuring
CHRAC	Fly, Hu	Chromosome associate
ACF	Fly	ATP- Utilizing
ISW1/2	Yeast	Imitation of SWI
RSF	Hu	Restructuring
M1-2/NurD	Hu	Nucleosome R deacetylase

Chromatin remodeling complexes:

SWI/SNF: contain 11 subunits, 2MD,  has helicase activity, slide nucleosomes, ATP mediated chromatin remodeling factor, involved in regulation of cell cycle, cell differentiation, transcriptional regulation, cancer inducing; involved in immunity, acts at 120 loci (yeast), 150 complexes per cell,

SAGA/PCAF; The number of subunits~20, 1.8 MD, found in EK (eukaryotes) included in GcN5/ADA, involved in acetylation facilitating transcription,

SAGA = Spt-Ada-Gcn5-acetyltransferase complex

NURF = 4 subunits -0.5MD, found in Droso, involved in chromatin remodeling, consisting of subunits 215, 140, 55 and 38KD subunits. Nucleosome stimulated ATPase (Nucleosome Remodeling Factors).

RSC = 15 subunits 3MD involved in restructuring complexes of chromatin, acts at 700 loci

CHRAC/CAF1= subunit2, 0.5Md, helps in H3/$ binding to DNA, Chromosome Accessibility Complex).

ACF = ATP dependent chromatin assembly and remodeling Factor.

Trithorax= huge complex involved in activation of some HOX genes during development,

PCG= Polycomb group complex involved in repression of some HOX genes during development,

SWI = It is a yeast mating switch complex and contain ATPase units.

CRC = Chromosome remodeling complexes General.

SNF- Sucrose non Fermentor associated with SWI.

BAH- components are proteins associated with bromodomain binding factors.  Bromodomain consists of 110 a.a sequence which binds to modified histone tails mostly acetylated and recruits more HATS.

ISWi- it is an imitation of SWI

RPDs-Some of the RNAP associated transcription complexes are called RPDs.

RPD3- is a histone deacetylase.

NURD = Nucleosome Remodeling Deacetylase,

CAF = Chromosome associated factor

The remodeling complexes are grouped-

1.     SWI/SNF complexes (include ATPases sw2/snf2, RSC, Brahma BRM (Brahma related protein) BRG1-Brahma related gene 1.

2.     ISWI- imitation switch components, ACFs ATP utilizing chromatin assemble and remodeling factors, CHRAC-chromatin accessibility complex. NURF- Nucleosome remodeling factor and NSF-Nucleosome remodeling and spacing factor.

3.     Mi2 complexes –they include human NURD also contain HDAC1 and HDAC2.

4.     HP- many HPs chromodomain proteins. 

It is known some these factors are associated or bound to transcriptional activators or repressors.  Having ATPases factors use ATP energy they distort binding of DNA to Histone and push the histone complexes along the DNA; they free the DNA from nucleosomal structure, so a segment of DNA is left free for the assembly of Transcription factors and their associated components.  Methylation of certain histone tails recruits HP1 proteins which compacts chromatin to condensed heterochromatic state.

Note: ACF= ATP-utilizing chromatin assembly and remodeling factor; CHRAC = chromatin accessibility complex; HDAC= Histone deacetylases; HMT= histone methyl transferase; ISWI-2= imitation of switch protein; Mi2NURD= Nucleosome remodeling complex; NUA4= Nucleosome acetyl transferase H4; NuRF = Nucleosome remodeling factor; PCAF= p300/CBP associated factor; RSC= Remodel structure of chromatin; RCF= Remodeling and spacing factor; SAGA= Spt-Ada-GCN5-acetyl transferase; SET= SET domain containing protein; SMRT/NCOR= silencing mediator of retinal and thyroid receptor/nuclear receptor co repressor; STAGA= Spt3-TAFII-31-GCN5L acetylase; SUV39H1= Suppressor of variegation 3-9homologue-f;  SWI/SNF switching defective/sucrose Non Fermenter.

Chromatin is a complex of DNA-histones as nucleosomal thread superimposed by nonhistones.  Such a thread is often exists, in certain regions of the chromosomes, as condensed and relaxed threads.  It is believed that relaxed region is more open for transcription than the condensed ones.  Remodeling means the chromatin condensed to be decondensed; and decondensed chromatin to be condensed; it is done by chromatin remodeling proteins.

This remodeling of chromatin is performed by certain multi-subunit protein complexes that are associated with chromatin.  It is also important to remember that there are several thousands of proteins, called site specific identifier proteins, bound to chromosomal DNA they form part and parcel of chromatin thread. Some such bound proteins are perpetuated and some may be added or removed with time and state of the cell.  Multi subunit complexes which are ATP dependent provide fluidity to chromatin. The chromatin is also associated with chromosome remodeling multi subunit protein complexes. Most of them ATP driven, so they remodel the nucleosomes to move from one site downstream so a region containing promoter is made available for regulatory proteins to bind and act. 

One such complex is SWI/SNF, they were discovered in yeast and they make HO genes to express. (SWI means switching mating defective). And SNF means Sucrose Non Fermenter. The SWI/SNF complex is 110KD consists of 11 subunits.  It is able to express 3% of the yeast genes.

Another complex called RSC (Remodel the Structure of Chromosome) is 100 times more abundant than SWI/SNF and they share two subunits of SWI/SNF.  RSC contain homologs of SWI/SNF, called as Sth1, they are called as swi2/snf2 in SWI/SNF.  SWI/SNF are ATPases and two of the subunits of RSC have bromodomain so they bind to acetylated Histone tails. 

Chromatin remodeling complexes and mol.wt and subunits:

 

                        Chromatin Remodeling Proteins;

 

Remodeling Protein domains:

 

 

Chromatin activating factors: GCN4, GCN5, GCN5L ADA, SAGA, SWI, SNF, NuRF, CAF1, NAP1, CRC, NUA4HAT,Trithorax, hTAFII250, GRIP, TIP60complex, RSC, CPB/p300, PCAF, Src1, ACTR and ELP3

Chromatin inactivating factors: NURD, SIN3 family, MBP, MBD1/2/3/4, MeCp2, Histone deacetylases 1, 2, 6, 7, 9, 10, 38 ,45, Histone methylases, HP1 family, RAP, RIF, AIR1, 2, 3 and 4, SUMO l, SW6, Chp2, Clr3 (Chromodomain).

 

Post-translational modifications of the core histone tail that stick out from the nucleosomes have been directly linked to the regulation of chromatin structure, a concept known as the histone code. Modifications of the core histones include acetylation, methylation, ubiquitination and phosphorylation.  Such modifications alter the interactions of histones with DNA and the recruitment of chromatin associated proteins. The best characterized histone modifications are acetylation and methylation. Acetylation and methylation of histone tails are carried out by histone acetyl transferases and histone methyl transferases. Acetylation is primarily associated with active gene expression, but methylation at different positions perform silencing and activation.

Histone acetylation results in the relaxation of the basic chromatin structure through increased charge repulsion and by serving as binding sites for protein complexes of chromatin-modifying and transcriptional activators. Histone methylation can be found in both heterochromatin and euchromatin too. H3 methylation of K4 is found in activated gene promoters. Trimethylation of histone H3 on lysine residue 9, (H3K9me3), is bound by heterochromatin protein 1 (HP1), which results in chromatin compaction and heterochromatin formation. This pattern of histone modifications causes gene silencing or activation can be inherited to their daughter cells, a phenomenon called epigenetics or it can happen during the course of life span.

Chromosomal State during Gene expression:

Regulation of gene expression in eukaryotes is more complex and intrinsic.  The genome is organized into a nucleoprotein complex of different orders. Chromosomes bear genes of different types such as coding for proteins, rRNA, tRNA and other Nc RNAs such as RNAi and few others.  They are expressed differentially during development and also in tissue specific manner during and after development.  Even after development genes are expressed in tissues in response a variety of signals.   

Chromosomes go through condensation and decondensation differentially during cell cycle and even after cell cycle. Chromosomes undergo structural changes, from relaxed state at interphase or G˚ to highly condensed state at metaphase.  At interphase substantial number of genes is expressed in tissue specific manner.  At metaphase the chromosome is condensed to such an extent all genes are shut off.  Once the cell derivatives differentiate and develop into specific cell types, chromosomes in relaxed form found attached to the inner surface of the nuclear membrane protein matrix; the attachment is to heterochromatic loci. Most of it is in the MARs and SARs region of the DNA (300-1000bp long) that contain repeat ~AT rich sequences. The Scaffold Associated Region (SAR) binding polypeptide SATB1 is found associated with MARs. The nucleus consists of pervasive nuclear matrix proteins (Nuclear matrix is analogous to the cell cytoskeleton), whose composition and nature is not yet clear. The relaxed euchromatin region is accessible to transcriptional complex, it does not mean that all those genes present in euchromatin region are expressed, it is not so.

It is logical to expect that chromosomal loci where gene to be expressed requires unwinding of nucleosomal 30nm structures into relaxed form and at least some part of the DNA of the said gene is to be free from histones for the binding of regulator proteins and its related factors. 

Whether regions that are active in gene expression or not can be tested by DNase1 treatment or micrococcal nuclease, which on partial digestion of the DNA wherever it is free DNA is digested completely and wherever nucleosomes are found, only the linker DNA is digested.  Nucleosome bound regions show ladders and nu free regions are completely digested.

DNase I hypersensitivity, and computational approaches have been employed to generate maps of histone modifications, open chromatin, nucleosome positioning, and transcription factor binding regions of mammalian genomes. Given the importance of non‐promoter elements in gene regulation and the recent explosion in the number of studies devoted to them, many scholars have focused on these elements to get insights on gene regulation.  

 

 

                                                PAGE of Proteins

Chromatin state can be observed when micrococcal nuclease is used to digest chromatin and digested pattern can be observed on gel electrophoretic image.  So the DNA of a gene is in active state should be free from histones, and mostly it is of promoter regions; it is the promoter region where Transcriptional apparatus and its associated factors bind upstream of InR.

Chromatin remodeling at the site of gene expression:

David Allis concept of Histone code provides an input where active chromatin, means any gene(s) in that region are expressed or expressing.  Such chromatin shows its H3 tail is acetylated at K9, K14 and H4 acetylated at K5 and its H3-K4 and H4 R3 are methylated (in general).   But condensed chromatin i.e. inactive is associated with H4-K12 acetylated, and H3’s K9 is methylated. Nucleosomes are also disposed for phosphorylation at Serine10 and 28.   The combination of modifications at specific amino acids in the N-tails H3 and H4 have intrinsic ability to change the shape of chromatin to open for transcription or close to transcription at specific loci or sites.  This state of histone modified chromatin varies from loci to loci.

Histone modifications during early cell division, cell determination and differentiation provide those epigenetic markings assisted by the binding of specific factors to specific sites in the regions provides the identity of the gene or genes to be expressed or not to be expressed in specific tissues or in specific environment or inducer or repressor signaling.  This epigenetic inheritance of histones during determination and differentiation of cells is yet be discerned beyond doubt. It also provides markers for the association of gene specific or tissue specific factors or both, thus provides the inheritable constitutional input for the next generation of cells or tissues, which can respond to various inputs.  To give a simple example of yeast GAL genes regulation; GAL1, GAL7 and GAL 10 are located nearby on the chromosome 11 and their gene products are involved in utilization of Galactose in the absence of Glucose.  The promoter elements of the said genes located at specific loci are bound by GAL4.  The GAL4 binding remains at the location for any number of generations of cell lineages and the said genes remain repressed.

GAL4 is actually an activator, but GAL 80 by binding to GAL4 suppresses GAL4’ activators function.  This is an eukaryotic unicellular system; this structure and functional features may apply to all multicellular system.  When GAL4 is activated by release of GAL80, it recruits SAGA complex which facilitates transcription by remodeling nucleosomal structure and facilitate transcription.

 

Fig: A model for SAGA functioning as a co activator for Gal4 by facilitating TATA-binding-protein (TBP) binding to the TATA box of the GAL1 gene. (1) Under noninducing conditions, Gal4 is bound to the UAS_G via its DNA-binding domain (DB), and the Gal4 activation domain (AD) is blocked by Gal80. (2) After the addition of galactose, the Gal3 inducer is activated and alters the Gal80–Gal4 complex such that the Gal4 activation domain is no longer blocked by Gal80. This change allows the Gal4 activation domain to recruit SAGA to UAS_G. The presence of the Gal3 protein at the promoter is suggested by the formation of a Gal3–Gal4–Gal80 complex in vitro and in vivo (Chasman and Kornberg 1990; Leuther and Johnston 1992; Parthun and Jaehning 1992; Platt and Reece 1998; Sil et al. 1999). However, other data have suggested that Gal3 is cytoplasmically localized (Peng and Hopper 2000).  Once recruited to the promoter, SAGA, mainly via an Spt3-–TBP interaction, recruits TBP to the TATA box to allow transcriptional initiation; nucleosomes are not shown.

 

It also suggests that cell types generated during development contain specific factors associated with chromatin, which on stimulus can be activate gene expression.  Epigenetic is the hall mark of gene regulation in eukaryotic (EuK) and prokaryotic (PK) systems.  In PK specific operons are repressed and some operons are expressed, because of the binding of repressors and activators respectively and perpetuate the same for many generations. It is very important to remember that majority of the genes are expressed as housekeeping genes, which are required as sine quo non components.

Dynamic Histone Acetyl Transferases / Deacetylases and Methylases/demethylases and Ubiquitinases:

HATs: Histone Acetylases:

In avian and mammalian cells, transcriptionally active chromatin regions have core histones undergoing high rates of acetylation and deacetylation, while in repressed chromatin regions the rate of reversible acetylation and methylation is slow. Histone acetylation is a dynamic process. Transcriptionally active chromatin has core histones that are rapidly hyper acetylated (t1/2 =5 to 12 min for monoacetylated H4) and rapidly deacetylated (t1/2 = 3 to 7 min). A second population is acetylated (t1/2 = 200-300 min for monoacetylated H4) and deacetylated at a slower rate (t1/2 = 30 min for monoacetylated H4). 

Histone acetylation manipulates higher order chromatin and nucleosome structure. The tails of H3 and H4 are important in fiber-fiber interactions, suggesting that acetylation of the H3 and H4 tails will prevent chromatin fibers from interacting (repulsion) with each other. Also, histone acetylation has a profound effect on the solubility of chromatin in 150 mM NaCl or 3 mM MgCl₂. Acetylation of the core histones destabilizes histone-DNA contacts and has a role in maintaining the unfolded structure of nucleosomes. Thus, dynamic histone acetylation confers positional information for gene expression; this takes place in the chromatin of 300nm where DNA loops of 30nm are found. 

Dr. Dave Allis' group was first to purify a nuclear histone acetyl transferase (HAT A) and to clone its cDNA (Tetrahymena nuclear HAT p55). HAT p55 was found to be homologous to yeast GCN5, a transcriptional adaptor/co activator with HAT activity. While GCN4 acts as a gene activator the GCN5 acts as components of co activator responsible for acetylation. GCN4 as a pioneer factor binds DNA in sequence specific manner (promoter).  This pivotal discovery revealed how HATs were directed to specific chromatin regions for transcriptional activation.

HATs are components of multisubunit transcriptional co activators. It has been found that most of the genes expressed are found in chromatin loci where their specific histone tails are acetylated. And those genes remained suppressed or not activated are found to have methylated or deacetylated or the combination of both.  This need not be the case in situations.

Vincent Allfrey (in 1960s) discovered histone acetylation and deacetylation involved in transcriptional activation and inactivation in EuK (eukaryotic) systems. In 1990 HAT and HDATs proteins involved were identified and they found to act at specific positions of the Histone tails and some of them are well characterized. 

All HATs use acetyl-coA as group donors.  As there a large umber of them they grouped as five HAT families.

GNAT family:  Gcn5 related N-acetyltransferase; GCN5 was first found in

Yeast, it is well characterized; its related members are Gen5L, and PCAF, p300/CBP –and its associated factors; p300 and CBP means cAMP response element binding protein, they are homologous to trans-activational factors.  Hat1 is found in cytoplasm and acetylate histones before they are transported into the nucleus.

MYST family: named after founding members- Moz, Ybf2/Sas3, Sas2 and Tip60.

P300/CBP family: Very common, specially found to operate in many steroid activated gene regulation.

TAF Family; they are associated with TBP protein in RNA pol assembly complex.  TAF1 (also called earlier as TAFII 250) is the largest protein of TFIID complex that binds as the first component in the assembly of PIC.

 

The SRC family: steroid receptor co activators.

They have a variety o functions in transcriptional activation and also implicated in regulation of cell cycle and transcriptional regulation.

Most of the HATs act as multiple subunit complexes of 10-20 subunits, they form complexes such as SAGA (spt/Ada/Gen5L acetyl transferase), chromosome associated factors PCAF complex, STAGA (spt3/TAF/Gen5L acetyl transferase, where its HAT is Gen5L), ADA a transcriptional adaptor, TFIID –contain TAF1, TFTC -TBP free TAF-containing complex, NuA3 and NuA4 (Nucleosomal acetyl transferases of H3 and H4.

It is also interesting to find most of the above said complex does contain some common subunits such as Gcn5, Ada2 and Ada3 which are common to 14 subunit SAGA complex. Likewise Tra1 is common for both SAGA and NuA4,. Tra is a homolog of phosphoinositide 3-kinase (pI3K), it interacts with specific transcriptional activators such as MYC. It is also a fact that several HATs contain one or more TAFs.  For example SAGA contains TAF5, 6,9,10 and 12.  PCAF contains TAF 9, 10 and 12 but contain their close homologs PAF65b and 65a.

 

 

The BRG1 chromatin remodeling protein can associate with numerous chromatin-modifying complexes including transcription coactivators and corepressors. BRG1 (or hBrm) is the central catalytic subunit of SWI/SNF-BAF or -PBAF chromatin remodeling complexes, which have been implicated in the transcriptional activation or repression of a variety of genes. Nuclear receptors can associate with many of these complexes through direct interaction with BAF subunits such as BAF250, BAF60a and BAF57. BRG1 can be found in complexes with transcription coactivators and histone modifying enzymes such as WINAC and NUMAC. Conversely, BRG1 can be assembled in complexes known to repress transcription and induce gene silencing including NCoR and mSin3A/HDAC complexes.

