Histone H3


Table of contents

Silencing of polycomb target genes is associated with methylation of histone H3 Lys 27

Polycomb group complexes 2 and 3 are involved in transcriptional silencing. These complexes contain a histone lysine methyltransferase (HKMT) activity that targets different lysine residues on histones H1 or H3 in vitro. However, it is not known if these histones are methylation targets in vivo because the human PRC2/3 complexes have not been studied in the context of a natural promoter because of the lack of known target genes. RNA expression arrays and CpG-island DNA arrays were used to identify and characterize human PRC2/3 target genes. Using oligonucleotide arrays, a cohort of genes were identified whose expression changes upon siRNA-mediated removal of Suz12 [Drosophila homolog Su(z)12], a core component of PRC2/3, from colon cancer cells. To determine which of the putative target genes are directly bound by Suz12 and to precisely map the binding of Suz12 to those promoters, a high-resolution chromatin immunoprecipitation (ChIP) analysis was combined with custom oligonucleotide promoter arrays. Additional putative Suz12 target genes were identified by using ChIP coupled to CpG-island microarrays. HKMT-Ezh2 and Eed, two other components of the PRC2/3 complexes, colocalize to the target promoters with Suz12. Importantly, recruitment of Suz12, Ezh2 and Eed to target promoters coincides with methylation of histone H3 on Lys 27 (Kirmizis, 2004).

Identification of mammalian PcG target genes has remained elusive for two main reasons. First, the majority of the previous PcG studies focused mostly on the biochemical purification and in vitro characterization of the activities of the PcG complexes and second, the lack of DNA-binding domains within PcG proteins makes the search for their target loci difficult. In this present study, the first known direct target genes of mammalian PcG complexes has been identified. To do so, RNAi was first used to identify genes deregulated by the loss of Suz12 protein in colon cancer cells. Next, Suz12 was shown to bind directly to the promoter of one of these genes (MYT1). Other members of the PRC2/3 complexes were shown to colocalize with Suz12 at the MYT1 promoter. Most importantly, recruitment of Suz12, Ezh2, and Eed to the MYT1 promoter was shown to correlate with methylation of H3-K27. To demonstrate that this silencing mechanism is not unique to MYT1, other Suz12 target genes were identified using a ChIP assay coupled to a CpG island microarray. Similarly to MYT1, the other target promoters of Suz12 are bound by the PRC2/3 components and are characterized by H3-K27 methylation. Thus, the first identified human PcG target genes all appear to be regulated by the histone methylase activity of the PRC complexes (Kirmizis, 2004).

The Suz12 target gene MYT1 was originally cloned from a human brain cDNA library on the basis of its ability to bind cis-regulatory elements of the glia-specific myelin proteolipid protein (PLP) gene and is suggested to be the prototype of the C2HC-type zinc finger protein family. More recently, the Xenopus ortholog of MYT1 (X-MYT1) was identified as a transcriptional activator because it could induce expression of an N-tubulin promoter reporter construct in transient transfection assays. Dominant-negative forms of X-MYT1 inhibited normal neurogenesis, suggesting that X-MYT1 is essential for inducing neuronal differentiation. Intriguingly, a recent report shows that the Xenopus ortholog of Ezh2 (XEZ) is expressed exclusively in the anterior neural plate during early Xenopus embryogenesis, and it was postulated that XEZ might be involved in delaying anterior neuronal differentiation (Barnett, 2001). Based on the current findings, it is possible that Ezh2 delays neuronal differentiation, via the PRC2/3 complexes, by repressing the activity of the MYT1 gene. In addition to MYT1, four additional promoters were identified as being robustly bound by components of the PRC2/3 complexes; each of these promoters is also characterized by high levels of H3-K27. Although a link between components of the PRC2/3 complexes and Wnt1, the cannabinoid receptor (CNR1), or the potassium channel KCNA1 have not been previously reported, it is intriguing to note that these mRNAs are expressed at very low levels in most human tissues, suggesting that they may be generally silenced by the PRC complexes. In support of this hypothesis, some of these target genes were shown to be bound by PRC2/3 components in other cell lines, such as the human MCF7 and mouse F9 (Kirmizis, 2004).