 

 

Numerous studies have been conducted using various techniques which have identified BRG1-interacting proteins. For the purpose of this review, these BRG1-associating proteins have been grouped into two categories according to their transcriptional consequence: activation or repression. BRG1 has been reported to associate with numerous proteins implicated in transcriptional activation including various NRs such as AR (Marshall et al., 2003), ERα (Ichinose et al., 1997), GR (Fryer and Archer, 1998), PPARγ (Debril et al., 2004), PR (Vicent et al., 2006) and VDR (Kitagawa et al., 2003). Tumor suppressor proteins have also been found associated with BRG1, including BRCA1 (Bochar et al., 2000), p53 (Lee et al., 2002), and FANCA (Otsuki et al., 2001). Other proteins which are reported to interact with BRG1 include β-catenin (Barker et al., 2001), CARM1 (Xu et al., 2004), EVI-1 (Chi et al., 2003), Mef2D (Ohkawa et al., 2006), p130(RB2) (Giacinti and Giordano, 2006), Smad3 (Xi et al., 2007, 2008) and STAT proteins (Ni and Bremner, 2007; Pattenden et al., 2002). Proteins involved in transcriptional repression also interact with BRG1 and include GR (Bilodeau et al., 2006), HDACs (Underhill et al., 2000), HP-1 (Nielsen et al., 1999), Mbd3 (Datta et al., 2005), Mi-2β (Shimono et al., 2003), mSin3A (Sif et al., 2001), Rb (Giacinti and Giordano, 2006), PRMT5 (Pal et al., 2003), REST (Ooi et al., 2006), SMRT (Jung et al., 2001) and SYT-SSX (Ito et al., 2004; Perani et al., 2003). This list highlights a number of BRG1-interacting proteins which are considered transcription coactivators, corepressors or tumor suppressor proteins.

 

Look at the TAF6, 9 and 12 are the structural homologs of H3, H4 and H2b respectively. Thus, one can say they actually form an associated complex to interact with TBP.

Most of the HATs target their components to promoters and they found to be so in active genes.  The complex of HATs and their specificity is more intrinsic and it is only now people started to understand the activation of specific gene or group of genes,

The million-dollar question what makes specific HATs identify which loci or genes to be acetylated or is it a general phenomenon?  The answer lies in the type of HAT and the type of the site identified by certain factors, what are they? Very interesting aspect of HAT associated transcriptional co activators contain (mostly all) contain a ~110 residue module called a bromodomain.  Any protein that contains this domain binds to Acetylated Lysine moiety of Histone tails.

For example, GCN5 consists of HAT domain followed by a Bromodomain.  In the case of TAF1, it has N-terminal kinase domain followed by a HAT domain then two successive tandem Bromodomain.  The TAF1 targets specific acetylated histone tails. It contains two nearly identical anti parallel 4 helix bundles, where one finds pockets to recognize acetylated tails and bind to them.

An example to illustrate the activity of a gene shows that the N-terminal tail of H4 contains K residues at 5, 8, 12 and 16; it has been found the acetylation of these said residues increases the transcriptional activity of the said gene.  So, the question is what makes these specific sites to be acetylated among the millions of nucleosomes and how the acetylases determine the sites to which bind are promoter elements or other activating sites. Perhaps some specific DNA bound factor provides the site identity for acetylation.

It is also assumed that TAFs1 with its Bromodomain serves to target TFIID to the promoters that are found in the nucleosome containing sequence of a promoter or sequences in the vicinity of a promoter,

The critical feature is that the recruiting HAT-containing co activators complex to a specific upstream element bound by a protein.  Once such proteins bind to DNA, the HATs in them can promote acetylation of the nearing nucleosomal Histones and loosen the complex for the assembly of transcriptional complex to initiate transcription or to wait for other activators to act either to activate or suppress the activation.

In all probabilities chances of binding these acetylase complexes to specific sites or regions has to be identified that is already bound to DNA.  The logic is simple for all nucleosomes, irrespective the gene promoter they have in them, there should be an identity factor that is bound to DNA in sequence specific manner and such sites have to perpetuate during development.  Without such markers, it neighs possible for the HATs to identify the sites.

Histone Deacetylases (HDACs):

In 1996, Dr. S. Schreiber's group was first to clone a mammalian histone deacetylase (HDAC1). The study revealed that mammalian HDAC1 was related to yeast transcription regulator RPD3, providing a link between transcription regulation and histone deacetylation. Several HDACs have since been reported, including HDAC2 (the mammalian homologue of RPD3) and mammalian HDAC3. Mammalian HDAC1 and HDAC2, but not HDAC3, are in large multiprotein complexes containing mSin3, N-CoR or SMRT (corepressors), SAP18, SAP30, RbAp48, and RbAp46.

Several signal transduction pathways are regulated by the HDAC corepressor complex. For example, the Sin3A-N-CoR/SMRT-HDAC1, 2complexes is recruited by unliganded nuclear receptors and the Mad family of bHLH-Zip proteins.  An exciting development is the realization that methyl-CpG- binding protein 2 (MeCP2) binds to Sin3, recruiting more HDAC1/2 complex which could act on neighboring histones for deacetylation. These reports suggest that histone tail methylation at arginine and lysine residues and DNA methylation and histone deacetylation are coupled events in the formation of repressive chromatin structures and gene silencing. 

 

Composition of HDAC repressor complexes: HDACs lack intrinsic repressor activity and require co-factors for optimal HDAC activity. The co-repressor proteins involved in the major HDAC complexes NuRD (nucleosome remodeling and deacetylase), Sin3 (Switch insensitive 3), Co-REST (Co-repressor of REST (RE1 silencing transcription factor)) and N-CoR and SMRT complexes are shown. NuRD and sin3 complexes share the retinoblastoma associated protein (RbAp)46 and 48 proteins and also contain distinct sets of proteins. Abbreviations: Co-REST, Co-repressor of REST (RE1 silencing transcription factor); MBD3, Methyl CpG binding domain 3; Mi2, Mi2 autoantigen; MTA-2, Metastasis-associated gene family, member 2; N-CoR, Nuclear receptor co-repressor; NuRD, Nucleosome remodelling and deacetylating; RbAp46, Retinoblastoma associated protein of 46 kDa; SAP18, Sin3 associated protein of 18kDa; SDS3, Suppressor of defective silencing 3; Sin3, Switch insensitive 3; SMRT, Silencing mediator for retinoid and thyroid receptors; ZNF217, Zn finger factor 217 kDa. Adcock et al. Respiratory Research 2006 7:21   doi:10.1186/1465-9921-7-21.

It is reported that HDAC1 is associated with MAR- DNA in human breast cancer cells. These results suggest that HDAC1 may have a role in the organization of nuclear DNA. It is interesting to note that attachment region binding protein (ARBP), a nuclear matrix protein that binds to MARs, is homologous to MeCP2. Thus, N-CoR-Sin3A-HDAC1 complex could be recruited to the nuclear matrix and to MAR-DNA by MeCP2/ARBP.

Several nuclear factors can recruit HDAC directly without the assistance of the mSin3, N-CoR and SMRT. HDAC 1, 2 and 3 bind to YY1. Hypo phosphorylated Rb and E2F form a complex with HDAC1. The recruitment of the E2F-RB-HDAC1 complex is partly responsible for the repression of the cyclin E promoter in G1 phase of the cell cycle. 

Aberrant recruitment of histone modifying enzymes is seen in cancer. For example, PML-RAR (PLZF-RAR) and AML-1-ETO, oncoproteins in acute promyelocytic or myeloid leukemia generated by chromosomal translocations, recruit SMRT-mSin3A-HDAC1 and N-CoR-mSin3A-HDAC1, 2 complexes, SMRT-mSin3A-HDAC1 complex is recruited by the BTB/POZ domain found in the oncoproteins LAZ3/BCL6.

Isolation of novel mammalian PRMTs by molecular cloning and novel substrates (histones and non-histone proteins) and determining the corresponding methylation sites to define novel gene regulatory pathways in which this posttranslational modification is involved is important. With respect to the histone code hypothesis, it is now known whether site-specific arginine methylation interferes/cross-talks with other post-translational modifications and how this modification is "translated" into chromatin alteration on the molecular level. Discovery of reverse arginine methylation by specific demethylase therefore it is a dynamic process as it has been shown to be the case for other histone modifications.

Histone Deacetylases by removing acetyl group allow other components to bind or otherwise makes the region inaccessible for the assembly of transcriptional components including PIC or BTA and other upstream factors that regulated the gene expression; thus repress the expression a gene.  Deacetylation of specific lysine otherwise of histone tail, provides an opportunity for other enzymes to join and modify the tails in different and generate different codes.  Example, methylases that methylate histone tail at specific amino acid residues, which need not be the same residue that is deacetylated.   They can be different.  Among the many; ten HDAs have been identified with certainty. There are ten HDAs in yeast, 17 in humans.  HDAs have a family of proteins such as Class I and Class 2 etc.  There are at least three classes- such as Class I, Class II and Class III; most of them are multisubunit complexes.

In humans the HDACs consist of the said three classes: Class I-contain HDAC 1, 2, 3 and 8.  Class II consists of HDACs 4, to 7 and 10. And the Class III contains Sirutins (SIRT1-7) (SIR for Silent information regulators).  Most of them are multisubunit complexes, where some are common to all the classes and some are specific to each of the classes.  Some of the components are –Sin 3, NurD (nucleosome remodeling histone deacetylases), Co rest (co repressor of RE1 silencing transcription factor), NCOR (nuclear hormone receptor co repressor), SMRT (silencing mediator of retinoid and thyroid hormone receptor), Most of them serve as transcriptional co repressors at different sites and with different composition.  For example, REST on binding to its target, recruits Co-Rest and SIN3, which together repress the expression of the gene where they bind.  Such methylated complexes produce compaction of the chromosomal loci and make it heterochromatic, which can be discerned Giemsa staining.

Histone Methylases and Demethylase:

Lysine and Arginine residues in H3 and H4 tails are the targets for methylation by Histone methyl transferases (HMTs).  They use S Adenosyl Methionine, SAM as the methyl donor.  The enzymes have SET domain [(Su (var) 3-9, E (Z), Trithorax], contain catalytic sites.  Interestingly no demethylase has been identified, which means methylation is not reversible (?).  But in some cases, trimethylation of a histone tail is demethylated to di methylated sites.  Recent investigations have shown that demethylase exist.  Methyl groups are removed by specific demethylases.

 

Methylated histone tails are recognized by proteins containing Chromodomain.  For example methylated H3 at lysine 9 is recognized by Chromodomain containing Heterochromatin 1 (HP1) protein.  The binding of HP1 recruits other proteins to control chromatin structure and gene expression.  The said enzymes have active sites as clefts or grooves where only such tails bind with specific methylated amino acid (s).  This binding can lead to spreading to nearby chromatin to be silenced.  This happens due to to other domain called Chromodomain shadow domain of HP1 is associated with methyl transferase (recruits HMT Suv 39h protein complex) that methylates the neighboring histone tails, which recruits more HP1 proteins.

Thus heterochromatin spreads.  However, the spreading heterochromatization is often checked by insulators found in the pathway of heterochromatization. ex – chick b-globin clusters recruit HATs that acetylates H3 lys9 at nearby nucleosomes, thus it checks the spreading of heterochromatization.  Question is what makes the methylation mediated HP1 binding leads to heterochromatization or what we call it as condensation of chromatin?

Histone Ubiquitination and Transcription:

Ubiquitination to protein via lysine ligation leads to proteasome mediated protein degradation. But in yeast ubiquitination of H2B Lys 123 mediated by ubiquitin ligase i.e. Rad6 and E3 Bre1 are responsible for methylation of H3 K4 and k79.  This modification results in silencing genes located near telomere.  It is suggested that H2B ubiquitination (not for destruction) functions as master switch that controls selective methylation and silencing telomeric gene silencing.  It is interesting to find that TAF1 subunit of TF IID is involved in post translational modification of has been found in Drosophila.  It appears that ubiquitination of certain Histones sites is emerging as regulators.

 

 

Model for sumoylation function in transcription: Horizontal line represents a gene with a TATA box-containing promoter and ORF; ovals represent histone octamers/nucleosomes. Through a co activator, a DNA-bound activator can recruit a histone acetyltransferase (HAT) that acetylates histones and promotes chromatin structure amenable to transcription. This acetylation can potentially recruit SUMO-conjugating enzymes (E2/E3) capable of modifying either histones or activators to give an attenuating effect. A corepressor and HDAC activity could then be recruited by a DNA-bound repressor (possibly even with SUMO contributing to the interaction), deacetylation of histones, and making way for the addition of repression-specific methylation marks, such as H3 K9-methyl, by an HMT. Finally, methylated histones (and possibly SUMO) would recruit HP1, contributing to chromatin structure in a static repressed state.

Histone Modifications

The SUMO modification of proteins in general is required for normal chromosome condensation and mitosis. Disruption of sumoylation pathway leads to mitotic defects and embryonic development failure characterized by inability of cells to properly condense chromatin. We pursue a hypothesis that mitotic chromatin condensation and heterochromatic gene silencing are intrinsically linked at the biochemical level, both being dependent on SUMO modification of structural proteins and enzymatic machinery shared by these processes.

Possible Roles of modified Histones (from Harper):

1. Acetylation of histones H3 and H4 is associated with the activation or inactivation of gene transcription. 2. Acetylation of core histones is associated with chromosomal assembly during DNA replication.3. Phosphorylation of histone H1 is associated with the condensation of chromosomes during the replication cycle.
4. ADP-ribosylation of histones is associated with DNA repair.
5. Methylation of histones is correlated with of activation and repression gene transcription. 6. Mono-ubiquitylation is associated with gene activation, repression, and heterochromatic; gene silencing. 7. Sumoylation of histones (SUMO; small ubiquitin-related modifier) leads to transcription repression.

 

Chromatin Remodelling and Regulation of Gene Expression:

In prokaryotes bacterial circular DNA is also compacted by histone like proteins; but DNA in eukaryotes is compacted into chromatin; this compaction is essential for the DNA is very long as 3.2x10^9bp in humans or more in some 10^12bp.  Such a long and so thin 10.5A thick thread cannot be subjected to such changes during cell division, repair and recombination.  The DNA has to go through opening and closing at different sites may be in thousands during transcription.  In order to protect the DNA from shear and wear it is stabilized and strengthened by histones, which compacts DNA to 30nm thread and with the association of scaffold proteins it is further strengthened and compacted to 300nm fibre, which remains so through the cellular changes except DNA replication.  Rest of the time this 300nm fibre very often undergoes regional compaction at different loci or regions.  Such compactions are called heterochromatin and uncompacted 300n fibre is called euchromatin, which is engaged in transcription.  The 300nm fibre is bound by thousands of nonhistone proteins; that makes chromosome visible structure.  This chromatin exists in relaxed state at interphase where its HC domains remain bound to inner nuclear membrane matrix proteins. Some region of interphase chromatin are relaxed and involved in transcription, but there are regions where the 300nm chromatin is further condensed by heterochromatization, which can be constitutive (pericentric and telocentric regions) or facultative (its position varies in different tissue types.  The heterochromatic condensation is greater than metaphase condensation.  Amount of DNA per unit in HC is more than metaphase compacted chromatin. The HC regions can be visualized by differential staining (Giemsa) of metaphase chromosomes.

 

DNA methylation: Fingerprints of (epi) Genome:

DNA methylation takes place in specific DNA sequences such as 5’-CpGpCpGpCpG-3’.  Such 5-CpG-3’ repeats can be found 500 base pairs long and are located mostly within gene 5’promoters’ regions of genes.  Such repeat sequences are called CpG islands.  Such groups called islands can be used as finger prints of a genome. Methylation of Cytosine at 5’ position takes place by Cytosine methyl transferase using S-Adenosylmethionine (SAM) as the donor.  Such CpG islands are also found in other regions such as coding regions and non-coding regions too.  Such CpG repeat islands are located in different parts of the genome and it is a fixed pattern of the genome.  But such repeats in gene promoter region get methylated 5’C-CH3-G3’. 

Developmental Biology; We are studying common mechanisms of chromatin remodeling, DNA methylation and transcriptional regulation in lung development that might be activated in lung diseases, Directed by Wellington V. Cardoso, MD, PhD; http://www.bumc.bu.edu/

 

DNA methylation is performed a group of methylases called DNMTs.  There are DNMT1, DNMT3a and DNMT3b.  The DNMT1 performs methylation of hemimethylated DNA strand during replication. There are enzymes which remove such methyl groups from CpG islands.  Such DNMTs inhibitor is Azacytidine. Treatment with Azacytidine makes the genes which are inactive become active. Methylation of 5-CpG-3 leads to the recruitments of specific proteins such as MeCP2, which recruits HDAC.  Such methylation of 5’-CpG-3 islands in promoter regions renders the gene inactive.  This is achieved by recruiting HDACs and heterochromatin binding proteins such as HP1 etc.  This makes the gene promoter inaccessible for transcriptional factors and enzymes.

In most cases, methylation of DNA is a fairly long-term, stable conversion, but in some cases, such as in germ cells, when silencing of imprinted genes must be reversed, demethylation can take place to allow for "epigenetic reprogramming." The exact mechanisms for demethylation are not entirely understood; however, it appears that this process may be mediated by the removal of amino groups by DNA deaminases (Morgan et al., 2004). After deamination, the DNA has a mismatch and is repaired, causing it to become demethylated. In fact, studies using inhibitors of one DNMT enzyme showed that this enzyme was involved in not only DNA methylation, but also in the removal of amino groups; http://www.nature.com/scitable/topicpage.

 

Methylation modification of DNA at the 5-carbon position of cytosine by DNMTs where SAM donates the –CH3 group and is converted to SAH.  This reaction is potentially reversible by a yet to be defined DNA methylase.

A permissive state for transcription includes histones acetylated by HAT as well as methylated at H3K4 (H3K4me). Unmethylated CpGs are bound by transcription factors and RNA polymerase II thereby blocking interaction with DNMT. Environmental influences potentially trigger reversal of acetylation by HDAC and removal of methylation by LSD1. In this state, CpGs are vulnerable to methylation by DNMT and are bound by MeCP2, which recruits HDAC. HDAC maintains a deacetylated state of the histone, locking the chromatin in a repressed state that prohibits transcription factor binding. Presently the precise order of these events is unclear.