Polycomb group (PcG) genes are required for stable inheritance of epigenetic states throughout development, a phenomenon termed cellular memory. In Drosophila and mice, the product of the E(z) gene, one of the PcG genes, constitutes the ESC-E(Z) complex and specifically methylates histone H3. It has been argued that this methylation sets the stage for appropriate repression of certain genes. This study reports the isolation of a well-conserved homolog of E(z), olezh2, in medaka. Hypomorphic knock-down of olezh2 resulted in a cyclopia phenotype and markedly perturbed hedgehog signaling, consistent with a previous report on oleed, a medaka esc. Cyclopia was also found in embryos treated with trichostatin A, an inhibitor of histone deacetylase, which is a transient component of the ESC-E(Z) complex. The level of tri-methylation at lysine 27 of histone H3 is substantially decreased in both olezh2 and oleed knock-down embryos, and in embryos with hedgehog signaling perturbed by forskolin. It is concluded that the ESC-E(Z) complex per se participates in hedgehog signaling (Shindo, 2005).

To address the molecular mechanisms underlying Polycomb group (PcG)-mediated repression of Hox gene expression, this study focused on the binding patterns of PcG gene products to the flanking regions of the Hoxb8 gene in expressing and non-expressing tissues. In parallel, the distribution of histone marks of transcriptionally active H3 acetylated on lysine 9 (H3-K9) and methylated on lysine 4 (H3-K4) was followed, and of transcriptionally inactive chromatin trimethylated on lysine 27 (H3-K27). Chromatin immunoprecipitation revealed that the association of PcG proteins, and H3-K9 acetylation and H3-K27 trimethylation around Hoxb8 were distinct in tissues expressing and not expressing the gene. Developmental changes of these epigenetic marks temporally coincide with the misexpression of Hox genes in PcG mutants. Functional analyses, using mutant alleles impairing the PcG class 2 component Rnf2 (Homolog of Drosophila Ring) or the Suz12 mutation decreasing H3-K27 trimethylation, revealed that interactions between class 1 and class 2 PcG complexes, mediated by trimethylated H3-K27, play decisive roles in the maintenance of Hox gene repression outside their expression domain. Within the expression domains, class 2 PcG complexes appeared to maintain the transcriptionally active status via profound regulation of H3-K9 acetylation. The present study indicates distinct roles for class 2 PcG complexes in transcriptionally repressed and active domains of Hoxb8 gene (Fujimura, 2006; full text of article).

The main outcome of this study was to show that binding of a specific, Rnf2-containing form of the class 2 PcG complex, as well as H3-K27 trimethylation marking inactive chromatin, correlates with the maintenance of transcriptional silencing of a Hox gene in developing embryos. Moreover, the results demonstrated that genetic impairment of both PcG binding, and H3-K27 trimethylation leads to Hox gene derepression, and that H3-K27 trimethylation is required for PcG binding. In addition, the establishment of differential PcG binding and histone marks in expressing and non-expressing embryonic tissues occur in the same developmental time window as when Hox genes are deregulated in PcG mutants (Fujimura, 2006).

Rnf2 association to known regulatory elements of the Hoxb8 gene is seen predominantly in transcriptionally silent anterior embryonic tissues, whereas the binding of other PcG class 2 members, Phc1 and Cbx2, is observed at all AP levels, irrespective of transcriptional status. This implies that different forms of class 2 PcG complexes bind to the Hoxb genomic region in embryonic domains where the gene is transcriptionally active and repressed. This is reminiscent of previous findings in the Engrailed/Inv/GeneVI complex in Drosophila SL-2 cells, where the Pc protein is exclusively associated with transcriptionally silent genes, while Ph and Psc are present irrespective of the transcriptional status. Therefore the complete, 'perfect' form of the class 2 PcG core complex may mediate transcriptional repression more efficiently than form(s) lacking the Rnf2 component. If this is the case, incorporation of the Rnf2 component into the complex might be a limiting process to mediate transcriptional repression and regulate its stability. It is also possible that the role of Rnf2 is mediated through its E3 ubiquitin ligase activity directed to histone H2A (Fujimura, 2006).

Transcriptional repression of Hox genes in the developing embryo has been shown to correlate with the association of Rnf2-containing class 2 PcG complexes and H3-K27 trimethylation. De-repression of Hox genes in Rnf2 and Suz12 mutant cells reveal the requirement of both Rnf2 association and H3-K27 trimethylation in the mediation of this transcriptional repression. Since Rnf2 association to Hox genes is reduced in Suz12 mutant ES cells and Rnf2 mutation alters Hox expression without changing local levels of H3-K27 trimethylation, H3-K27 trimethylation mediated by class 1 PcG complexes at Hox genes may facilitate subsequent binding of Rnf2-containing PcG complexes. Recruitment of Rnf2-containing PcG complexes may in turn prevent the access of nucleosome remodeling factors, such as SWI/SNF complex, leading to the formation of a repressed chromatin status. Therefore, molecular circuitry underlying PcG silencing of Hox genes seems to have been evolutionarily conserved between Drosophila and mammals. It is also notable that Cbx2, a homologue of Drosophila Pc, binds to Hoxb8 in transcriptionally active embryonic tissues, despite the lack of histone H3 trimethylated at K27. This is consistent with biochemical data that have shown the association of purified or reconstituted PcG complexes with the nucleosomal templates lacking histone tails. The implication of these findings is that there are at least two different means by which class 2 PcG complexes bind to the chromatin, and that the association, which involves trimethylated H3-K27, mediates the repression at the Hox genes in vivo (Fujimura, 2006).

The maintenance of regionally restricted expression of Hox genes is likely to involve H3-K9 acetylation and H3-K4 methylation. This study has shown that these modifications of the histone tail increases craniocaudally along the axis. Although the transcriptionally active posterior tissues of 9.5 dpc and older embryos are more heavily acetylated at H3-K9 than the anterior, non-Hox expressing tissues, some acetylation of H3-K9 at Hoxb8 is seen in anterior regions where Hoxb8 expression is repressed at early and later developmental stages. De-repression of Hoxb8 expression upon depletion of Rnf2 in MEFs derived from the cranial part of 9.5 dpc embryos suggests the involvement of Rnf2-containing class 2 PcG complexes to mediate this transcriptional repression. Therefore, these data suggest that the associations of Rnf2-containing PcG complexes and acetylated H3-K9 may counteract each other and cooperate to maintain the anterior boundaries of Hoxb8 expression at mid-gestational stages and later. This is consistent with the antagonistic properties of Mll and Bmi1 mutations. Moreover, the establishment of the differential binding of the Rnf2 and H3-K9 acetylation at Hoxb8 during embryogenesis temporally coincides with de-repression of that Hox gene in Bmi1/Rnf110 and Phc1/Phc2 double homozygotes, and loss of its transcription in Mll homozygotes. Intriguingly, class 2 PcG complexes, which lack the Rnf2 component, are also involved in the maintenance of H3-K9 acetylation in embryonic tissues where Hox genes are expressed. This is consistent with predominant subnuclear localization of several PcG proteins in the perichromatin compartment where most pre-mRNA synthesis takes place. The molecular mechanisms underlying this positive action remain unaddressed (Fujimura, 2006).

In conclusion, class 2 PcG gene products play distinct roles in embryonic territories, which are silent or active for Hoxb8 transcription, by forming complexes of different composition. Interaction between class 1 and class 2 PcG complexes mediated by trimethylated H3-K27 play decisive roles in Hox gene repression outside their expression domains, as seen in Drosophila. In addition, within the Hox expression domain, class 2 PcG complexes are involved in maintaining a transcriptionally active status, independent of H3-K27 trimethylation (Fujimura, 2006).

Polycomb group proteins are essential for early development in metazoans, but their contributions to human development are not well understood. The Polycomb Repressive Complex 2 (PRC2) subunit SUZ12 was mapped across the entire nonrepeat portion of the genome in human embryonic stem (ES) cells. It was found SUZ12 is distributed across large portions of over two hundred genes encoding key developmental regulators. These genes are occupied by nucleosomes trimethylated at histone H3K27, are transcriptionally repressed, and contain some of the most highly conserved noncoding elements in the genome. PRC2 target genes are preferentially activated during ES cell differentiation and the ES cell regulators OCT4, SOX2, and NANOG cooccupy a significant subset of these genes. These results indicate that PRC2 occupies a special set of developmental genes in ES cells that must be repressed to maintain pluripotency and that are poised for activation during ES cell differentiation (Lee, 2006).

Polycomb-repressive complex 2 (PRC2)-mediated histone methylation plays an important role in aberrant cancer gene silencing and is a potential target for cancer therapy. S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep) induces efficient apoptotic cell death in cancer cells but not in normal cells. DZNep effectively depletes cellular levels of PRC2 components EZH2, SUZ12, and EED and inhibits associated histone H3 Lys 27 methylation (but not H3 Lys 9 methylation). By integrating RNA interference (RNAi), genome-wide expression analysis, and chromatin immunoprecipitation (ChIP) studies, a prominent set of genes were identifed, selectively repressed by PRC2 in breast cancer, that can be reactivated by DZNep. The preferential reactivation of a set of these genes by DZNep, including a novel apoptosis affector, FBXO32, contributes to DZNep-induced apoptosis in breast cancer cells. These results demonstrate the unique feature of DZNep as a novel chromatin remodeling compound and suggest that pharmacologic reversal of PRC2-mediated gene repression by DZNep may constitute a novel approach for cancer therapy (Tan, 2007).