Chromatin structure regulates transcriptional activity: Histone-deacetylase inhibitors: novel drugs for the treatment of cancer; Nucleosomes consist of DNA (black line) wrapped around histone octomers (purple). Post-translational modification of histone tails by methylation (Me), phosphorylation (P) or acetylation (Ac) can alter the higher-order nucleosome structure. Nucleosome structure can be regulated by ATP-dependent chromatin remodellers (yellow cylinders), and the opposing actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Methyl-binding proteins, such as the methyl-CpG-binding protein (MECP2), target methylated DNA (yellow) and recruit HDACs. a | DNA methylation and histone deacetylation induce a closed-chromatin configuration and transcriptional repression. b | Histone acetylation and demethylation of DNA relaxes chromatin, and allows transcriptional activation, Ricky W. Johnstone; http://www.nature.com/

 

DNA Methylation: The diagram shows a representative region of genomic DNA in a normal cell. The region shown contains repeat-rich, hypermethylated pericentromeric heterochromatin and an actively transcribed tumour suppressor gene (TSG) associated with a hypomethylated CpG island (indicated in red). In tumour cells, repeat-rich heterochromatin becomes hypomethylated and this contributes to genomic instability, a hallmark of tumour cells, through increased mitotic recombination events. De novo methylation of CpG islands also occurs in cancer cells, and can result in the transcriptional silencing of growth-regulatory genes. These changes in methylation are early events in early tumorigenesis   © 2005 Nature Publishing Group  Robertson. DNA methylation and human disease. Nature Reviews Genetics 6, 598’

 

There are many ways that gene expression is controlled in eukaryotes, but methylation of DNA (not to be confused with histone methylation) is a common epigenetic signaling tool that cells use to lock genes in the "off" position. In recent decades, researchers have learned a great deal about DNA methylation, including how it occurs and where it occurs, and they have also discovered that methylation is an important component in numerous cellular processes, including embryonic development, genomic imprinting, X-chromosome inactivation, and preservation of chromosome stability. Given the many processes in which methylation plays a part, it is perhaps not surprising that researchers have also linked errors in methylation to a variety of devastating consequences, including several human diseases.  Experiments with 5-azacytidine provide early clues to the role of Methylation in gene expression. DNA methylation occurs at the cytosine bases of eukaryotic DNA, which are converted to 5-methylcytosine by DNA methyltransferase (DNMT) enzymes. The altered cytosine residues are usually immediately adjacent to a guanine nucleotide, resulting in two methylated cytosine residues sitting diagonally to each other on opposing DNA strands.  Methylation can be observed by staining cells with an immuno fluorescent labeled antibody for 5-methylcytosine. In mammals, methylation is found sparsely but globally, distributed in definite CpG sequences throughout the entire genome, with the exception of CpG islands, or certain stretches (approximately 1 kilobase in length) where high CpG contents are found. Note the methylation of these sequences can lead to inappropriate gene silencing, such as the silencing of tumor suppressor genes in cancer cells.  results of immunoprecipitation studies using human cells suggest that DNA methylation and histone methylation work together during replication to ensure that specific methylation patterns are passed on to progeny cells (Sarraf & Stancheva, 2004).

 

In an interestingly coordinated process, proteins that bind to methylated DNA also form complexes with the proteins involved in deacetylation of histones. Therefore, when DNA is methylated, nearby histones are deacetylated, resulting in compounded inhibitory effects on transcription. Likewise, demethylated DNA does not attract deacetylating enzymes to the histones, allowing them to remain acetylated and more mobile, thus promoting transcription. In most cases, methylation of DNA is a fairly long-term, stable conversion, but in some cases, such as in germ cells, when silencing of imprinted genesmust be reversed, demethylation can take place to allow for "epigenetic reprogramming." The exact mechanisms for demethylation are not entirely understood; however, it appears that this process may be mediated by the removal of amino groups by DNA deaminases (Morgan et al., 2004). After deamination, the DNA has a mismatch and is repaired, causing it to become demethylated. In fact, studies using inhibitors of one DNMT enzyme showed that this enzyme was involved in not only DNA methylation, but also in the removal of amino groups. Theresa Phillips, Ph.D. (Write Science Right) © 2008 Nature Education. http://www.nature.com/

 

Silent Chromatin in the Middle – “Centromere”:

Central Centromere is bordered by a distinct heterochromatin a dominant region of the chromosome microscopically visible.  It looks like a not stainable gap as if there is no DNA in that region.  Centromere plays a par excellent role in chromosomal movement during mitosis and meiosis.  This acts as core structural component for the binding of Kinetochore complex and for the tractile fibers to attach and pull sister chromatids or homologous chromosomes to their respective poles.  The central region of centromere is bound by histone octamers but one of the histones is different called CEP-A/B.  This part of the nucleosomes is free from heterochromatization; how this region is left free from heterochromatization while the borders are distinctly heterochromatic very condensed structure?  This is designed for the binding of kinetochore during metaphase. Is the early kinetochore element preventing such heterochromatization?  On either side of central centromere, one finds a distinct heterochromatin.  Its HC formation is similar to telomere where H3K9 deacetylation and its methylation leads to the binding of HP1; this spreads to neighbor nucleosomes.

 

 

Structurally the CEN region appears as a gap (non-stainable), but on either side of the CEN region one finds darkly stainable chromatin called constitutive heterochromatin; none of the genes in the region are active but silent.  Structurally the CEN region is made up of satellite DNA for that matter most of constitutive heterochromatin is made up of satellite DNA (?). Centromeric (CEN) chromatin is embedded in heterochromatin and contains blocks of histone H3 nucleosomes interspersed with blocks of CENP-A nucleosomes, the histone H3 variant that provides a structural and functional foundation for the kinetochore. The spectrum of histone modifications present in human and Drosophila melanogaster CEN chromatin is distinct from that of both euchromatin and flanking heterochromatin.

 

A model for DNA bundling by the CENP-B dimer in centromeric chromatin: Orange ribbons with arrowheads indicate 171-base pair α-satellite repeats. Green circles show the centromeric nucleosomes, which may contain histones (H2A, H2B, and H4) and CENP-A. The crystal structures of the dimerization and DNA-binding domains of CENP-B are connected by the Pro-rich region (yellow dashed lines), the central transposase-like domains (pink dashed circles), and the Asp/Glu-rich region-blue dashed lines

 

This is the CENP-B that is associated with CEN DNA

 

 

The kinetochore can be thought of as three sets of subcomponents. a) The chromosomal DNA-inner kinetochore protein interface. b) The inner kinetochore-mitotic spindle interface. c) The kinetochore protein-cell cycle machinery interface. APC anaphase-promoting complex; CEN, centromeric DNA; SCF, ubiquitin-ligase complex. Copyright 2007 Nature Publishing Group, Kitagawa, K., et. al., The spindle-assembly checkpoint in space and time, Nature Reviews Molecular Cell Biology 2, 678-687.

 

http://www.cbs.dtu.dk/

 

 

           

Centromere DNA consists of tandem repeats of LINES and SINES and AT rich DNA.

 

 

 

Tandem repeat sequence of DNA at telomeres and CEN regions

 

 

 

Figure: Proposed structure for centromere DNA at the kinetochore region. (Top) Bi-oriented sister chromatids adopt a cruciform structure. Centromere-flanking chromatin is held together by intra-strand Cohesins bridges and chromosome arms by interstrand cohesin’s bridges. The transition between these two regions in budding yeast is mobile and on average 7 kb from the centromere core. (Bottom) The Cse4-containing nucleosome (orange circle) and flanking nucleosomes (green circles) are proximal to the microtubule plus-end. http; Current Biology.

 

 

Figure: Model for three-dimensional organization of centromeric (CEN) chromatin in D. melanogaster and humans.

Incorporation of the two-dimensional and three-dimensional histone modification patterns extends our understanding of the chromatin composition and organization of the CEN region, and suggests that interspersed CENP-A/CID and H3 Lys4-dimethyl nucleosome blocks comprise a unique chromatin state that is distinct from the flanking heterochromatin. Associations between similarly modified nucleosome blocks are proposed to contribute to the formation of distinct three-dimensional structures in CEN and flanking chromatin. Interspersed CENP-A/CID and distinctly modified H3 and H4 may mediate formation of the 'cylindrical' three-dimensional structures observed in metaphase chromosomes.  H3 Lys9-diMe chromatin, which recruits heterochromatin proteins such as HP1 and cohesion proteins such as RAD21/SCC1, is present in the inner kinetochore space between mitotic sister chromatids and in regions that flank CEN chromatin. This arrangement may position CENP-A toward the pole-ward face of the mitotic chromosome and facilitate recruitment of outer kinetochore proteins, and promote HP1 self-interaction and proper chromosome condensation and cohesion. Cohesins are presented as ringed structures, in accord with recent models^.

 

 

Fig: Model for the kinetochore. CenH3 hemisomes (red/gray disks) are separated by extended linker DNAs and so are decondensed relative to surrounding heterochromatin (blue disks). Asymmetric CenH3 nucleosomes assemble in random orientations [CenH3/H4 (red) and H2A/H2B (gray)]. Only one unit of a CenH3-rich block is shown. During mitotic condensation, heterochromatin packs tightly as a result of its homogeneity. Intervening blocks of CenH3 chromatin cannot pack into this crystal-like structure because of its smaller size, long linkers, and heterogeneity in its relative orientation, resulting in extruded loops of uncondensed CenH3 nucleosomes that serve as the foundation for kinetochore formation. The flanking gray cones represent pericentric regions flanking the primary constriction.

 

 

 

 

 

Schematic depicting centromeric chromatin composition in relation to the cell cycle. CENP-A–containing nucleosomes (red) are interspersed with canonical H3-containing nucleosomes (green) after replication in S phase, and this mixed set of nucleosomes is the substrate for nucleating kinetochore assembly in mitosis and is maintained as cells exit in anaphase. CENP-A assembly initiates in telophase and proceeds through early G1 (presumably concurrent with removal of H3 nucleosomes). CENP-A– and H3-containing nucleosomes are stylized as single nucleosomes but may represent continuous alternating arrays of one or the other type. In mitosis, CENP-A nucleosomes may coalesce to form a rigid interface for kinetochore formation as proposed previously (Zinkowski et al., 1991; Blower et al., 2002; Black et al., 2004,2007a,b

 

Telomeric Region and its Repression:

Telomeric silencing requires proteins such as RAP1 that binds to DNA and Sir (Silent information Regulator) proteins bind to RAP (Ras related Protein); RAP acts as nucleation centre.  Sir2 is deacetylase protein. Proteins such as Sir1, 3 and 4 associate as a complex in telomeric region and silence the genes, if any. And HMR (yeast mating Region?) region in yeast also performs in similar fashion.  Telomeric tail lacks histones.  In the proximal region Histone H3 and H4 tails Sir3 and Sir4 bind to histone tails and maintain the tails in deacetylated state.  Hypo-acetylated regions are associated with Sir2; such complexes bound to each other and prevent the entry of any transcriptional regulator proteins.

Repression of certain gene loci such as HML and HMR is due to condensation of chromatin in promoter-regulator regions.  Adenine methylases gene introduced into yeast cell methylates GATC sequences in MAT region but not HML and HMR for they are already compacted for the enzyme to act. 

Chromosomes with glowing telomeric ends

 

 

Any gene placed near telomere gets silenced.  RAP1 and SIR proteins bind to telomere region for repression. RAP1 binds to Telomere single stranded DNA (extended) sequences, which are tandemly repeated.  SIR2 is HDAC, RAP1 binds to DNA, SIR3 and SIR4 bind to RAP1 and Sir2 binds to Sir4; These RAP1 and Sir proteins bind to hypo acetylated histones; this association spread to neighbor histone bound telomeric regions.  Sir3 and 4 bind to hypoacetylated H3 and H4 tail and Sir2 maintains them in deacetylated state. Fluorescence labeled probes show that telomeric regions are condensed state like other Heterochromatin bands.  A similar programme is operated in pericentric region; however, there is predominance of H3H4 modification operates in CEN region.

Chromatin- compacted and silent

Silent chromatin at the ends: lessons from yeasts: Marc Bühler and Susan M Gasser.

Heterochromatin in S. cerevisiae, S. pombe, P. falciparum and human.

 

 

Figure Hypothetical model for heterochromatin assembly at P. falciparum chromosome ends. This is a general view of the known chromatin components at P. falciparum sub telomeres. The spreading of heterochromatin along the different TAREs into adjacent coding regions probably involves PfHP1, PfSir2 and PfKMT1 in cooperation. The role of PfOrc1 in this process remains unknown.

 

In most organisms, histones H3 and H4 are among the most conserved proteins. Their amino terminal NH2 terminal tails, which are necessary for the formation of the nucleosomal core, are fairly conserved in terms of their sequence, particularly at residues that are susceptible to specific covalent modifications. The amino terminal ends of histones H3 and H4 of P. falciparum contain sites susceptible to post-translational changes. Several modifications have been identified in P. falciparum by mass spectrometry analysis of histones, Western blot assays performed with antibodies specific to methylated and acetylated histones, and ChIP on ChIP assays. Those modifications are: acetylation of histone H3 at residues K9, K14, K18, and K27 and of histone H4 at K5, K8, K12 and K16; methylation of histone H3 at K4, K9 and K36 and histone H4 at K20, as well as the sumoylation of histone H4. Recently, a comprehensive mass spectrometry analysis of P. falciparum histones identified 44 new post-transcriptional modification sites in these proteins, most of them associated with a transcriptionally permissive state.

 

a, Telomeric heterochromatin is nucleated by the interaction of Sir2–Sir4 with telomere-binding proteins. b, After the deacetylation of histone tails by Sir2, Sir3 is recruited. c, Propagation of heterochromatin caused by further rounds of histone deacetylation and binding of Sir protein. d, HTZ1-containing nucleosomes represent a barrier and protect active regions from being silenced.

Schematic model of silencing mechanism at yeast telomeres:

 Fig: Multiple copies of Rap1 bind to a simple repeated sequence at each telomere region, which lacks nucleosomes (top). This nucleates the assembly of a multiprotein complex (bottom) through protein-protein interactions between Rap1, Sir2, Sir3, Sir4, and the hypoacetylated amino-terminal tails of histones H3 and H4 of nearby nucleosomes. Asterisks represent hyperacetylated histone amino-terminal tails. The heterochromatin structure encompasses ≈4 kb of DNA neighboring the Rap1- binding sites, irrespective of its sequence. The actual structure of the higher-order heterochromatin is not yet understood. See text. [Adapted from M. Grunstein, 1997, Curr. Opin. Cell Biol. 9:383.]

Telomere consists of repeat sequence of TTAGGG with single stranded 3’ tail. The length of this sequence shrinks and expands in time.  This sequence is protected from exonuclease activity by heterochromatization. The 3’ single stranded tail is bound by RAP1 protein which gets associated with Sir proteins.  Posterior to this tail one finds dsDNA associated nucleosomal structure, which is also bound by Sir3 and Sir4 to hypoacetylated and methylated histone tail and Sir2 a deacetylase is associated with Sir4; interaction between Sir proteins leads to looping and heterochromatization (compaction).

Silencing at the telomeres

Telomeres can be found at the ends of all 16 chromosomes in yeast for that matter in all chromosomes and in all organisms. They consist of a 300-350 bp region with irregular TG_1-3 repeats, which are disrupted by X- and Y-elements of variable size (Palladino and Gasser, 1994). The Rap1 binds on average every 18 bp in the telomeric repeats and is a structural core component of the telomeres. The Rap1 binding region is also referred to as the telosome and is characterized by its nuclease resistance.

Rap1 in turn recruits Sir2, Sir3 and Sir4 to the telomeres, where Sir3 and Sir4 bind to the N-termini of histone H3 and H4. The Sir proteins spread in a histone-dependent manner up to 2.8 kb into the core telomeric heterochromatin. (Strahl-Bolsinger, et al., 1997) have proposed a model in which telosomal Rap1 folds back onto subtelomeric regions. This allows a condensation at telomeric heterochromatin due to the interaction between Rap1 and the Sir’s, as well as among the Sir proteins themselves.

Silencing at the telomeres and subsequent condensation prevents the chromosome ends from being degraded and improves their replication (Palladino, et al., 1993). Additionally, the positioning of the telomeres to the nuclear periphery is maintained.

Telomeres: protecting chromosomes against genome instability

Roderick J. O'Sullivan & Jan Karl Seder

 

G1 phase telomeric chromatin has hallmarks of constitutive heterochromatin: the trimethylation of Lys9 of H3 (H3K9me3) and of Lys20 of H4 (H4K20me3) and heterochromatin protein 1 (HP1) binding (see the figure, part a, right). Sub telomeric, chromatin can be distinguished by a regular nucleosomal distribution, extensive DNA methylation and histone post-translational modifications that are distinct from those at telomeres (part a, left). The replication of DNA during S phase coincides with the disruption and restoration of the parental chromatin identity. Newly synthesized histones are acetylated at Lys5, Lys12 and Lys16 of H4 and at Lys9 and Lys56 of H3. The removal of these acetyl groups at telomeres by SIRT proteins seems to be important for regulating the association of accessory proteins to telomeres, as exhibited by the relationship between the acetylation of Lys9 of H3 (H3K9ac), SIRT6 and Werner syndrome ATP-dependent helicase (WRN) function.

 

Changes in chromatin structure have been shown to occur at dysfunctional telomeres. These changes are often similar to those exhibited at sites of DNA damage, such as phosphorylation of H2AX, changes in H4K20me2 levels and recruitment of tumour suppressor p53-binding protein 1 (TP53BP1), implying that there is a general 'epigenetic' stress response (see the figure, part c). However, it is still unclear whether changes seen at dysfunctional telomeres are proactive or merely responsive to changes in telomeric architecture. Nuclear reprogramming also leads to dramatic changes in telomeric chromatin and telomere length, emphasizing the dynamic and developmentally regulated nature of chromosome ends. POT1= protection of telomeres 1; TERRA= telomeric repeat-containing RNA; TRF2= telomeric repeat-binding factor 2.

 

Figure: Human telomere components and structure. A) Graphic representation of the telomeric DNA in human cells, which is normally composed of 4-12 kb of G-rich repeats (TTAGGG in red, AATCCC in blue), culminating in a 100-200 nt 3' overhang. Open and t-loop configurations are shown. B) Graphic representation of telomeric DNA with associated proteins. The six subunit “shelterin” or “telosome” complex coats the length of the duplex telomeric DNA via the direct interaction of TRF1 and TRF2 with telomeric DNA. TIN2 interacts with both TRF1 and TRF2 but not with telomeric DNA. TPP1 (formerly PTOP, PIP1 or TINT1) bridges TIN2 with POT1. POT1 interacts specifically with single-stranded G-rich telomeric DNA, presumably at the chromosome end when the telomere is in open configuration, or at the single-stranded region within the t-loop junction. Telomeres are also assembled into chromatin.