Genetic studies have demonstrated that Bmi1 promotes cell proliferation and stem cell self-renewal with a correlative decrease of p16INK4a expression. Polycomb genes EZH2 and BMI1 repress p16 expression in human and mouse primary cells, but not in cells deficient for pRB protein function. The p16 locus is H3K27-methylated and bound by BMI1, RING2, and SUZ12. Inactivation of pRB family proteins abolishes H3K27 methylation and disrupts BMI1, RING2, and SUZ12 binding to the p16 locus. These results suggest a model in which pRB proteins recruit PRC2 to trimethylate p16, priming the BMI1-containing PRC1L ubiquitin ligase complex to silence p16 (Kotake, 2007).

The mammalian pRB family proteins, pRB, p107, and p130 (also known as pocket proteins), play a key role in controlling the G1-to-S transition of the cell cycle and maintaining differentiated cells in a reversible quiescent or permanent senescent arrest state. The pocket proteins are hypophosphorylated in cells exiting mitosis as well as in quiescent cells, where they bind to and negatively regulate the function of the E2F family transcription factors. In cells entering the cell cycle, extracellular mitogens first induce the expression of D-type cyclins, which bind to and activate CDK4 and CDK6, leading to the phosphorylation of pRB family proteins, causing functional inactivation by E2F dissociation, thereby promoting a G1-to-S transition. Inhibition of CDK4 and CDK6 by the INK4 family of CDK inhibitors (p16INK4a, p15INK4b, p18INK4c, and p19INK4d) retains pRB family proteins in their hypophosphorylated, growth-suppressive states and prevents G1-to-S progression. Disruption of the INK4-RB pathway, consisting of INK4-cyclinDs-CDK4/6-RB-E2Fs, deregulates G1-to-S control and represents a common event in the development of most, if not all, types of cancer (Kotake, 2007).

Among the major challenges toward a better understanding of G1 control by the INK4-pRB pathway is how different INK4 genes are regulated, thereby linking G1 control to different cellular pathways. INK4 proteins are relatively stable, and the primary regulation of INK4 is through transcriptional control. The expression of each of the INK4 genes is distinctly different during development, in different adult tissues, and in response to different cellular conditions. There have been only a few reports wherein a transcriptional regulator has been demonstrated to bind to an INK4 promoter by either gel shift or chromatin immunoprecipitation (ChIP) assay. Identification of factors that directly bind to INK4 promoters holds the key to linking different cellular pathways to G1 control by the INK4-pRB pathway, but these links remain disproportionately poorly understood in comparison with knowledge of the function of the INK4-pRB pathway (Kotake, 2007).

To elucidate the molecular mechanisms regulating p16 expression, whether p16 gene expression is directly regulated by BMI1, an oncogene that encodes a transcriptional repressor of the Polycomb group (PcG) of proteins, was directly tested. Deletion of Bmi1 retards cell proliferation, causes premature senescence in mouse embryonic fibroblasts (MEFs), and reduces the number of hematopoietic stem cells, with an associated up-regulation of p16 (and to a lesser extent of p19Arf). Codeletion of p16 (or p16-Arf) partially rescues the proliferative defects of Bmi1-null cells, providing genetic evidence supporting a functional interaction between the Bmi1 and p16 genes. However, whether BMI1 directly binds to and regulates the transcription of the p16 gene has not been demonstrated. A notable feature of p16 is its high level of expression in virally transformed cells and its inverse correlation with pRB function, suggesting a negative regulation of p16 gene expression by pRB. Therefore whether pRB and BMI1 collaboratively repress p16 expression was also examined (Kotake, 2007).

These results provide the first biochemical evidence supporting a direct regulation of p16 transcription by the PRC2 histone methyltransferase complex and the BMI1-RING2-containing PRC1 histone ubiquitin ligase complex. Both H3K27 methylation at and BMI1/RING2 binding to the p16 locus require the function of the pRB family proteins, linking for the first time H3K27 methylation and the function of BMI1 with the pRB proteins. The detailed biochemical mechanism by which pRB family proteins collaborate with BMI1 to repress p16 transcription is yet to be determined. In repeated attempts, binding of pRB to the p16 locus could not be detected. The simplest model suggested by these results is that the pRB family proteins are either involved in regulating the enzymatic activity or the recruitment of PRC2 to the p16 locus. H3K27 methylation by PRC2 would then facilitate recruitment of the BMI1-containing PRC1L complex to ubiquitinate H2A, leading to p16 silencing (Kotake, 2007).