                                                          Telomeric structural organization

Heterochromatins:

 

 

 

Heterochromatin in General:

Chromatin

The basic unit of chromatin organization is the nucleosome, which comprises 147 bp of DNA wrapped around a core of histone proteins. Nucleosomes can be organized into higher order structures and the level of packaging can have profound consequences on all DNA-mediated processes including gene regulation. Euchromatin is associated with an open chromatin conformation and this structure is permissible for transcription whereas heterochromatin is more compact and refractory to factors that need to gain access to the DNA template. Nucleosome positioning and chromatin compaction can be influenced by a wide-range of processes including modification to both histones and DNA.

The nucleosome, made up of four histone proteins (H2A, H2B, H3, and H4); they are the primary building blocks of chromatin.  Originally it was thought to function as a static scaffold for DNA packaging.  Histones, more recently been, have shown to be dynamic proteins; undergoing multiple types of post-translational modifications. Two such modifications, methylation of arginine and lysine residues are major determinants for formation of active and inactive regions of the genome. Arginine methylation of histones H3 (Arg2, 17, 26) and H4 (Arg3) promotes transcriptional activation and is mediated by a family of protein arginine methyltransferases (PRMTs), including the co-activators PRMT1 and CARM1 (PRMT4). In contrast, a more diverse set of histone lysine methyltransferases has been identified, all but one of which contain a conserved catalytic SET domain originally identified in the Drosophila Su[var]3-9, Enhancer of Zeste, and Trithorax proteins. Lysine methylation has been implicated in both transcriptional activation (H3 Lys4, 36, 79) and silencing (H3 Lys9, 27, H4 Lys20).  Histone H3 and H4 acetylation is common in active chromatin.  These modifications for gene activation or repression are mostly found in the upstream of the promoter and promoter regions.

Euchromatin in certain regions become heterochromatin and vice versa; histone deacetylation, histone methylation and the binding of corepressor like HP1 leads to heterochromatization; on the contrary demethylation and acetylation, phosphorylation, and specific H3K4 methylation and loss of H1 lead to euchromatization.

The figure shows activation and inactivation by specific histone modification and association of repressor and activator complexes.  Histone modification show changes in the compaction and relaxation of nucleosomal thread. Binding of activator or repressor to certain sequences act as nucleating centers for gene activation or repression respectively.

 

Schematic depiction of the role of histone acetylation in chromatin remodeling: Acetylation of histone carboxyl-terminal tails is believed to promote unpacking of nucleosomes from 30-nm chromatin fibers. Histone PTM enzymes or agents that can influence the state of chromatin packing are indicated as favoring either side of this reversible process. Possible roles of phosphorylation and ubiquitination are less well understood because these effects may be dependent upon modifications of specific amino acids or influenced in the context of other modifications concurrently existent histones.

Oncogenic fusion proteins such as AML1–ETO, CBFB–MYH11 and PML–RARA recruit transcriptional co-repressor complexes (including nuclear receptor co-repressor 1 (NCOR1) and NCOR2) that result in the loss of histone acetylation and the acquisition of repressive histone modification marks, such as histone H3 lysine 9 (H3K9) methylation and H3K27 trimethylation, as well as DNA methylation, and thereby a closed chromatin structure. This leads to the transcriptional silencing of various target genes, including genes that are crucial for haematopoietic differentiation. Epigenetic or transcriptional therapy (targeting the fusion proteins, components of the co-repressor complexes and downstream effectors such as microRNAs) has the potential to reverse these changes, leading to histone acetylation, acquisition of active marks such as H3K4 methylation, an open chromatin structure with subsequent transcriptional activation and differentiation of the leukemic clone. Ac, histone acetylation; AML1, acute myeloid leukemia 1; CBFB, core binding factor-β; CpG, cytosine residues that precede guanosine; DNMT, DNA methyltransferase; HDAC, histone deacetylase; HMT, histone methyltransferase; PML, promyelocytic leukemia; RARA, retinoic acid receptor-α; RNApol II, RNA polymerase II; TF, transcription factor

One of the main experimental techniques that has allowed the elucidation of chromatin structure and function is chromatin immunoprecipitation (ChIP). The first step in ChIP analysis is to cross-link the chromatin associated protein to DNA in live cells. The cells are then lysed and DNA complexes are sheared into small fragments, and the protein of interest is immunoprecipitated. Consequently, DNA sequences that interact with the protein of interest are enriched in this immunoprecipitation stage. Protein–DNA cross-links are subsequently reversed and the amount of DNA precipitated is quantified, usually by real-time PCR or microarray analysis. Thus, ChIP analysis, using antibodies to specific chromatin-associated proteins, together with an analysis of individual histone modifications, enables the characterization of endogenous chromatin structure at individual genomic regions.

 

 

Methylation and Acetylation of Histone in Specific Regions of the Nucleosomes is the Key for Gene Expression:

Localized gene silencing:

In eukaryotes, DNA is wrapped around histones to form nucleosome, a fundamental unit of chromatin. The native chromatin is further compacted into higher-order structure and plays a critical role in various chromosomal processes, such as gene regulation, recombination, replication, chromosome condensation and the proper segregation of chromosomes. The organization of higher-order chromatin structure has been linked to the post-translational modifications of histones, such as acetylation, phosphorylation and ubiquitination. How these histone modifications participate in the modulation of chromatin structure has remained elusive. In their recent publication in Science, HFSP Long-term Fellow Jun-ichi Nakayama and colleagues provide the first in vivo evidence that methylation of histone H3 plays a central role in the transcriptional repression and organization of large chromosomal domains. Their work also defined a highly conserved pathway wherein histone deacetylases and histone methyltransferases act cooperatively to establish a “histone code essential for higher order of chromatin assembly

 

Figure; Both H3K4me3 and H3K27me3 marks are critical for the maintenance and differentiation of stem cells. H3K4me3 is the epigenetic mark for transcriptionally active chromatin, and H3K27me3 is the epigenetic mark for transcriptionally inactive chromatin. In stem cells and progenitor cells, many genes important for developmental control are marked by both H3K4me3 and H3K27me3 marks and therefore are poised for transcription activation or repression. Thus, the enzymes that regulate the levels of H3K4me3 and H3K27me3 are important for the homeostasis of the stem cells.

 

Pathway Description of acetylation and deacetylation: Result- activation and deactivation of Genes in specific.

Protein acetylation plays a crucial role in regulating chromatin structure and transcriptional activity. Many transcriptional co activators possess intrinsic acetylase activity, while transcriptional co repressors are associated with deacetylase activity. Acetylation complexes (such as CBP/p300 and PCAF) or deacetylation complexes (such as Sin3, NuRD, NcoR and SMRT) are recruited to DNA-bound transcription factors (TFs) in response to signaling pathways. Histone hyper acetylation by histone acetyltransferases (HATs, also called KATs for lysine acetyltransferases) is associated with transcriptional activation, whereas histone deacetylation by histone deacetylases (HDACs or KDACs) is associated with transcriptional repression. Histone acetylation stimulates transcription by remodeling higher order chromatin structure, weakening histone-DNA interactions, and providing binding sites for transcriptional activation complexes containing proteins that possess bromodomain, which bind acetylated lysine. Histone deacetylation represses transcription through an inverse mechanism involving the assembly of compact higher order chromatin and the exclusion of bromodomain-containing transcription activation complexes. Histone hyper acetylation is a hallmark of silent heterochromatin. Site-specific acetylation of a growing number of non-histone proteins, including p53 and E2F, has been shown to regulate their activity, localization, specific interactions as well as stability/degradation, therefore controlling a variety of cellular processes, such as transcription, proliferation, apoptosis and differentiation. It is becoming clear that crosstalk between acetylation and other modifications, such as methylation and phosphorylation in histone and non-histone proteins, plays a critical role in the final output signals. Some modifications are required in a combinatorial order to exert an effect, while others are mutually exclusive. In this context, the different putative combinations likely work to diversify cellular signaling networks, providing a much broader way to modulate responses. At an organismal level, acetylation plays an important role in immunity, circadian rhythm and memory formation. Protein acetylation is becoming a favorable target in drug design for numerous disease conditions. However, most of the available activators and inhibitors are as yet broad and non-specific. Recent studies have provided detailed analysis on the function and structure of the different KATs and KDACs, therefore we could expect the development of better and more specific modulators in the near future.

Dynamics and Interplay of Nuclear Architecture, Genome Organization, and Gene Expression:

Schematic overview of histone modifications:

 

(A) Active chromatin marks. Nucleosomes encompassing the transcribed region of a gene—a promoter, enhancer, and insulator, respectively—are shown (structure of gene and regulatory elements are represented below). The N-terminal “tails” of histone H3 are shown in dark gray, and the tails of H4 are in light gray. H3/K4 methylation and H3/K9 mono-methylation are enriched at the enhancer, the promoter, and the 5′ end of the active gene. H3/K27 and H4/K20 mono-methylation is enriched over the transcribed region, whereas H3/K36 trimethylation peaks at the 5′ of the active gene. Note that active genes are also enriched in H3, H4, and H2A acetylation (not shown).

 

(B) Poised chromatin marks. Four nucleosomes encompassing the transcribed region of a gene poised for transcription and one nucleosome each on a promoter element, an enhancer element, and an insulator are shown. The promoter and the transcribed region are enriched in the repressive mark H3/K27 trimethylation, whereas the region around the transcription start is also enriched in the active mark H3/K4 trimethylation. This combination of active and repressive marks can poise genes for activation and forms a so-called “bivalent domain.”

 

(C) Inactive chromatin marks. Modifications of histones H3 and H4 in nucleosomes encompassing a repressed or silenced gene are shown. The coding sequence and promoter of the inactive gene are enriched in H3/K9 and H3/K27 di- and trimethylation. The 5′ end of the gene and the promoter region are marked by H3/K79 trimethylation, whereas the insulator element carries activating marks. Data are based on Barski et al. (2007). (D) Scheme of a protein-coding gene with exons shown as light-gray boxes and introns as white boxes. Cis-acting regulatory sequences (enhancer, promoter, and insulator) are represented by black and dark- gray boxes.

 

After-effects of DNA and Histone methylation:

 

DNA methylation induces the binding of MeCP2 to CH3pG which attracts HDACs histone deacetylation such modifications which characterizes histones in both heterochromatin and repressed euchromatin.  MeCP2 binds to methylated DNA, which recruits HDAC that causes deacetylation of histones; deacetylation in turn leads to methylation of histone tails; in turn leads to extension of deacetylation and methylation; finally, HP1 family of proteins bind, interact with one another and compacts the chromatin called heterochromatin.  Histone methyl tails interact (hydrophobically) for compaction.  The packing of nucleosomes is tight, there is no scope for any other proteins to bind, thus the HC region is inactive.

(Ac= Acetyl; Me= Methyl; MeCP2= Methyl-CpG binding Protein 2; HDAC= Histone De-Acetylase).

 

Another mode of HC formation is Histone H3K9 methylation induces DNA CpG methylation which leads to heterochromatization (HC) of the chromatin and repression gene expression; SUVAR39H is a methyltransferase which specifically methylates the Lysine 9 of histone H3. Such a methylation creates a binding site for the Heterochromatin Protein HP1 which recruits a DNA methyl transferase, which in turn capable of methylation CpG in DNA (Me= Methyl; Methyl H3-K9= Methyl on Lysine 9 of Histone H3; HP1=Heterochromatin Protein 1; DNMT=DNA Methyl transferase.

 

The assumption that acetylation, methylation does not alter the charge of arginine and lysine residues and is unlikely to directly modulate nucleosomal interactions required for chromatin folding is not without doubts.  Methylation of histone tails provide hydrophobic interaction tails and provide scope for interaction and compaction, but acetylation of histone tails not only repel the binding of DNA but also repel histone tails each other and make histones move away from each other. While the mechanisms by which arginine methylation regulates transcription are unknown, lysine methylation coordinates the recruitment of chromatin modifying enzymes. Chromodomains (HP1, PRC1), PHD fingers (BPTF, ING2), Tudor domains (53BP1), and WD-40 domains (WDR5) are among a growing list of methyl-lysine binding modules found in histone acetyltransferases, deacetylases, methylases and ATP-dependent chromatin remodelling enzymes. Lysine methylation provides a binding surface for these enzymes, which then regulate chromatin condensation and nucleosome mobility in order to maintain local regions of active or inactive chromatin. In addition, lysine methylation can block binding of proteins that interact with unmethylated histones or directly inhibit catalysis of other regulatory modifications on neighbouring residues. The presence of methyl-lysine binding modules in the DNA repair protein 53BP1 suggests roles for lysine methylation in other cellular processes.

Histone methylation is crucial for proper programming of the genome during development and mis-regulation of the methylation machinery can lead to diseased states such as cancer. Until recently, methylation was believed to be an irreversible, stable epigenetic mark that is propagated through multiple cell divisions, maintaining a gene in an active or inactive state. While there is no argument that methylation is a stable mark, recent identification of histone demethylases such as LSD1/AOF2, JMJD1, JMJD2 and JHDM1 has shown that methylation is reversible and provides a rational for how genomes might be reprogrammed during differentiation of individual cell lineages.

 

First Step in Gene Activation:

Genes are located as segment of DNA with an encoded sequence, but camouflaged by the binding of DNA around histones octamers and H1 to linker DNA.  Binding of pioneering factors to activator sequence such as TATA box or upstream sequences including enhancers is the first step.  These DNA sequences of a gene or genes are found in 300nm chromatin domains.  In the nucleus in interphase these 300nm chromatin segments are open as 30nm structures or 11nm threads without H1.  Most of these euchromatin chromonemal threads are found in the nuclear sap not at the periphery of nuclear membrane where heterochromatin regions are anchored to membrane matrix proteins.  The inward projected chromatin (11nm-30nm) has sites for the binding of activators for activation and repressor for inactivating gene.  In the nuclear sap is filled with such threads in the form of network (appears to be so).  If the chromonemas are 30nm thick, they are relaxed to 11nm by the binding of chromosome remodeling factors, which bring about relaxation using ATP dependent helicase activity. 

Using another paradigm, most of the genes to be activated in cell specific manner are already bound by nucleating factors at TATA box or upstream sequences.  These are recognized by TFs such as TFIID or Enhancers likeSP1 in the upstream and recruit factors required for loosening of the chromatin by histone acetylases.  Histone acetylating factors are very dynamic while the methylating factors exhibit slow turnover.  Most of the loci where genes are transcribing actively nucleosomal histones are hyper acetylated and the region is free of histones or histones are slide away from the site of activation.

 

DNA (white ribbon) is wrapped around a histone protein core (yellow discs) in chromatin. Transcription occurs when an activator binds a specific region of DNA and recruits transcription factors (TAFs, TBP, and others) and protein complexes (SWI/SNF and others) that modify histones or remodel chromatin

 

Diagram below, shows gene expression or activation starts with the binding of an Activator:

a | In the first step, a histone H3 kinase is recruited to the promoter by the transcriptional activator to phosphorylate Ser10 on histone H3. b | A histone H4 HAT (histone acetyltransferase) complex is recruited to the promoter to acetylate a lysine residue on histone H4. c | The Gcn5 HAT complex is recruited to the promoter, possibly through multiple interactions including an interaction between the bromodomain of Gcn5 with acetyl-lysine modified histone H4 and an interaction between a phosphorylated histone H3 tail with the HAT domain. This recruitment results in the acetylation of Lys14 on histone H3. d | The effect of these post-translational histone modifications is to recruit RNA polymerase II and the GTFs (general transcription factors) to DNA for transcriptional activation, Ac-acetylation, P-phosphorylation.

 

Steps in Gene activation:

 

Formation of the multiprotein complex at the promoter of a gene; The depicted model shows acetylated chromatin (acetylation = red spheres) that is selectively remodeled by ATP-dependent chromatin remodeling complexes (blue sphere and black sphere) and thus allowing the binding of DNA-binding trans activators close to the promoter of the gene. Then the RNA polymerase II, general initiation factors, and mediators bind at the promoter followed by the binding of RNA polymerase II elongation factors to the multiprotein complex, finally leading to mRNA transcription. The proteins that form the multiprotein complex are repressed by spheres in different shapes and colors.

 

Transcriptional Bursting and Transcriptional Factories: from Wikipedia

 

Transcription takes place in "bursts" or "pulses". This phenomenon has recently come to light with the advent of new technologies, such as MS2 tagging, to detect RNA production in single cells, allowing precise measurements of RNA number, or RNA appearance at the gene. Widespread techniques, such as Northern Blotting, Microarrays, RT-PCR and RNA-Seq measure bulk RNA levels from homogenous population extracts.

The reality is that transcription is irregular, with strong periods of activity, interspersed by long periods of inactivity. Averaged over millions of cells, this appears continuous. But at the individual cell level, there is considerable variability for most of the genes show very little activity at any one time.

What do the repressive and permissive states represent? An attractive idea is that the repressed state is a closed chromatin conformation whilst the permissive state is an open one. Another hypothesis is that the fluctuations reflect transition between bound pre-initiation complexes (permissive) and dissociated ones (restrictive). Bursts may also result from bursty signaling, cell cycle effects or movement of chromatin to and from transcription factories.

In nuclear sap many genes from different loops emanating from the scaffold are found transcribing in clusters where factors and RNAPs are all found in active state, often called transcription explosion.  Such sites are visible.

What do the repressive and permissive states represent? An attractive idea is that the repressed state is a closed chromatin conformation whilst the permissive state is an open one. Another hypothesis is that the fluctuations reflect transition between bound pre-initiation complexes (permissive) and dissociated ones (restrictive). Bursts may also result from bursty signaling, cell cycle effects or movement of chromatin to and from transcription factories.

In nuclear sap many genes from different loops emanating from the scaffold are found transcribing in clusters where factors and RNAPs are all found in active state, often called transcription explosion.  Such sites are visible.

 

Polycomb group of proteins and Trithorax group of proteins:

Polycomb and Trithorax genes are involved in HOX gene regulation during the development of Drosophila. Polycomb and Trithorax proteins are involved in specific gene(s) repression and activation respectively in specific cell types and maintain the repression or activation in lineage of cells. 

Polycomb proteins:

PcG a multi-protein complex and consist of two group of complexes PcR1 and PCR2.  The PCR2 complex associates with specific proteins and binds to specific cognate DNA sequences in promoter regions at very early stage of differentiation (Stem cells).  PCR2 consists of a methylases domain called SET.  Once the PCR2 binds it starts methylating H3K27.  This leads to the recruitment of PCR1and it binds to methylated histone octamers through dimeric PCR1 which contain Chromodomain sites specific to H3K27. Methylation leads to the binding of HP1 protein which propagates more methylation more recruitment of HP1.  Interaction between HP1 proteins, compact the chromatin more than Condensin compaction of metaphase chromatin. This complex maintains and perpetuates in cell lineage for hundreds of years.  This association takes place at replication stage where newly replicated DNA associates with histones modified or to be modified and the same is perpetuated in its lineage of cells.

 

Polycomb-mediated gene expression changes during differentiation.

Polycomb silencers control cell fate, development and cancer:

Anke Sparmann & Maarten van Lohuizen.

 

Gene expression changes during differentiation.

 

a-     Gene expression changes at Polycomb group (PcG) target sites could occur by several possible mechanisms. a | Following commitment to a certain cellular fate, a subset of genes specifying this fate becomes activated through displacement of PcG proteins from their promoter regions. This dynamic function of PcG proteins indicates that the permanent silencing of specific genes might require a second epigenetic mark, possibly provided by DNA methylation. Gene silencing could follow two possible routes.

b | Certain loci seem to be pre-programmed for repression in non-differentiated cells through binding of inactive PcG complexes. Cellular differentiation triggers PcG activation by as yet unidentified mechanisms, possibly involving post-translational modifications of Polycomb repressive complex (PRC) components. c | Repression of genes can also be achieved by de novo recruitment of PcG proteins, although the mechanisms that target PcGs to the appropriate genomic regions remain ill defined. DNMT= DNA methyl transferase.

PcG proteins are epigenetic factors essential for development that execute their repressive function in 2 distinct multiprotein complexes named Polycomb Repressive Complex (PRC) 1 and 2. PRC1 and PRC2 repress transcription respectively by Ubiquitylating Histone H2A lysine (K) 119 and by tri-methylating (me3) Histone H3K27. Deregulation of both PRC1 and PRC2 activities is a common feature of human tumors strongly suggesting that PcG proteins play an active role in cancer formation. Several aspects of PcG mediated transcriptional regulation are poorly understood. This includes the upstream signaling and the factors that regulate PcG activities, the molecular mechanisms by which H3K27me3 and H2AUbq lead to transcriptional silencing and the mechanisms that specify PcG recruitment to target genes during development.

 

 

PRC2 mediated  histone methylation enzymes and silencing the cromatin.

 

 

Epigenetic gene silencing by Polycomb protein complexes:

Polycomb silencers control cell fate, development and cancer

Anke Sparmann & Maarten van Lohuizen.

 

 

Binding of the PRC2 (Polycomb repressive complex 2) initiation complex to the Polycomb group (PcG) target genes induces enhancer of zeste homologue 2 (EZH2)-mediated methylation (me) of histone proteins, primarily at lysine 27 of histone H3 (H3K27). PRC1 is able to recognize the trimethylated H3K27 (H3K27me³) mark through the chromodomain of Polycomb (PC). This interaction might bring neighboring nucleosomes into the proximity of the PRC2 complex to facilitate widespread methylation over extended chromosomal regions²⁰. Although the precise mechanisms for PRC-mediated stable gene silencing are still poorly understood, they are proposed to involve direct inhibition of the transcriptional machinery, PRC1-mediated ubiquitylation (Ub) of H2AK119, chromatin compaction and recruitment of DNA methyltransferases (DNMTs) to target gene loci by PRC2. Pol. II RNA Polymerase II.

 

 

 

 

Repression:

 

UMED6 a repressor binds to specific site such as URS in sequence specific manner, its repressor domain associates with SIN3 a multiprotein complex that includes RPD3.

 

Deacetylation and methylation specific H3/H4 at promoter region promote repression. Repressor direct deacetylases and methylases to octamer histones in promoter regions and prevent the binding of GTF.  Unacetylated histones fail to interact with similar histones and favor folding nucleosome into condensed state. Unacetylated histones bind to phosphate groups of DNA.  RPD3 one of the RNAP component has homology for HDAC. RPD3 has deacetylase domain and acts at promoter regions; it depends upon two other proteins such as UMED6 and Sin3.

 

Model for Repression of chromatin

 

Sin3 is multi protein complex contains RPD3, also binds to UMED6.

RPD3 positions in the promoter and removes acetyl groups from neighboring histones; Chip method has shown that in the vicinity of promoter where UME6 is bound histones are deacetylated, Deacetylation prevents the binding of GTF at TATA region and represses the gene expression. In this model repressor directs RPD3 and SIN3 to deacetylase to the site, where both act as co repressors. This has been observed in many mammalian systems.  SIN3 and its homologues interact with repressor domain of their repressor complex. In higher systems repressor is also associated with histone methylases (HMT) subunits that methylases H3K9; such groups facilitate the binding of HP1, a co repressor.

 

KAP1 (histone Lysine methylases) a co repressor complex a class of its kind consists of 200 or more Zif containing proteins (TFs), A trans gene in the mouse fibroblasts is repressed through KAP1 a co repressor associated with Histone in most of the cells,

 

In addition, DNA methylation of C in CpG containing group of genes, it triggers chromatin condensation locally or in the region of CpG loci and active gene of similar kind are devoid of methylation to CpG.  Methylated C*pG are bound by MeCP2 which in turn interacts with mSIN3 a co repressor, this leads to more deacetylation in the region of their binding and prevent the binding of TFs.

Histone each octamers contain eight tails that are reasonably long and they touch each other; this can lead to compaction due to interaction of hydrophobic methyl group.

 

Mutants of UMED3 and SIN3 show derepression of genes where UMED6 is supposed to bind. Repressed genes are associated with methylated H3K9 and HP1 and active genes don’t contain them.

 

Polycomb group proteins	Trithorax group proteins
Binds and methylates H3K27 at promoter-extends the methylation	Binds and methylates H3K4 at promoter,
PRC1 binds to methylated H3 tails and extends to others	This leads to binding of HATs and remodeling proteins
This prevents binding of TC	This promotes binding of TC
This leads to the binding more PRC1 and methylation of H3K27	Promotes transcription and prevent H3K9 methylation, prevent HP1 binding and prevent PRC1 binding

 

Deacetylation of N’-terminal histone tails, leads to methylation and chromatin contraction and stop transcription

Mechanism of Deacetylation and hyperacetylation

 

Trithorax group of proteins:

Trithorax group of proteins recruit acetylases and specific methylases that methylates H3K4 (3) in promoter region.  This leads to the binding of more histone acetylases and chromatin remodeling proteins that promotes transcription.  This prevents methylation at H3K9 and prevent HP1 binding to H3K27 and prevent PRC 1 binding to H3K27 CH3.  Such nucleosomes with H3K4CH3 bind to newly replicated DNA and TrX-G that binds to H3K4 leads to perpetuation.

The Trithorax protein contains a zinc finger domain, homologous to many transcriptional activators, including a human homolog involved in oncogenic transformation in cases of acute leukemia. Polytene chromosomes are especially thick chromosomes in the fly's salivary glands in which the DNA has been duplicated a thousand times over. TRX protein can be immunolocalized on polytene chromosomes at 16 binding sites that overlap those occupied by Polycomb group proteins. This overlap suggests that Trithorax is involved in a multiprotein complex made up of both gene silencing (Polycomb) and activating (Trithorax) subunits. In the absence of certain members of the Pc-G (Enhancer of zeste or Posterior sex combs proteins), TRX protein becomes disassociated from its chromosomal binding site, providing further evidence of the likelihood that a large protein complex is involved in activating and repressing genes (Kuzin, 1994).

The intron/exon structure of the trx gene and the large alternatively spliced TRX mRNAs are capable of encoding only two protein isoforms. TRX proteins differ only in a long Ser- and Gly-rich N-terminal region, encoded by exon III, present only in the larger trx isoform. Trithorax has a novel variant of the highly conserved nuclear receptor type of DNA binding domain (Stassen, 1995). Five different transcripts can be identified. Each one uses the same first exon. Transcript M has no second or third exon, and a short 3'UTR. Transcript ME also has no second or third exon, and a long 3'UTR. These two transcripts code for the low molecular weight protein isoform. Transcripts E1, E2 and L differ in their use of second and third exons as follows: E1 uses the second and not the third; E2 uses the third and not the second, and L uses both. E1and L, both uses the third exon, code for the high molecular weight protein isoform, while E2 codes for the low (Sedkov, 1995 and Stassen, 1995).

 

 

 

Activation of GCN4

 

GCN4 binds to its specific UAS sequence in the promoter; its activation domain interacts with co activators such as GCN5 complex which has histone acetylase activity.  GCN5 in turn acetylates histone tails; this facilitates the binding of TFs.

This kind of operation is common in most of the eukaryotes; most unusual and specific action of activators is that they direct methylases and HAT to specific sites of a specific histone, such as H3K4.

 

Eukaryotes contain co activator complexes which perform acetylation of histone, one such complex is SAGA which functions with GCN4.   The GCN5 is one of the subunits of SAGA complex. Trimethylation H3K4 recruits SAGA complex; it is chromosome remodeling complex.  Activator GCN4 associates with GCN5, and GCN5 has histone acetylase activity.

 

HAT was isolated and purified from Tetrahymena for the first time and a similar HAT called GCN5 was purified from yeast cells; they are homologous proteins,

GCN4 contains DNA binding domain (DB) and Activator domain (AD). GCN4 mediated activity leads to Hyperacetylation histone tails in the promoter regions, which results in opening of DNA in the region of promoters and facilitates the binding of TFs and GTF later. Acetyl groups contain negative charges and DNA with negative charges phosphate groups repulse each other thus octamers free from the DNA or pushed along the DNA and free the DNA; this provides region for TFs to bind.   This promoter region is less condensed and sensitive DNase1.

Specific histone tail acetylation leads to the binding of proteins containing Bromo domain such as TFIID which binds to Histone acetylated region of TATA Box.

Nucleosomes in all promoter regions of active genes are hyper acetylated. Histone of each octamers contains eight modified tails which are reasonably long and they can touch each other.  Acetyl groups of one octamer interact with acetyl groups of one another; they repel each other and also repel acidic charges of DNA, which certainly facilitates the stretching the linker DNA and freeing from histones or loosening histone from DNA; perhaps free itself from the linker protein so as to free for TFs to bind.

 

 

 

“Mechanism of transcriptional activation by GCN4:

Transcriptional activators stimulate assembly of preinitiation complexes (PIC) at their target promoters by removing repressive chromatin structures and recruiting general transcription factors (GTFs) and RNA Polymerase II (Pol II). Activators carry out these functions indirectly by recruiting co activators. One class of co activators is the ATP-dependent nucleosome remodeling complexes, including SWI/SNF and RSC, that expose (or obscure) protein binding sites in promoter DNA. Another class is the histone acetyltransferases (HATs), such as SAGA and NuA4. Histone acetylation destabilizes chromatin structure and also stimulates recruitment of other co activators harboring bromodomain. Similarly, it is thought that histone methyltransferases enhance recruitment of co activators containing chromodomains (CHD) or plant homeodomain (PHD) fingers. A third group of co activators serve as adaptors to help recruit TATA binding protein (TBP) or Pol II itself, a function generally ascribed to SAGA (for TBP) and the Mediator complex (for Pol II). Mediator further stimulates phosphorylation of Ser5 in the heptad repeats of the C-terminal domain (CTD) of the largest subunit of Pol II (RPB1) by the CDK KIN28 in TFIIH. Transcriptional activation also leads to increased association of certain cofactors with the coding sequences, including the Paf1 complex (Paf1C), which interacts with Pol II and promotes recruitment of histone methyltransferases that target histone H3 on Lys4 (by SET1 complex) and Lys36 (by SET2). Paf1C also promotes Ser2 phosphorylation of the RPB1 CTD in elongating Pol II and thereby stimulates polyadenylation and transcription termination.  We are studying the mechanism of transcriptional activation of amino acid biosynthetic genes by GCN4 We showed previously that the activation domain (AD) of GCN4 contains 7 hydrophobic clusters that make additive contributions to transcriptional activation in vivo ³³ and stimulate GCN4 binding to SAGA, SWI/SNF, RSC, Mediator and CCR4/NOT complexes in cell extracts ^34-36. We also obtained Gcn- mutations impairing activation by GCN4 in one or more subunits of all 5 of these cofactors ^35,36 and showed that GCN4 recruits them all to its target gene ARG1 in vivo ³⁶. More recently, we showed that mutations in one or more subunits of these cofactors reduce recruitment of TBP and Pol II by GCN4 to the promoters at ARG1, ARG4 and SNZ1, implicating all five complexes in stimulating PIC assembly. Interestingly, deletion of certain SAGA subunits has a greater impact on recruitment of Pol II versus TBP. Thus, even though TBP binding to the TATA element is required for Pol II recruitment, (which we demonstrated by analyzing a TATA element deletion at ARG1), it appears that SAGA also promotes Pol II recruitment independent of stimulating TBP recruitment.

In addition to reducing TBP and Pol II recruitment, the arg1-TATA mutation lowers recruitment of other GTFs (TFIIB, -IIA, -IIE, -IIF) but has no effect on recruitment of SAGA, Mediator or SWI/SNF to the UAS ³⁸. Thus, recruitment of these co activators by GCN4 is independent of PIC assembly. Consistent with this, our kinetic ChIP analysis showed that, on induction of GCN4 by amino acid starvation, recruitment of cofactors to the UAS precedes TBP and Pol II recruitment to the ARG1 promoter. Despite nearly simultaneous recruitment of SWI/SNF, Mediator, and SAGA to the UAS, we observed strong interdependency in their recruitment by GCN4. Thus, SWI/SNF recruitment is stimulated by SAGA (HAT and non-HAT functions) and Mediator, and recruitment of SAGA is promoted by Mediator and RSC. Recruitment of Mediator is dependent on SAGA at ARG4 and SNZ1 but not at ARG1.

This extensive interdependency distinguishes GCN4 from the activator GAL4, which recruits SAGA and Mediator independently  and requires PIC assembly for SWI/SNF recruitment, and also from activator SWI5 that recruits SWI/SNF independently of Mediator and SAGA and requires SWI/SNF for SAGA and Mediator recruitment (at least in late mitosis).  Thus, yeast activators exhibit distinct patterns of cofactor interdependency.

Our kinetic analyses of PIC formation in co activator mutants confirmed that TBP recruitment per se is not sufficient for wild-type promoter occupancy by Pol II and suggested that all four co activators enhance Pol II recruitment downstream of TBP binding to the promoter. We further uncovered functions for SWI/SNF and SAGA in transcription elongation as mutations in these cofactors had greater effects on Pol II occupancy of coding sequences versus the promoter  (Fig. 14B).

Co-Activator enhancement of TBP

Together, these results provide a detailed picture of the GCN4 activation mechanism which differs significantly from those described for other activators, and they extend the range of known functions stimulated by these cofactors in vivo.

Recently, we found that SAGA is associated at high levels with the coding sequences of GCN4 target genes, and also with GAL1, during induction, and that SAGA association with the ORF requires both transcription and Ser5-CTD phosphorylation by KIN28. We further showed that GCN5, most likely in SAGA, functions in transcribed coding sequences to (i) enhance nucleosome eviction from the highly transcribed GAL1 gene; (ii) maintain high-level H3 acetylation in nucleosomes reassembled in the wake of elongating Pol II; (iii) promote Pol II processivity to an extent that increases transcriptional output from an ORF of extended (8kb) length; and (iv) stimulates H3-K4 trimethylation. Interestingly, GCN5 also opposes the effects of several histone deacetylase complexes that are likewise recruited by GCN4 to transcribed coding sequences, presumably to maintain the optimum level of H3 acetylation needed to prevent gene silencing (by hypoacetylation) or activation of cryptic promoters (by hypoacetylation) .

We made progress on the mechanism of Mediator recruitment by demonstrating that the tail sub complex containing GAL11/MED15, MED2, and PGD1/MED3 is an in vivo target of the GCN4 activation domain. Deleting each of these subunits impairs recruitment to ARG1 of all Mediator subunits tested. A stable tail sub complex released from Mediator in a sin4/med16 mutant can bind to the GCN4 activation domain in vitro. Importantly, the tail, but not head, subunits of Mediator are recruited by GCN4 in sin4 cells and the function of MED2 in promoting TBP recruitment to the promoter is maintained in the sin4 mutant. Hence, GCN4 can recruit the tail domain independently of the rest of Mediator, and the tail may provide an adaptor function for TBP recruitment.

Another example of gene(s) activation:

 

Mammalian systems contain such complexes of ~400KD called CBP/p300 which also functions similar to GCN5 is another example of an activator that is phosphorylated has many domains; one of its domains binds to CREBP which is bound to CRE elements and the other domain binds to the other activator domain; the third domain has HAT activity; the 4^th domain associates with other HAT complexes. CREBP is believed to direct CBP/p300 to acetylate specific nucleosomes. The largest subunit of TFIID contains HAT activity and perhaps it maintains acetylation of histone tails; maintenance of acetylation and methylation in promoter regions is very important for the gene to be active.

 

Methylation of histones at H3K9 or 27 (often trimethylated) in general leads to repression, but methylation of H3K4 in promoter region leads to activation of genes. Trithorax in Drosophila is an example which methylates H3K4 and activates specific HOX genes in specific cell lines, on the contrary Polycomb group of proteins repress a group of HOX genes by methylation of H3K9 or 27 or both in specific cell lines. Trithorax contain a domain called SET which assembles into multisubunit complex that methylates H3K4, which serves as site for the binding of another subunit of Trithorax called TRTX, thus they maintain methylation at H3K4. Such expressions and repression are detected by fluorescent labeled antibody techniques called Chip. In Drosophila 100 or more such sites have been detected.

 

Histone modifications vary greatly:

Acetyl groups on histone tails show rapid turnover, but methylation is stable and exchange is slow. Acetylation and deacetylation processes are in equilibrium by HATs and DHATs. In the regions where activators are bound acetylation predominates, where as in repressors’ bound deacetylation and methylation predominates.  Methylation is stable and turnover is very slow and they exhibit epigenetic inheritance.