The results also suggest a regulatory loop between p16 and the pocket proteins, with p16 acting as an upstream activator of the pocket proteins and the pocket proteins repressing p16 transcription as negative feedback. INK4 proteins are intrinsically stable and, once synthesized, stably bind to and inhibit the activity of CDK4/6 by both interfering with ATP binding and by reducing the cyclin-CDK4/6 surface. Without a mechanism for repressing INK4 expression, mitogen-induced cyclin D synthesis would not be able to compete off INK4 from CDK4/6, and displaced, monomeric cyclin D proteins would be rapidly degraded, leaving a constitutive activation of RB function and locking cells in a permanent G1-arrested state. Repression of p16 expression by pRB family proteins thus also constitutes a feedback loop to set up a balance between INK4-mediated inhibition and cyclin D-mediated activation of G1 progression. This function of p16, however, must be repressed in stem cells, which undergo continuous proliferation and self-renewal in vivo. It is speculated that one mechanism to achieve this is through expression of BMI1 in the stem cell compartment (Kotake, 2007).

Role of the polycomb protein EED in the propagation of repressive histone marks

Organization of chromatin by epigenetic mechanisms is essential for establishing and maintaining cellular identity in developing and adult organisms. A key question that remains unresolved about this process is how epigenetic marks are transmitted to the next cell generation during cell division. This study provides a model to explain how trimethylated Lys 27 of histone 3 (H3K27me3), which is catalysed by the EZH2-containing Polycomb Repressive Complex 2 (PRC2), is maintained in proliferating cells. It was shown that the PRC2 complex binds to the H3K27me3 mark and colocalizes with this mark in G1 phase and with sites of ongoing DNA replication. Efficient binding requires an intact trimeric PRC2 complex containing EZH2, EED and SUZ12, but is independent of the catalytic SET domain of EZH2. Using a heterologous reporter system, it was shown that transient recruitment of the PRC2 complex to chromatin, upstream of the transcriptional start site, is sufficient to maintain repression through endogenous PRC2 during subsequent cell divisions. Thus, it is suggested that once the H3K27me3 is established, it recruits the PRC2 complex to maintain the mark at sites of DNA replication, leading to methylation of H3K27 on the daughter strands during incorporation of newly synthesized histones. This mechanism ensures maintenance of the H3K27me3 epigenetic mark in proliferating cells, not only during DNA replication when histones synthesized de novo are incorporated, but also outside S phase, thereby preserving chromatin structure and transcriptional programs (Hansen, 2008).

Polycomb group proteins have an essential role in the epigenetic maintenance of repressive chromatin states. The gene-silencing activity of the Polycomb repressive complex 2 (PRC2) depends on its ability to trimethylate lysine 27 of histone H3 (H3K27) by the catalytic SET domain of the EZH2 subunit, and at least two other subunits of the complex: SUZ12 and EED. This study shows that the carboxy-terminal domain of EED specifically binds to histone tails carrying trimethyl-lysine residues associated with repressive chromatin marks, and that this leads to the allosteric activation of the methyltransferase activity of PRC2. Mutations in EED that prevent it from recognizing repressive trimethyl-lysine marks abolish the activation of PRC2 in vitro and, in Drosophila, reduce global methylation and disrupt development. These findings suggest a model for the propagation of the H3K27me3 mark that accounts for the maintenance of repressive chromatin domains and for the transmission of a histone modification from mother to daughter cells (Margueron, 2009).

The fate of a cell is specified by its gene expression profile, often set early in development and maintained throughout the lifetime of the cell by epigenetic mechanisms. The polycomb group of proteins functions by silencing inappropriate expression by maintaining a repressive epigenetic state1. It is thought that the PRC2-mediated trimethylation of lysine 27 on histone H3 (H3K27me3) has a crucial role in marking repressive chromatin domains, whereas PRC1 is important for effecting transcriptional repression. Thus, once established, H3K27 trimethylation is the epigenetic mark for maintaining transcriptional repression. Mechanisms are therefore required to maintain this mark in repressed chromatin domains in non-dividing cells and to restore it after the twofold dilution caused by DNA replication in dividing cells. However, it is not yet clear how PRC2 complexes recognize previously marked sites and how they accurately propagate these repressive marks to unmodified nucleosomes deposited during DNA replication (Margueron, 2009).