 

Hyper acetylation induced gene activation - Putative pathway whereby the new HDAC inhibitor may reverse silencing in cells from people with Friedreich's ataxia.

Breaking the silence in Friedreich's ataxia;

Richard Festenstein; Nature Chemical Biology 2, 512 - 513 (2006)

 

Hyper acetylation induced gene activation - Putative pathway whereby the new HDAC inhibitor may reverse silencing in cells from people with Friedreich's ataxia.

 

The chromatin organization of an active FXN gene is shown on the far left. Protruding from the nucleosomes are acetylated histone tails (Ac, acetyl). The presence of GAA repeats might nucleate heterochromatin formation, pushing the pathway to the right through several stages. An early step in this process may be the deacetylation of the lysines on histones, which would provide a substrate for histone methyltransferases (HMTases). Methylation of the histone H3 tail (on Lys9) would provide a binding site for HP1, consistent with a histone code for silencing⁹. If the nucleosomes were sufficiently close (possibly because of the repetitive nature of the DNA), then HP1 dimers might stabilize a condensed higher-order heterochromatin structure that prohibits access of the transcriptional machinery to the FXN locus. In this hypothetical scenario, the new HDAC inhibitor shifts the equilibrium toward a transcriptionally active FXN. Though consistent with the authors' data, the model awaits further investigation; in particular finding the target of the HDAC inhibitor.

 

An activator when binds to its specific site on DNA interacts with several co activators such as p300/CBP, PCAF and TAF 250.  All these have ability to acetylate H3 tails at K site.  This leads to loosening of DNA from histones and further opening of chromosomes.

 

 

This schematic model on increasing levels of transcriptional repression induced by DNA methylation and MeCP2 binding followed by corepressor and deacetylase binding with subsequent deacetylation and chromatin compaction was adapted with modifications from Jones and Laird (1999). Nucleosome core particles are shown as gray disks with DNA wrapped around as black ribbon. Acetylation, methylation, MeCP2 binding, corepressor and deacetylase binding are represented by red, green, blue, gray, and dark blue spheres, respectively.

 

Give ways in which eukaryotic gene repressor proteins can operate:

Gene activator proteins and gene repressor proteins compete for binding to the same regulatory DNA sequence. (B) Both proteins can bind DNA, but the repressor binds to the activation domain of the activator protein thereby preventing it from carrying out its activation functions. In a variation of this strategy, the repressor binds tightly to the activator without having to be bound to DNA directly. (C) The repressor interacts with an early stage of the assembling complex of general transcription factors, blocking further assembly. Some repressors also act at late stages in transcription initiation, for example, by preventing the release of the RNA polymerase from the general transcription factors. (D) The repressor recruits a chromatin remodeling complex which returns the nucleosomal state of the promoter region to its pre-transcriptional form.

 

Figure For simplicity

Certain types of remodeling complexes appear dedicated to restoring the repressed nucleosomal state of a promoter, whereas others (for example, those recruited by activator proteins) render DNA packaged in nucleosomes more accessible. However the same remodeling complex could in principle be used either to activate or repress transcription: depending on the concentration of other proteins in the nucleus, either the remodeled state or the repressed state could be stabilized. According to this view, the remodeling complex simply allows chromatin structure to change. (E) The repressor attracts a histone deacetylase to the promoter. Local histone deacetylation reduces the affinity of TFIID for the promoter and decreases the accessibility of DNA in the affected chromatin. A sixth mechanism of negative control—inactivation of a transcriptional activator by heterodimerization—was illustrated in Figure for simplicity, nucleosomes have been omitted from (A)-(C), and the scale of (D) and (E) has been reduced relative to (A)-(C).B

Mammalian X-chromosome inactivation by heterochromatization: NCBI

 

 X-chromosome inactivation begins with the synthesis of XIST (X-inactivation specific transcript) RNA from the XIC (X-inactivation center) locus. The association of XIST RNA with the X chromosome is correlated with the condensation of the chromosome. Both XIST association and chromosome condensation gradually move from the XIC locus outward to the chromosome ends. The details of how this occurs remain to be deciphered.

How an entire chromosome transcriptionally inactivated? X-chromosome inactivation is initiated and spreads from a single site in the middle of the X chromosome, the X-inactivation center (XIC). In the figure portions of the X chromosome that are removed from the XIC and fused to an autosome escape inactivation. In contrast, autosomes that are fused to the XIC of an inactive X chromosome are transcriptionally silenced. The XIC (a DNA sequence of approximately 10⁶ nucleotide pairs) can therefore be considered as a large regulatory element that seeds the formation of heterochromatin and facilitates its bi-directional spread along the entire chromosome. Encoded within the XIC is an unusual RNA molecule, XIST RNA, which is expressed solely from the inactive X chromosome and whose expression is necessary for X-inactivation. It does not get translated into protein; rather the XIST RNA remains in the nucleus, where it eventually coats the inactive X chromosome. The spread of XIST RNA from the XIC over the entire chromosome correlates with the spread of gene silencing, indicating that XIST RNA participates in the formation and spread of heterochromatin In addition to containing XIST RNA, the X-chromosome heterochromatin is characterized by a specific variant of histone 2A, by hypoacetylation of histones H3 and H4, by methylation of a specific position on histone H3 and by methylation of the underlying DNA, a topic we will discuss below. Presumably all these features make the inactive X chromosome unusually resistant to transcription.

Many features of mammalian X-chromosome inactivation remain to be discovered. How is the initial decision made as to which X chromosome to inactivate? What mechanism prevents the other X chromosome from also being inactivated? How does XIST RNA coordinate the formation of heterochromatin? How is the inactive chromosome maintained through many cell divisions? We are just beginning to understand this mechanism of gene regulation that is crucial for the survival of our own species.

X-chromosome inactivation in females is only one way that sexually reproducing organisms solve the problem of dosage compensation. In Drosophila, all the genes on the single X chromosome present in male cells are transcribed at two-fold higher levels than their counterparts in female cells. This male-specific “up-regulation” of transcription results from an alteration in chromatin structure over the entire male X chromosome. As in mammals, this alteration involves the association of a specific RNA molecule with the X chromosome; however, in Drosophila, the X-chromosome-associated RNA increases gene activity rather than blocking it. The male X chromosome also contains a specific pattern of histone acetylation which may help to attract the transcription machinery to this chromosome (NCBI Book)

Senescence-Associated Heterochromatin Foci ^;

Rugang Zhang, Wei Chen, and Peter D. Adams^*

 

Senescence is characterized by an irreversible cell proliferation arrest. Specialized domains of facultative heterochromatin, called senescence-associated heterochromatin foci (SAHF), are thought to contribute to the irreversible cell cycle exit in many senescent cells by repressing the expression of proliferation-promoting genes such as cyclin A. SAHF contain known heterochromatin-forming proteins, such as heterochromatin protein 1 (HP1) and the histone H2A variant macroH2A, and other specialized chromatin proteins, such as HMGA proteins. Previously, we showed that a complex of histone chaperones, histone repressor A (HIRA) and anti-silencing function 1a (ASF1a), plays a key role in the formation of SAHF. Here we have further dissected the series of events that contribute to SAHF formation. We show that each chromosome condenses into a single SAHF focus. Chromosome condensation depends on the ability of ASF1a to physically interact with its deposition substrate, histone H3, in addition to its co chaperone, HIRA. In cells entering senescence, HP1, but not the related proteins HP1 and HP1ß, becomes phosphorylated on serine 93. This phosphorylation is required for efficient incorporation of HP1 into SAHF. Remarkably, however, a dramatic reduction in the amount of chromatin-bound HP1 proteins does not detectably affect chromosome condensation into SAHF. Moreover, abundant HP1 proteins are not required for the accumulation in SAHF of histone H3 methylated on lysine 9, the recruitment of macroH2A proteins, nor other hallmarks of senescence, such as the expression of senescence-associated ß-galactosidase activity and senescence-associated cell cycle exit. Based on our results, we propose a stepwise model for the formation of SAHF.

FIG. A:   Stepwise model for the formation of SAHF in senescent human cells. This model indicates the key steps in SAHF formation, after initiation by senescence triggers. Dashed lines are steps which are currently poorly defined (see text for details). Abbreviations: HMT, histone methyltransferase; Me, methylated lysine 9 histone H3; Ac, acetylated histone. HMGA proteins are shown in mature SAHF, but their point of entry is not known.

 

Silencing of rRNA Locus:

 

 

 

 

 

 

 

 

 

 

 

It has been observed nucleolar loci exist in certain chromosomes and not in all chromosomes.  They are inherited by parents, i.e., male and female.  It so happens, the nucleolar DNA that opens up for transcription is either from one parent or the other.  So, one set of rRNA genes are kept silent and the other is expressed; which is similar to X chromosome inactivation.

 

In hybrids, genes inherited from both parents are typically expressed, producing intermediate phenotypes for characters such as flower or leaf morphology. This is true for Arabidopsis thaliana (A.t.), Arabidopsis arenosa (A.a) and their hybrid, A. suecica (A.s.), whose flowers are shown at the top left corner of this page. However, some genes are expressed from the chromosomes inherited from only one parent. An example is nucleolar dominance an epigenetic phenomenon in hybrids which describes the formation of a nucleolus (or nucleoli) on the chromosomes inherited from only one of the progenitors, regardless of whether that progenitor was the maternal or the paternal parent. Nucleolar dominance occurs in both the plant and animal kingdoms and is due to the expression of only one parental set of rRNA genes

In 1997 we found that nucleolar dominance in Brassica hybrids or A. suecica can be reversed by chemical inhibitors of DNA methylation or histone deacetylation (see Figure below), which led to the realization that nucleolar dominance is due to the selective silencing of one set of rRNA genes rather than the selective activation of the other. Because rRNA genes are clustered by the hundreds, spanning millions of base pairs of chromosomal DNA, nucleolar dominance is one of the most extensive gene silencing phenomena known.

Further study has shown that rRNA gene silencing involves concerted changes in DNA methylation and histone modification and we have proposed a model whereby DNA and histone modifications are each upstream of one another in a self-reinforcing, circular pathway (see Figure below). Changes in DNA methylation, histone acetylation and histone methylation are critical to the on-off switch mechanism that controls the number of active rRNA genes, both in hybrids displaying nucleolar dominance and in non-hybrids that regulate the effective dosage of their rRNA genes in response to the physiological needs of the cell

 

rRNA genes:

The high number of genes does only in part reflect the cellular demand for rRNAs, as only a fraction of these repeats is used for rRNA synthesis at any given time. In metabolically active human or mouse cells, approximately half of the ∼400 rRNA gene copies are transcriptionally active and the other half is silent. Once initiated, RNA polymerase I is capable of elongating through reconstituted chromatin without apparent displacement of the nucleosomes,

In some plants the SC /NOR from one of the parents remains inactive just like human X chromosomes.

 

Another silencing locus in S. cerevisiae is the highly repetitive rDNA locus on chromosome XII. The rDNA locus encodes the ribosomal RNAs, which are not translated into proteins, but establish the ribosome together with ribosomal proteins. One rDNA unit consists of ~9.1 kb, which is tandemly repeated in 100-200 copies per cell. Silencing at this locus is thought to regulate the access of RNA polymerase I, but prevents RNA polymerase II from transcription (Shou, et al., 2001).

At the rDNA locus, Sir2 assembles together with Net1, Cdc14, Nan1 and PolI into the so-called RENT (regulator of nucleolar silencing and telophase exit) complex. In sir2Δ cells, the rDNA silencing is decreased and the chromatin structure is less compact (Smith and Boeke, 1997), whereas the recombination rate is increased (Gottlieb and Esposito, 1989). Thus, Sir2 improves rDNA silencing via its deacetylation activity.

The structural core component of the RENT complex is Net1, which recruits the other RENT-proteins to the rDNA locus. Net1 binds within the RENT complex to PolI, the RNA polymerase I, and stimulates its enzymatic activity (Shou, et al., 2001). Whether Net1 directly binds to DNA is still unknown, but it is possible that other still unknown proteins contribute to rDNA silencing. Interestingly, when NET1 is overexpressed, it is also associated with the HMR-E silencer, whereas net1-1 acts indirectly in HMR silencing by releasing Sir2 from the nucleolus (Kasulke, et al., 2002).

RNA interference (RNAi) in heterochromatin formation.

(1) The DNA base sequence is read, and double-stranded RNA is produced. (2) The double-stranded RNA is cleaved to produce shorter RNA, which in turn binds to protein to form a complex. (3) The complex returns to the region where double-stranded RNA is produced, and complementarily binds to the RNA being transcribed. The complex attracts methylases Clr4 and HP1 protein to form heterochromatin.

Heterochromatin is gene-poor, relatively inaccessible to DNA-binding factors, and transcriptionally silent.  At centromeres and other heterochromatic domains in organisms ranging from the fission yeast Schizosaccharomycs pombe to humans, repetitive DNA sequences are transiently transcribed, which produces double-stranded RNAs by inter- or intramolecular pairing of repeat-containing RNAs. These double-stranded RNAs enter the RNA interference pathway, involving cleavage and processing by the Dicer ribonuclease and the association of the products, termed siRNAs, with a complex termed RITS. The RITS complex then localizes to repetitive DNA in the nucleus, presumably by pairing interactions between siRNAs and DNA or nascent RNA, which in turn results in targeting of histone methyltransferases to nucleosomes at the repeat sequences (Michael Bugler). This leads to HP1 protein binding and heterochromatization;

 

Model showing RNAi-mediated nucleation and spreading of heterochromatin.

Roles of the Clr4 methyltransferase complex in nucleation, spreading and maintenance of heterochromatin:

Ke Zhang, Kerstin Mosch, Wolfgang Fischle & Shiv I S Grewal

 

RNAi factors such as RITS, RDRC and Dicer, involved in processing of repeat transcripts (red line) into siRNAs, are required for targeting of ClrC to the heterochromatic repeats. siRNA-bound Ago1 is likely to specify the targeting of RITS to nascent repeat transcripts. RITS then facilitates ClrC loading. Rik1 might also directly associate with the repeat transcript and/or some part of the elongating RNAPII complex, thus promoting ClrC loading to nucleate heterochromatin. After the initial methylation of H3K9 by ClrC, Clr4 bound to H3K9me could modify adjacent nucleosomes creating additional binding sites for ClrC and other chromodomain proteins including Swi6 and Chp2 (HPs), which in turn mediate recruitment of factors such as SHREC, thereby promoting higher-order chromatin organization. Swi6 could further contribute to long-range heterochromatin spreading by promoting higher-order chromatin organization by forming oligomers and stabilizing the ClrC binding to chromatin. Boundary DNA elements block inappropriate spreading of heterochromatin into euchromatic regions. Green flag, histone acetylation; red lollipops, H3K9me.

 

Schematic model showing the role of RITS and RNAi in transcriptional and post-transcriptional silencing;

RNAi machinery is involved in the initial targeting of Clr4 protein to heterochromatin nucleation center (blue box), producing dsRNAs through a potential interaction between Clr4 with RITS (bar symbol) or with an unknown complex containing siRNAs (top). After the initial methylation of H3-Lys9 by Clr4, RITS can be tethered to chromatin through the interaction of Chp1 chromodomain and methylated H3-Lys9. Once bound to chromatin, RITS probably recruits Rdp1 and Dcr1 to process nascent transcripts into siRNAs. Newly produced siRNAs can trigger further recruitment of Clr4, creating a feedback loop to stably maintain heterochromatin (bottom). Swi6 bound to methylated H3-Lys9 recruits histone-modifying activities, including Clr4 that create additional binding sites for Chp1 chromodomain on adjacent nucleosomes, leading to RITS spreading.

 

Silent chromatin assembly in budding and fission yeast.

Silent chromatin assembly in budding and fission yeast. (A) Cis-acting DNA sequences (nucleation sites, yellow boxes) are necessary to nucleate assembly of silent chromatin. Trans-acting proteins that directly bind the nucleation sites are indicated. Nucleation sites at fission yeast centromeres are likely to exist, although they have not been identified to date (yellow boxes). Bidirectional transcription (indicated by black arrows) of cendg/dh/H-like sequences (red boxes) is thought to produce dsRNA, which is processed into siRNAs by the RNAi machinery in S. pombe. siRNAs are required at least for the initiation of heterochromatin assembly at the silent mating-type locus and in addition for the maintenance of heterochromatin at centromeres. (B) Sir3 and Sir4 have dimerization capacity that results in the spread of the SIR complex outward from the nucleation site. Sir3 contributes to the specificity for deacetylated histone tails, whereas Sir4 enhances the affinity of the complex through its ability to bind DNA. Sir2-mediated deacetylation keeps telomeric nucleosomes hypoacetylated creating a high-affinity binding site for Sir3. (C) In S. pombe, the RITS complex promotes Clr4-mediated H3K9 methylation by associating with nascent transcripts through siRNA base pairing, and with methylated H3K9 through the chromodomain of its Chp1 subunit. Low levels of H3K9 methylation are maintained in RNAi mutant cells by a yet to be identified alternative pathway (putative nucleation element, yellow box). Primary siRNAs originating from dsRNA formed by bidirectional transcription of a centromeric sequence could prime further dsRNA synthesis and secondary siRNA generation by recruiting the RDRC complex to the nascent transcript. This would allow the spreading of H3K9me away from the nucleation site. H3K9^me is bound by the chromodomain proteins Chp1, Chp2 and Swi6. The binding of Chp2 to H3K9^me results in the recruitment of the SHREC complex, which in turn deacetylates H3K14. For unknown reasons this reduces RNA Pol II occupancy

RNAi-mediated heterochromatin assembly in fission yeast.

 

Transcription and RNAi in heterochromatic gene silencing

Marc Bühler & Danesh Moazed

(a) Biochemically purified fission yeast complexes required for RNAi-mediated silencing. RITS, RDRC and Dicer are described in the text. ARC is an Ago1 chaperone complex that carries duplex siRNA, and the CLRC complex, which contains the Clr4 H3K9 methyltransferase, is required for H3K9 methylation and siRNA generation (reviewed in ref. 16). (b) The nascent-transcript model proposes that the RITS complex mediates heterochromatin formation by associating with nascent transcripts via siRNA base-pairing, and with methylated H3K9 via the chromodomain of its Chp1 subunit. dsRNA synthesis and siRNA generation occur in association with specific chromosome regions and may underlie cis restriction of siRNA-mediated silencing. The chromosome-associated siRNA synthesis loop is essential for the spreading of H3K9 methylation and silencing at the centromere. The coupling of the siRNA synthesis loop to H3K9 methylation forms a stable feedback loop that epigenetically maintains heterochromatin.