The histone lysine methyltransferase (HKMT) activity of the PRC2 complex resides in the SET-domain-containing protein EZH2, but activity requires the other subunits of the core complex; the zinc-finger-containing SUZ12 and the WD40 repeat proteins EED and RbAp48 (also known as CAF1). In certain contexts, the PHD-domain-containing protein PHF1 plays an important part in modulating the HKMT activity of PRC2. This work has examined the structure and biochemistry of EED, and determined the role of its homologue ESC in Drosophila development. From this it was established that the EED subunit of PRC2 binds to repressive methyl-lysine marks, ensuring the propagation of H3K27 trimethylation on nucleosomes by allosterically activating the methyltransferase activity of the complex (Margueron, 2009).

A truncated version of EED (residues 77 to 441, hereafter DeltaEED) was crystallized and selenomethionine-substituted DeltaEED was used to solve the structure. The WD40-repeats of DeltaEED fold into a seven-bladed beta-propeller domain with a central pocket on either end. Unaccounted electron density was noticed in one of these pockets; the crystallization mixture included a non-detergent sulphobetaine additive, NDSB-195, which was built into the extra electron density. Because the quarternary amine of the sulphobetaine resembled a trimethylated lysine side chain it was reasoned that EED might bind to trimethylated lysine residues on the N-terminal tails of histones (Margueron, 2009).

Histone lysine residues methylated in vivo include H3K4, H3K9, H3K27, H3K36, H3K79, H4K20 and H1K26. The binding affinity of DeltaEED to trimethylated versions of these lysine residues was measured by fluorescence competition assays using synthetic peptides. DeltaEED bound to H1K26me3, H3K9me3, H3K27me3 and H4K20me3 peptides with dissociation constant (Kd) values ranging from 10 to 45 muM, and the binding became approximately fourfold weaker for each successive loss of a methyl group from the methyl-lysine. Notably, DeltaEED did not bind appreciably to H3K4me3, H3K36me3 or H3K79me3 'marks' associated with active transcription. These results were validated by isothermal titration calorimetry, and there is good agreement between the two independent methods (Margueron, 2009).

Next, the structure was solved of DeltaEED co-crystallized with H1K26me3, H3K9me3, H3K27me3 and H4K20me3 peptides. The peptides in the four co-crystal structures adopt similar, largely extended structures and all exploit the aromatic cage of DeltaEED to recognize the trimethyl-lysine residue. This is the first example of such a binding site on a beta-propeller domain and it consists of three aromatic side-chains, Phe 97, Tyr 148 and Tyr 365. The trimethyl-ammonium group of the lysine is inserted into this cage and is stabilized by van der Waals and cation-pi interactions. A fourth aromatic side-chain (Trp 364) interacts with the aliphatic moiety of the lysine side chain by hydrophobic interactions. Adjacent to the methyl-lysine pocket, DeltaEED makes two hydrogen-bond interactions with carbonyls on the peptides. First, the main-chain carbonyl of the methyl-lysine residue forms hydrogen bonds with the side chain of Arg 414. Second, the main-chain carbonyl of the residue immediately amino-terminal of the methyl-lysine on the peptide makes a hydrogen bond with the main-chain amide of Trp 364. The residues flanking the methyl-lysine residue, at the -1 and +1 positions, are oriented away from the protein, whereas the next residues, at the -2 and +2 positions, make important contacts. Comparison of the four complexes suggests an important role for two distinct hydrophobic interaction sites. H1K26, H3K9 and H3K27 each have an alanine residue two amino acids N-terminal to the lysine (-2), which fits into a small pocket on the surface of EED formed by the hydrophobic moieties of Trp 364, Tyr 308 and Cys 324. The size of this pocket is sufficient to accommodate an alanine residue but not larger hydrophobic residues. In the case of H4K20 peptide (the only one of the four that bound to DeltaEED and lacks an alanine at -2), its binding is facilitated by an alternative hydrophobic interaction between the leucine residue in the +2 position of the peptide with a second hydrophobic pocket formed by residues Ile 363, Ala 412 and the gamma-carbon of Gln 382 of EED. It seems that the ability to exploit one of these two small hydrophobic pockets is an important component of the specificity of EED towards the methyl-lysine marks associated with repressive chromatin. However, the affinity of EED for these modified peptides is relatively modest, and it is likely that this interaction only becomes physiologically relevant in association with the histone-binding activity of other components of the PRC2 complex, as suggested by earlier work on Drosophila PRC2 (Margueron, 2009).

To probe the physiological role of the aromatic cage of EED, site-directed mutants of several of the cage residues were created. Mutations of Phe 97, Trp 364 and Tyr 365 to alanine produced well-behaved protein, and competition experiments showed that the Trp364Ala and Tyr365Ala mutations had no detectable binding to H1K26me3 peptides, whereas DeltaEED Phe97Ala bound about eightfold more weakly than wild-type DeltaEED to histone peptides. As a control for the effect of mutation of an aromatic residue on the EED structure that is not involved in the aromatic cage, the mutation Tyr358Ala was generated; binding by this mutant was reduced by about twofold (Margueron, 2009).

Next, nucleosome arrays were used reconstituted with chemically modified histones that carry a single modification of the four possible methylation states of H3K27, H3K36 or H3K9. The nucleosome arrays were incubated with full-length His-tagged EED protein followed by nickel-nitrilotriacetic acid (Ni-NTA) pull-down assays. Western blotting for H3 and EED demonstrated an interaction between EED and nucleosomes containing H3K27me3. This interaction was specific as EED was not able to interact with chromatin reconstituted with histones containing the different levels of H3K36 methylation, but did bind to chromatin trimethylated on H3K9. Interestingly, the truncated DeltaEED protein tested in the peptide-binding experiments also failed to interact with nucleosomes. Presumably, the diminished binding is due to the absence of a previously characterized H3-binding site within the N terminus of EED13, which may act together with the methyl-lysine-binding site to achieve stable binding (Margueron, 2009).

Given that other subunits of PRC2 contact histones and thus modulate chromatin binding, the nucleosome-binding experiment was repeated using a PRC2 complex purified from insect cells co-infected with baculovirus expressing each of the subunits. Although, as expected, the reconstituted PRC2 complex showed some binding to unmodified chromatin, the complex bound considerably tighter to chromatin carrying the H3K27me3 or the H3K9me3 modification. Interestingly, PRC2 reconstituted with EED carrying the Phe97Ala or Tyr365Ala substitution does not show binding to chromatin under these conditions, with either methylated or unmodified nucleosomes. Together, these results demonstrate that the aromatic cage in EED is critical for the PRC2 complex to bind to repressive marks, through its specific recognition of defined (repressive) trimethylated-lysine residues (Margueron, 2009).

Because a probable function for the binding of PRC2 to trimethylated lysine would be to contribute to the propagation of the H3K27me3 mark, HKMT assays were performed using recombinant oligonucleosomes in the presence of methylated peptides. The addition of unmodified or monomethylated H3K27 peptides did not significantly affect the enzymatic activity of PRC2, but trimethylated peptides activated it by about sevenfold. Stimulation of enzymatic activity by the H3K27me3 peptide reached a plateau around 100 muM, and half-maximum stimulation is achieved at 30-40 muM, which is in good agreement with the dissociation constant determined for DeltaEED and the H3K27me3 peptide and gives a strong indication that the binding event observed with purified, truncated EED is closely correlated with the allosteric activation mechanism. The Michaelis parameters were determined for PRC2 in the presence of variously methylated H3K27 peptides. During titrations of S-adenosyl-methionine (SAM) a marked increase was observed in the maximum reaction rate (Vm) in the presence of the H3K27me3 peptide. A similar result was observed with titration of nucleosomes. Notably, in both cases the substrate concentration required to achieve the half-maximal reaction rate (Km) is not significantly affected by the incubation with peptides (Margueron, 2009).

To ascertain whether the observed stimulation was EED-mediated, mutant PRC2 complexes containing EED(Phe97Ala) or EED(Tyr365Ala) were reconstituted. These mutant recombinant PRC2 complexes retain a similar basal activity to wild-type, but neither mutant recombinant PRC2 was stimulated by the addition of H3K27me3 peptides. The data also show that the H3K9me3, H4K20me3 and H1K26me3 peptides were all able to stimulate PRC2 activity to some extent, whereas the H3K4me3 and H3K36me3 peptides were ineffectual. However, it was noticed that the binding affinity to EED and stimulation of PRC2 activity do not strictly correlate (that is, H3K9me3 has a good binding affinity for EED but stimulates PRC2 activity relatively poorly). To investigate the role of histone sequence in binding/activation the arginine residue at the -1 position (present in all four histone peptides that activate the methyltransferase activity of PRC2) was first mutated to alanine. Remarkably, although the binding of this mutant peptide to DeltaEED is only reduced by about 1.5-fold, it is no longer able to activate PRC2 HKMT activity, demonstrating that repressive-histone-peptide binding to the aromatic cage of EED is necessary, but not sufficient for PRC2 activation. To further test this model a series of chimaeric and mutant peptides were made that show that the lysine at -4, the alanine at -3 and the arginine at -1 are not important for binding to EED but are key to the activation of PRC2. It is proposed that these are the residues that mediate an interaction with another part of the PRC2 complex that leads to its activation (Margueron, 2009).