 

Left: generalized RNAi pathway. Double-stranded RNA (dsRNA) produced, for example, by convergent transcription, serves as a substrate for the enzyme Dicer, which cuts dsRNA molecules into short (approx. 21–22 nucleotide long) fragments. An RNA-dependent RNA polymerase (RdRP) amplifies these small RNAs. The RNAi silencing complex (RISC) contains the ARGONAUTE (AGO) protein, mediates the annealing of the small RNA strands to the cognate mRNA and induces degradation or blocks translation. Right: action of RNAi on DNA. A specialized RISC complex (RITS) targets loci homologous to small RNAs for epigenetic suppression, presumably through recognition of DNA sequence or of nascent RNA, leading to recruitment of histone methylases, which add methyl groups to lysine residues (in particular in positions K9 and K27) on histone 3 (H3). Methylated H3K9 may recruit heterochromatin protein 1 or DNA methylases, which transfer methyl groups to the DNA and ultimately lead to heterochromatin formation. CH3 (inverted): methylated DNA; CH# methylated histones.

 

 

Heterochromatin: A Rapidly Evolving Species Barrier

Stacie E. Hughes¹ and R. Scott Hawley

 

Nearly 100 years ago, biologists divided regions of chromosomes into two types, euchromatin and heterochromatin, on the basis of their appearance (reviewed in. The initial classification of DNA was based on the observation that euchromatic regions changed their degree of condensation during the cell division cycle, whereas heterochromatic regions remained highly condensed throughout the majority of the cell cycle. Although the biological significance of heterochromatin remained obscure for many years, it is now apparent that heterochromatin plays a number of biological roles, including a recently identified role in speciation. In addition to differences in the timing of chromosome condensation, numerous other differences have been identified between euchromatin and heterochromatin. Euchromatin is enriched with unique coding sequences, and the genes within the euchromatin are typically actively transcribed. Heterochromatin, on the other hand, is considered to be gene poor, being primarily composed of arrays of highly repetitive simple sequences, such as satellite sequences and/or transposable elements. Heterochromatin is enriched at the centromeres and telomeres of chromosomes.

As heterochromatin rapidly changes, the mechanisms that maintain it may well diverge as populations become isolated by various mechanisms. If those mechanisms change in such a way that the heterochromatin of population A can no longer be maintained by the maintenance proteins in population B, then the heterochromatin itself becomes a barrier between those populations as speciation proceeds. Many more questions await investigation, both in terms of the system of hybrid in viability described above and in terms of assessing the degree to which the safe-guarding of heterochromatic integrity underlies other examples of speciation. But one thing is clear: if any part of heterochromatin is indeed “junk,” then it is “junk” that both needs to be taken good care of and “junk” that sets one species apart from its neighbors.

 

 

Chromatin signature:

Defining the chromatin signature of inducible genes in T cells

Pek S Lim¹¤, Kristine Hardy¹¤, Karen L Bunting², Lina Ma¹, Kaiman Peng³, Xinxin Chen³ and Mary F Shannon¹

To facilitate comparison of genes with similar basal expression levels, we used expression-profiling data to bin genes according to their basal expression levels.

1. We found that inducible genes in the lower basal expression bins, especially rapidly induced primary response genes, were more likely than their non-responsive counterparts to display the histone modifications of active genes, have RNA polymerase II (Pol II) at their promoters and show evidence of ongoing basal elongation. There was little or no evidence for the presence of active chromatin marks in the absence of promoter Pol II on these inducible genes.

2. In addition, we identified a subgroup of genes with active promoter chromatin marks and promoter Pol II but no evidence of elongation. Following T cell activation, we find little evidence for a major shift in the active chromatin signature around inducible gene promoters but many genes recruit more Pol II and show increased evidence of elongation.

Bivalent - genes marked with both active (histone H3 lysine 4 trimethyl (H3K4me3)) and repressive (histone H3 lysine 27 trimethyl (H3K27me3)) histone modifications,

Furthermore, many of these bivalent genes are found to have RNA polymerase II (Pol II) located at their promoters in what is proposed to be a poised state.  The existence of a bivalent state has also been shown on some genes in other types of stem cells and in more differentiated cells, implying that this chromatin state may be involved in controlling genes that respond to developmental or environmental signals in all cell types.

 

It has long been known that certain inducible genes, such as the heat shock genes] and some oncogenes, have Pol II paused or stalled close to the start of gene transcription and that an increased elongation rate plays a role in their response to signalling.

 

More recently, genome-wide studies in mouse and human embryonic stem cells and differentiated human cells have identified large numbers of genes where Pol II is located at the promoter in the absence of ongoing transcription and these genes are often referred to as poised. In yeast, Pol II was constitutively bound to hundreds of promoter regions that are activated immediately following exit from stationary phase.

 

Recent genome-wide studies in Drosophila have also defined groups of genes with promoter-enriched Pol II, a feature that is postulated to facilitate rapid induction of transcription of these genes. Genes in the first class lack Pol II and are considered as inactive.

 

The second class includes active genes where Pol II can be detected at both the promoter and in the body of the gene, but it should be noted that, in general, the level of Pol II in the body of the gene is lower than at the promoter or the 3' end.

 

The third class consists of those genes where Pol II is detected at the promoter but not in the body of the gene and are considered potentially active. Genes in this third class are generally referred to as poised genes and are enriched for developmental control genes and genes that respond to developmental or environmental signals.

 

Recent evidence in Drosophila suggests that genes with promoter-proximal enrichment of Pol II can span a wide range of expression levels, supporting the idea that promoter proximal pausing is a common mechanism used to control transcription rate.

 

Using three approaches - ChIP combined with microarray technology (ChIP-on-chip), mining of ChIP-Seq data and ChIP with quantitative PCR (ChIP-qPCR) - for individual genes, we sought to define the chromatin signature of inducible genes in T cells.

 

These studies, and others on single genes, have led to the identification of a group of genes that are described as poised and potentially active

Even within the lowest basal expression bin (log₂ 3 to 4) in the human T cell data set, >65% of the genes had evidence of promoter Pol II.

Most of these genes (11 of 16) also had an active histone acetylation and methylation signature

 

Hargreaves et al. have recently proposed a model whereby constitutive transcription factors such as Sp1 recruit certain histone acetylases, such as p300, to primary response genes to maintain an active chromatin acetylation signature. Inducible transcription factors then recruit different acetylases that modify a different set of lysines on the histone proteins to provide a platform to generate an even more active gene. It would be of interest to examine these latter histone modifications in T cells.

Conclusions;

The results presented here show that inducible genes, especially primary response genes, are in a more active chromatin state than their non-responsive counterparts for a given basal expression level. Recent evidence suggests that the permissive state of primary response genes may be present throughout development to allow rapid expression of these genes in many cell types. It will be important to determine the molecular mechanisms that initiate and maintain this permissive state.

 

Bivalent chromatin genes:

 

Figure: Bivalent chromatin domains mark the promoters of developmentally important genes in pluripotent ES cells. PRC2 and TrxG proteins catalyze the tri-methylation of histone H3 on lysine 27 and 4, respectively. PRC1 is also recruited to many of these genes and can mono-ubiquitinylate histone H2A on lysine 119, a modification that is also thought to be important for gene silencing, possibly through blocking RNAPII elongation. As such, bivalent genes are said to be silent, yet poised for activation. H2AZ is highly enriched in a manner that is remarkably similar to PRC2 and may also be an important regulatory component at bivalent genes. Upon differentiation, the bivalent histone marks can be resolved to monovalent modifications in which the gene is “ON” or “OFF”. Bivalent domains can also be maintained or newly established in lineage-committed cells.

Gene(s) status in chromatin:

Genes exist in inactive state for certain repressor complexes (PcG) are bound in promoter region.  In some loci certain genes are made silent because localized heterochromatization. Thus, one can observe status of thousands of genes in chromatin in three forms.

1. Once such dormant regions are activated they recruit other factors including RNA polymerases and they remain there as bound structures waiting to be activated; this can be considered as dormant state ready to be activated. 

2. There are other situations where all these factors bound including RNAPs initiated transcription but after some distance of transcription they are halted for further stimulation as in HSP genes.  Such expressions are called poised state. 

3. The others are in transcription elongation mode called active state. 

 

S/MAR

They occur at the flanks of transcribed regions, S/MARS act as association points for common nuclear structural proteins. S/MARs are required for authentic and efficient chromosomal replication, transcription, recombination and chromosome condensation. S/MARs not only separate a given transcriptional unit (chromatin domain) from its neighbors, but also provide platforms for the assembly of factors enabling transcriptional events within a given domain. While the number of S/MARs in the human genome has been estimated to approach 64 000 (chromatin domains) plus an additional 10 000 (replication foci), in 2007 still only a minor fraction (559 for all eukaryotes) had met the standard criteria for an annotation in the S/MARt database SMARtDB.  From Wikipedia, the free encyclopedia:

 

Nowadays the nuclear matrix is seen as a dynamic entity the description of scaffold-attachment elements (SARs) (Laemmle and coworkers); Subsequent work demonstrated the existence of constitutive (SAR-like) and the facultative (MAR-like) functional elements depending on the context. Constitutive S/MARs were found to be associated with a DNase I hypersensitive site in 'all' cell types (whether or not the enclosed domain was transcribed), DNase I hypersensitivity of the facultative type depended on the transcriptional status. They act as permanent domain boundaries in all cell types^. The major difference between these two functional types of S/MARs is their size: the constitutive elements may extend over several kilobase pairs whereas facultative ones are at the lower size limit around 300 base pairs.

 

S/MARs not only separate a given transcriptional unit (chromatin domain) from its neighbors, but also provide platforms for the assembly of factors enabling transcriptional events within a given domain. 

 

DNases, Topoisomerases, Poly(ADP-ribosyl) polymerases and enzymes of the histone-acetylation and DNA-methylation apparatus are found associated with this region. While the number of S/MARs in the human genome has been estimated to approach 64 000 (chromatin domains) plus an additional 10 000 (replication foci), in 2007 still only a minor fraction (559 for all eukaryotes) had met the standard criteria for an annotation in the S/MARt database SMARtDB

 

Mechanisms of action of HMGA proteins:

Roles of HMGA proteins in cancer

Alfredo Fusco & Monica Fedele

 

HMGA proteins directly bind to the DNA, modifying its conformation and consequently facilitating the binding of a group of transcriptional factors (TF). They interact with both DNA and TFs to generate a multiprotein stereo specific complex bound to DNA (see figure part a). HMGA proteins have been shown to participate in this way in the regulation of many genes, the best studied being the interferon (IFN)- gene. The activation of IFN- expression is due to a multifactor complex that assembles in the nucleosome-free enhancer region of the gene, which includes the factors nuclear factor B, interferon regulatory factor, activating transcriptional factor2/JUN and the HMGA1a protein.^.

Finally, the HMGA proteins have the ability to alter chromatin structure (see figure part c). Indeed, they have been shown to be important elements that are associated with matrix- and scaffold-associated regions. The binding of HMGA proteins to these regions de-represses transcription by displacement of histone H1 by DNA^. These observations provided the first direct evidence that nuclear matrix bound transcription factors are also bound to MARs in situ. Further, the results provide evidence that the nuclear matrix is not simply a storage site for inactive transcription factors/cofactors.

(A) Active chromatin marks. Nucleosomes encompassing the transcribed region of a gene—a promoter, enhancer, and insulator, respectively—are shown (structure of gene and regulatory elements are represented below). The N-terminal “tails” of histone H3 are shown in dark gray, and the tails of H4 are in light gray. H3/K4 methylation and H3/K9 mono-methylation are enriched at the enhancer, the promoter, and the 5′ end of the active gene. H3/K27 and H4/K20 mono-methylation is enriched over the transcribed region, whereas H3/K36 trimethylation peaks at the 5′ of the active gene. Note that active genes are also enriched in H3, H4, and H2A acetylation (not shown).

 

(B) Poised chromatin marks. Four nucleosomes encompassing the transcribed region of a gene poised for transcription and one nucleosome each on a promoter element, an enhancer element, and an insulator are shown. The promoter and the transcribed region are enriched in the repressive mark H3/K27 trimethylation, whereas the region around the transcription start is also enriched in the active mark H3/K4 trimethylation. This combination of active and repressive marks can poise genes for activation and forms a so-called “bivalent domain.”

 

(C) Inactive chromatin marks. Modifications of histones H3 and H4 in nucleosomes encompassing a repressed or silenced gene are shown. The coding sequence and promoter of the inactive gene are enriched in H3/K9 and H3/K27 di- and trimethylation. The 5′ end of the gene and the promoter region are marked by H3/K79 trimethylation, whereas the insulator element carries activating marks. Data are based on Barski et al. (2007). (D) Scheme of a protein-coding gene with exons shown as light-gray boxes and introns as white boxes. Cis-acting regulatory sequences (enhancer, promoter, and insulator) are represented by black and dark- gray boxes.

 

Defining the chromatin signature of inducible genes in T cells:

Pek S Lim¹¤, Kristine Hardy¹¤, Karen L Bunting², Lina Ma¹, Kaiman Peng³, Xinxin Chen³ and Mary F Shannon¹

To facilitate comparison of genes with similar basal expression levels, we used expression-profiling data to bin genes according to their basal expression levels. We found that inducible genes in the lower basal expression bins, especially rapidly induced primary response genes, were more likely than their non-responsive counterparts to display the histone modifications of active genes, have RNA polymerase II (Pol II) at their promoters and show evidence of ongoing basal elongation. There was little or no evidence for the presence of active chromatin marks in the absence of promoter Pol II on these inducible genes. In addition, we identified a subgroup of genes with active promoter chromatin marks and promoter Pol II but no evidence of elongation. Following T cell activation, we find little evidence for a major shift in the active chromatin signature around inducible gene promoters but many genes recruit more Pol II and show increased evidence of elongation.

“In general, actively transcribed genes are associated with lysine acetylation on histones H3 and H4 and with methylation of histone H3 on lysine 4 (H3K4me). On the other hand, methylation of lysine 9 (H3K9me) or lysine 27 (H3K27me) on H3 is associated with repression. Many protein complexes responsible for adding or removing these modifications have been isolated and shown to play important roles in controlling gene expression

 

Of particular interest in this regard are recent genome-wide studies of histone marks in mouse pluripotent embryonic stem cells that have defined a class of developmentally regulated genes as 'bivalent' - genes marked with both active (histone H3 lysine 4 trimethyl (H3K4me3)) and repressive (histone H3 lysine 27 trimethyl (H3K27me3)) histone modifications. Furthermore, many of these bivalent genes are found to have RNA polymerase II (Pol II) located at their promoters in what is proposed to be a poised state. .

 

More recently, genome-wide studies in mouse and human embryonic stem cells and differentiated human cells, have identified large numbers of genes where Pol II is located at the promoter in the absence of on-going transcription and these genes are often referred to as poised. In yeast, Pol II was constitutively bound to hundreds of promoter regions that are activated immediately following exit from stationary phase. Recent genome-wide studies in Drosophila have also defined groups of genes with promoter-enriched Pol II, a feature that is postulated to facilitate rapid induction of transcription of the said genes..

 

 

These studies have led to the definition of three classes of genes based on Pol II location. Genes in the first class lack Pol II and are considered as inactive. The second class includes active genes where Pol II can be detected at both the promoter and in the body of the gene, but it should be noted that, in general, the level of Pol II in the body of the gene is lower than at the promoter or the 3' end. The third class consists of those genes where Pol II is detected at the promoter but not in the body of the gene and are considered potentially active. Genes in this third class are generally referred to as Poised genes and are enriched for developmental control genes and genes that respond to developmental or environmental signals.

 

Using three approaches - ChIP combined with microarray technology (ChIP-on-chip), mining of ChIP-Seq data and ChIP with quantitative PCR (ChIP-qPCR) - for individual genes, we sought to define the chromatin signature of inducible genes in T cells. To facilitate comparison of genes with similar basal expression levels, genes were binned according to their basal expression levels determined from expression profiling studies. Our results show that inducible genes in the lower basal expression bins, especially rapidly induced primary response genes, and were more likely to display the chromatin characteristics of active genes than their non-responsive counterparts.

Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals:

Mitchell Guttman^1,2, Ido Amit¹, Manuel Garber¹, Courtney French¹, Michael F. Lin¹, David Feldser³, Maite Huarte^1,6, Or Zuk¹, Bryce W. Carey^2,8, John P. Cassady^2,8, Moran N. Cabili⁷, Rudolf Jaenisch^2,8, Tarjei S. Mikkelsen^1,4, Tyler Jacks^2,3, Nir Hacohen^1,9, Bradley E. Bernstein^1,10,11, Manolis Kellis^1,5, Aviv Regev^1,2, John L. Rinn^1,6,11,12 & Eric S. Lander^1,2,7,8,12

There is growing recognition that mammalian cells produce many thousands of large intergenic transcripts, which are non-coding RNAs called LincRNAs.

 

However, the functional significance of these transcripts has been particularly controversial. Although there are some well-characterized examples, most (>95%) show little evidence of evolutionary conservation and have been suggested to represent transcriptional noise. Here we report a new approach to identifying large non-coding RNAs using chromatin-state maps to discover discrete transcriptional units intervening known protein-coding loci. Our approach identified ~1,600 large multi-exonic RNAs across four mouse cell types. In sharp contrast to previous collections, these large intervening non-coding RNAs (lincRNAs) show strong purifying selection in their genomic loci, exonic sequences and promoter regions, with greater than 95% showing clear evolutionary conservation. We also developed a functional genomics approach that assigns putative functions to each lincRNA, demonstrating a diverse range of roles for lincRNAs in processes from embryonic stem cell pluripotency to cell proliferation. We obtained independent functional validation for the predictions for over 100 lincRNAs, using cell-based assays. In particular, we demonstrate that specific lincRNAs are transcriptionally regulated by key transcription factors in these processes such as p53, NFκB, Sox2, Oct4 (also known as Pou5f1) and Nanog. Together, these results define a unique collection of functional lincRNA that are highly conserved and implicated in diverse biological processes. There is growing recognition that mammalian cells produce many thousands of large intergenic transcripts.

 

 

Linc RNAs

Science: 'Linc-Ing' a Noncoding RNA to a Central Cellular Pathway. ScienceDaily (Aug. 12, 2010)

Surprising breakthrough is now made even more compelling with the finding that dozens of these lincRNAs are induced by p53 (the most commonly mutated gene in cancer), suggesting that this class of genes plays a critical role in cell development and regulation. Furthermore, the researchers identify one lincRNA in particular (lincRNA-p21), and demonstrate its critical role in suppressing the reading of many genes across the genome following p53 activation.

 

Through a massive sequencing of ChIP data they uncovered a chromatin “signature” for actively transcribed regions between known protein coding genes, and used this signature to identify about 1700 previously un-annotated stretches of the genome at least five kb long.

 

Enhancers are distal regulatory sequences that control gene expression in development. Ãrom et al. now report in Cell that some long noncoding RNAs has functional properties of enhancers. Known enhancers are also transcribed in cells in which they are active, suggesting that noncoding RNAs are integral to enhancer action.

That surprising breakthrough is now made even more compelling with the finding that dozens of these lincRNAs are induced by p53 (the most commonly mutated gene in cancer), suggesting that this class of genes play a critical role in cell development and regulation.

 

This current work demonstrates that several dozen lincRNAs are targeted directly by p53, and lincRNA-p21 in particular responds to p53 signaling by suppressing multiple genes across the genome to drive apoptosis. “We were surprised to find that lincRNA-p21 appears to be functioning as a global repressor, regulating hundreds of genes in the p53 pathway,” said Huarte. “In other words,” added Rinn, “this lincRNA is playing defense for p53 to block other pathways in their efforts to interfere with p53’s critical job of tumor suppression by cell death.”

“In the same way that air traffic controllers organize planes in the air, lincRNAs organize key nuclear complexes in the cell,” said Rinn. “lincRNA-p21specifically binds to a protein called hnRNP-K and then guides hnRNP-K to its final destination to shut down any genes that interfere with p53.”

“lincRNAs could be those elusive ‘anti-factors’ that serve to shut genes down by reshuffling proteins around the genome.”

 

Histone acetylation and chromatin signature in stem cell identity and cancer

 

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Vivek Shukla^{, b}, Thomas Vaissière^a and Zdenko Herceg^{, ,} International Agency for Research on Cancer (IARC), 150 Cours Albert Thomas, 69008 Lyon, France

 

Unique properties of stem cells defined as “stemness” may be determined by acetylation and methylation of histones near gene promoters that regulate gene transcription; however,r these histone modifications elsewhere in the genome may also be important. In this review, we discuss new insights into possible mechanisms by which histone acetyltransferase (HATs) and histone acetylation in concert with other chromatin modifications may regulate pluripotency, and speculate how deregulation of histone marking may lead to tumorigenesis.

The chromatin signature of pluripotent cells:

HFSP Long-Term Fellow Nadine Vastenhouw and colleagues

 

Zebra fish embryos are used to study the chromatin status. Much of our knowledge of pluripotency originates from studies in cultured embryonic stem (ES) cells. Analysis of chromatin states across the ES cell genome has suggested that pluripotent cells have a unique chromatin signature. In particular, the promoters of many genes that regulate development contain repressive chromatin marks in combination with active marks. Such “bivalent” genes are thought to be transcriptionally inactive, yet poised for imminent activation. Permanently pluripotent cells such as ES cells do not exist in vivo, and it has therefore been unclear whether embryonic cells have the same chromatin profile as ES cells

 

The study found bivalent chromatin domains that were associated with developmental regulatory genes, suggesting that these domains are not simply an artifact of cultured cells. The research also indicated that many inactive genes in zebrafish and ES cells carried “monovalent” chromatin marks; i.e. active, but not repressive, chromatin modifications. Further studies suggested that these chromatin marks are only established during the maternal-zygotic transition and could be established in the absence of specific transcriptional regulators. Taken together, these results suggest that the chromatin signature of pluripotency is established during the maternal-zygotic transition to poise genes for activation. While these studies are fundamental in nature, they may also inform strategies to more efficiently reprogram differentiated cells into pluripotent cells, one of the goals of regenerative medicine.

 

Open chromatin encoded in DNA sequence is the signature of ‘master’ replication origins in human cells:

1.      Benjamin Audit¹,², Lamia Zaghloul¹,², Cédric Vaillant¹,², Guillaume Chevereau¹,², Yves d'Aubenton-Carafa³, Claude Thermes³ and Alain Arneodo¹,²,^*

 

For years, progress in elucidating the mechanisms underlying replication initiation and its coupling to transcriptional activities and to local chromatin structure has been hampered by the small number (approximately 30) of well-established origins in the human genome and more generally in mammalian genomes. Recent in silico studies of compositional strand asymmetries revealed a high level of organization of human genes around 1000 putative replication origins. Here, by comparing with recently experimentally identified replication origins, we provide further support that these putative origins are active in vivo. We show that regions ∼300-kb wide surrounding most of these putative replication origins that replicate early in the S phase are hypersensitive to DNase I cleavage, hypo methylated and present a significant enrichment in genomic energy barriers that impair nucleosome formation (nucleosome-free regions). This suggests that these putative replication origins are specified by an open chromatin structure favored by the DNA sequence. We discuss how this distinctive attribute makes these origins, further qualified as ‘master’ replication origins, privileged loci for future research to decipher the human spatio-temporal replication program. Finally, we argue that these ‘master’ origins are likely to play a key role in genome dynamics during evolution and in pathological situations.

In that context, an in silico analysis of the strand composition asymmetry (skew) profile of the human genome allowed us to identify 1060 putative replication origins, likely conserved in mammalian genomes, that border 678 large (mean length of 1.2 Mb) genomic domains labeled N-domains as their skew profile is shaped like an N  Recently, the localization of replication origins has been experimentally investigated along 1% of the human genome (ENCODE regions) by hybridization to Affymetrix ENCODE tiling arrays of purified small nascent DNA strands and of restriction fragments containing small replication bubble.

 

DNA hypomethylation is associated with N-domain borders:

Cytosine DNA methylation is a mediator of gene silencing in repressed heterochromatic regions, while in potentially active open chromatin regions, DNA is essentially unmethylated. DNA methylation is continuously distributed in mammalian genomes with the notable exceptions of CGIs, short unmethylated regions rich in CpGs and of certain promoters and transcription start sites (TSSs).

In conclusion, analyses of experimental and numerical open chromatin markers suggest the existence of ‘master’ replication origins likely to be active in germ line as well as somatic human cells.

 

Discovery and Annotation of Functional Chromatin Signatures in the Human Genome.  Gary Hon^1,2, Wei Wang^1,3*, Bing Ren.

Recent studies have observed that histone tails can be modified in a variety of ways. Analyzing a collection of 21 histone modifications, we attempted to determine what common signatures are associated with different classes of regulatory elements and whether they mark places of distinct function. Indeed, at promoters, we identified a number of distinct signatures, each associated with a different class of expressed and functional genes. We also observed several unexpected signatures marking exons that directly correlate with the expression of exons. Finally, we recovered many places marked by two distinct repressive modifications, and showed that they mark distinct populations of repetitive elements associated with distinct modes of gene repression. Together, these results highlight the rich information embedded in the human epigenome and underscore its value in studying gene regulation.

Transcriptional regulation in human cells is a complex process involving a multitude of regulatory elements encoded by the genome. Recent studies have shown that distinct chromatin signatures mark a variety of functional genomic elements and those subtle variations of these signatures mark elements with different functions. To identify novel chromatin signatures in the human genome, we apply a de novo pattern-finding algorithm to genome-wide maps of histone modifications. We recover previously known chromatin signatures associated with promoters and enhancers. We also observe several chromatin signatures with strong enrichment of H3K36me3 marking exons. Closer examination reveals that H3K36me3 is found on well-positioned nucleosomes at exon 5′ ends, and that this modification is a global mark of exon expression that also correlates with alternative splicing. Additionally, we observe strong enrichment of H2BK5me1 and H4K20me1 at highly expressed exons near the 5′ end, in contrast to the opposite distribution of H3K36me3-marked exons. Finally, we also recover frequently occurring chromatin signatures displaying enrichment of repressive histone modifications. These signatures mark distinct repeat sequences and are associated with distinct modes of gene repression. Together, these results highlight the rich information embedded in the human epigenome and underscore its value in studying gene regulation.

Indeed, at promoters, we identified a number of distinct signatures, each associated with a different class of expressed and functional genes. We also observed several unexpected signatures marking exons that directly correlate with the expression of exons. Finally, we recovered many places marked by two distinct repressive modifications, and showed that they mark distinct populations of repetitive elements associated with distinct modes of gene repression. Together, these results highlight the rich information embedded in the human epigenome and underscore its value in studying gene regulation.

 

Open chromatin encoded in DNA sequence is the signature of ‘master’ replication origins in human cells.

Benjamin Audit¹,², Lamia Zaghloul¹,², Cédric Vaillant¹,², Guillaume Chevereau¹,²,Yves d'Aubenton-Carafa³, Claude Thermes³ and Alain Arneodo¹,²

 

 For years, progress in elucidating the mechanisms underlying replication initiation and its coupling to transcriptional activities and to local chromatin structure has been hampered by the small number (approximately 30) of well-established origins in the human genome and more generally in mammalian genomes. Recent in silico studies of compositional strand asymmetries revealed a high level of organization of human genes around 1000 putative replication origins. Here, by comparing with recently experimentally identified replication origins, we provide further support that these putative origins are active in vivo. We show that regions ∼300-kb wide surrounding most of these putative replication origins that replicate early in the S phase are hypersensitive to DNase I cleavage, hypo methylated and present a significant enrichment in genomic energy barriers that impair nucleosome formation (nucleosome-free regions). This suggests that these putative replication origins are specified by an open chromatin structure favored by the DNA sequence. We discuss how this distinctive attribute makes these origins, further qualified as ‘master’ replication origins, privileged loci for future research to decipher the human spatio-temporal replication program. Finally, we argue that these ‘master’ origins are likely to play a key role in genome dynamics during evolution and in pathological situations.

Cell cycle regulation of chromatin at an origin of DNA replication;

Jing Zhou, Charles M Chau, Zhong Deng, Ramin Shiekhattar, Mark-Peter Spindler, Aloys Schepers and Paul M Lieberman

 

 

Model of cell cycle coordinated histone tail modification and chromatin remodeling at OriP.  An SNF2h/HDAC2 complex can be colocalized to DS during G1, simultaneously with histone H3 deacetylation, chromatin remodeling, and MCM loading.

 

Chromatin signature at Origin:

 

Model of cell cycle coordinated histone tail modification and chromatin remodeling at OriP. An SNF2h/HDAC2 complex can be co localized to DS during G1, simultaneously with histone H3 deacetylation, chromatin remodeling, and MCM loading.
Model of cell cycle coordinated histone tail modification and chromatin remodeling at OriP. An SNF2h/HDAC2 complex can be colocalized to DS during G1, simultaneously with histone H3 deacetylation, chromatin remodeling, and MCM loading.
Model of cell cycle coordinated histone tail modification and chromatin remodeling at OriP. An SNF2h/HDAC2 complex can be colocalized to DS during G1, simultaneously with histone H3 deacetylation, chromatin remodeling, and MCM loading.

M9 - Control of DNA replication by epigenetic modification and chromatin remodeling

To ensure genome stability, DNA replication is tightly controlled at its initiation stage and strictly limited to once-per-cell-cycle. Replication competence is gained only in the G1 phase of the cell cycle, a process called licensing, which is characterized by the formation of pre-replicative complexes (pre-RC). Pre-RCs are formed at specific sites called replication origins. Little is known how pre-RC assembly relates to the chromosomal context, and whether chromatin remodeling and posttranslational modification (PTM) are involved in this process.

Model of chromatin remodeling complexes and histone modifying enzymes at replication origins. Both enzymatic functions may influence pre-RC assembly at replication origins and origin selection in the context of chromatin.

 It is the aim of this proposal to study two aspects of the relationship between pre-RC formation at replication origins and in their chromatin environment. (i) Does Pre-RC assembly require the action of a specific chromatin-remodeling complex? (ii) Is Pre-RC formation during G1 associated with specific histone modifications? To address the first question, we will use a proteomic approach to identify pre-RC components associated with a SNF2h-containing remodeling complex. Using ChIP-on-Chip we will determine, whether all origins or a specific subset of them requires the activity of chromatin remodeling complexes. The second aspect focuses on the role of a specific histone acetyltransferase, Hbo1, and its cofactor ING5 and, on how the activity of this complex is integrated into cell cycle regulation of pre-RC formation.

Predictive chromatin signatures in the mammalian genome

Gary C. Hon1,2, R. David Hawkins2 and Bing Ren1,

 

 

Modification of histone H3.

 

Lysine residues on histone H3 can be mono-, di- or tri-methylated. Shown are modifications H3K4me1, H3K4me3 and H3K36me3, which mark:

1. H3K4me1active/poised enhancers,

2. H3K4me3 active/poised promoters,

3. H3K36me3actively transcribed regions, respectively. Me = methylation.

 

HMG-14 and Transcriptional Elongation

The nucleosomes that did remain appeared to be devoid of histone H1 and to contain instead nonhistone chromatin proteins HMG-14 and 17 proteins. HMG-14 is able to stimulate the elongation phase of transcription by RNA polymerase II. It does not promote the initiation of transcription and it does not affect transcription elongation on nucleosome-free DNA. It appears that the association of HMG-14 with nucleosomes is part of the process that maintains transcription on nucleosomes.

The recent findings from these studies reaffirm the idea that unique chromatin signatures correlate with cell type and function, and that a given chromatin state is a balance of multiple epigenetic marks and implies that cell fate can be experimentally manipulated through tipping this balance.

 

Heat shock protein chromatin signature:

After heat shock, the transcriptional activator, heat-shock factor (HSF), forms a stable trimeric complex that binds the Hsp70 promoter^{105, 106}. Heat shock stimulates nucleosome loss at the Hsp70 locus. Nucleosome loss is dependent on HSF, GAF and poly (ADP)-ribose polymerase (PARP)⁶⁴. PARP localizes at many sites along polytene chromosomes but only catalyzes formation of ADP-ribose polymers from donor NAD⁺ at the heat-shock loci after induction by heat shock.

a | At the top is a schematic of the Drosophila melanogaster 87A heat-shock locus; the example of Hsp70Ab is used below. Equivalent events occur at Hsp70Aa. The arrows at the Hsp70Aa and Hsp70Ab promoters indicate the direction of transcription through the gene. RNA polymerase II (Pol II, shown in orange) and GAGA factor (GAF or Trithorax-like) are bound at the promoters of the Hsp70 genes before heat shock.

Nucleosomes, loss proceeds outwards from the 5′ end of the Hsp70 genes ahead of Pol II, extending as far as the SCS and SCS′ boundary elements (region of loss is grey in top panel). The CG31211 and CG3281 genes are not transcribed (as shown by blunt-headed arrows at their promoters) although Pol II is bound at their promoters and nucleosomes are lost.

  Before heat-shock activation at Hsp70, GAF recruits co-activators, the GTFs and ATP-dependent nucleosome-remodeling complexes, thereby facilitating pre-initiation complex (PIC) formation at the promoter. At Hsp70, however, PIC formation is not sufficient to activate productive transcription elongation. The CDK7 subunit of TFIIH phosphorylates serine 5 (S5) of the carboxyl-terminal domain (CTD) and Pol II initiates transcription into the first 20–40 bases of the gene, at which it pauses at the promoter-proximal pause site. Pol II is held here by the negative elongation factor (NELF) and DRB sensitivity-inducing factor (DSIF), which is composed of SPT4 and 5.

Bb.  Heat shock induces binding of HSF, which recruits additional co-activators and nucleosome-remodeling activities. HSF is required but is not sufficient to recruit the pause release factor P-TEFb to Hsp70. Recruitment of Mediator by HSF, which occurs independently of PIC formation, might contribute to P-TEFb binding. Recruited P-TEFb phosphorylates S2 of the CTD, SPT5 and NELF. These phosphorylation cause NELF to dissociate from Pol II, releasing polymerase into productive transcription elongation. Although Pol II still pauses briefly at the promoter-proximal pause site under heat-shock conditions, the duration of these pauses are much shorter than at normal temperature.

Genetic Imprinting:

 

Taking human species as an example, sperms contain either one X or one Y chromosome.  The X-chromosome is genetically active and Y-chromosome mostly inactive with the exception of few genes which are expressed in males. 

 

                                                Genetic Imprinting.

 

In females, early in embryonic stage both X-chromosomes from Ma and Pa inherited, are active.  But as cell division continues and when cell differentiation initiates, at this point of time, in some tissues X from Pa becomes inactive and the chromosome from Ma remain active.  In some other tissues the chromosome from Pa is active and chromosome from Ma remains inactivated.  So, the females have mosaic of active and inactive X chromosomes in different tissues.  Allelic gene expression in such tissue expresses epigenetic variation. The X-chromosome that is inactivated when stained appears a dark body is called Bar body. The X-chromosomes carry several hundreds of genes and many of them are allelic.

  

Differentially, one of the two X chromosome in one tissue and the other, in other tissue, becomes inactive, which is attributed directly X inactivation transcript.  The gene for such transcript is Xic. The Xic gene produces Xist RNAs.  If one of the chromosome inactive the other X chromosomes remains active for another transcript acts as antisense RNA against Xist RNA.

 

Thus, an allele with one dominant and the other recessive, in one tissue the dominant gene is expressed and, in another tissue, the recessive character is manifested, which phenomenon is called genetic mosaics, or what is popularly called calico-women.  In another tissue it can reverse expression.  A good example is of coat colors in cats and other animals.  This is often referred to as epigenetic phenomenon.

 

Silencing of certain allelic genes in one tissue and expression of the other allele in another tissue is very common, one such gene allelic pairs of genes is IGF-II (insulin like growth factor-II).  It is an example for genetic imprinting. Molecular basis of development poses a situation for classical biologists what is determination and what differentiation is and how to differentiate between the two.

 

Somatic cells in Females

Determination in molecular term is the availability of certain specific factors and assembly of the same on to a set of promoters for transcription, which ultimately determine the fate of the said cell.    Inducing the expression of certain genes in a cell which changes the character of the cell and it will propagate so, is termed as determination.

 

Once the gene inducing factors are in place, expression of the genes, which are different from the earlier set, determines what is the phenotype or the character of the cell, which is different from the previous state; such cell or cells are called differentiated.

 

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