To evaluate the importance of EED binding to trimethylated marks in vivo, ESC, the EED homologue in Drosophila, was examined and the effect of mutating its aromatic cage was tested. The Drosophila PRC2 complex was reconstituted and it was shown that addition of H3K27me2/3 peptides to the HKMT assay resulted in a robust stimulation of PRC2 enzymatic activity. ESC is required throughout development, but in the early embryo the maternal stock of esc product is critical, as evidenced by the resultant derepression of homeotic genes in embryos produced by esc- mothers. At later stages of development, PRC2 activity is sustained through the overlapping participation of ESC and its close homologue ESCL. Overexpression of ESC in the ovaries (for example, in a female with one extra esc copy) can supply enough function to allow the development of esc embryos, producing flies that are virtually normal except for the eponymous extra sex combs in males. Mutations affecting the aromatic cage were constructed: Phe77Ala (equivalent to Phe 97 in EED) and Phe345Ala (equivalent to Tyr 365 in EED), as well as Tyr338Ala (equivalent to Tyr358Ala in EED) just preceding the aromatic cage, and Myc-tagged wild-type or mutant esc transgenes were expressed under the control of the esc promoter. Although the wild-type transgene rescued the extra sex comb phenotype almost completely, the aromatic cage mutant transgenes were ineffectual. Flies lacking both zygotic ESC and ESCL in these crosses produce larvae with poorly developed brain and imaginal discs, which die when they pupate. This lethality is completely rescued by one copy of the wild-type esc>Myc-ESC transgene. In contrast, none of the aromatic cage mutant transgenes were able to rescue the lethality even when present in two copies, although the esc>Myc-ESC(Phe77Ala) transgene alleviated the brain and imaginal disc phenotypes. Of note, zygotic expression of the Phe345Ala transgene impaired the contribution of wild-type esc indicating that this mutant acts as a dominant negative. The failure of the mutant Myc-ESC to rescue is not due to instability or the inability to be incorporated into a PRC2 complex: the ESC mutants were expressed at levels comparable to that of the wild type. Furthermore, immunoprecipitation experiments showed that the mutant ESCs co-immunoprecipitated with endogenous E(Z) as efficiently as the wild-type protein. To determine whether the mutant ESCs affected PRC2 function with respect to its gene targeting or activity, chromatin immunoprecipitation (ChIP) was performed followed by quantitative PCR. Immunoprecipitation using anti-E(Z) shows that wild-type Myc-ESC is nearly as effective as endogenous ESC (compare with the esc+ escl- chromatin), whereas PRC2 complex with Myc-ESC-bearing mutations in the aromatic pocket is recruited less efficiently to the Ubx polycomb response element (PRE). Chromatin immunoprecipitation with anti-H3K27me3 antibodies also shows that wild-type Myc-ESC is nearly as effective as endogenous ESC (yw) in trimethylating H3K27 in the Ubx upstream enhancer region (PBX, -30 kilobases (kb)), in the vicinity of the PRE (FM1, FM6, -23 kb) or at the Ubx promoter. Notably, the mutant ESCs are deficient in the extent of H3K27me3, and this decrease correlates with the phenotypes observed. Importantly, the observed effects are due to the aromatic cage, as a mutation of Tyr338Ala, which is not important for cage formation, had no effect. Finally, the global levels of H3K27 methylation were analyzed by western blot. An almost complete loss of H3K27me3 was observed in extracts from esc- escl- larvae expressing the mutant ESCs. Perhaps surprisingly, the H3K27me2 levels were equally strongly affected (Margueron, 2009).

In conclusion, chromatin domains are distinguished by the presence of a characteristic set of marks. When these marks are used to sustain an epigenetic state, eukaryotic cells must have the means of propagating these marks through cellular division and of ensuring that they obey appropriate boundaries during development. That PRC2 might recognize the chromatin mark it sets was anticipated by observations that PRC2 binds to H3K27me3, although that observation did not address the mechanism for the propagation of H3K27me3. This work shows the structural and functional basis for epigenetic self-renewal, and leads to the conclusion that PRC2 readout of H3K27me3 (and to a lesser extent other 'repressive' marks) is key to the propagation of this repressive mark (Margueron, 2009).

A combination of aromatic and hydrophobic residues is commonly used by proteins that recognize methylated lysine residues and has been found in chromo-, tudor- and plant homeo-domains (PHDs), but no such arrangement has previously been described for any WD40-repeat-containing protein. Sequence analysis across the family of beta-propeller domains leads to the conclusion that the ability of EED to specifically recognize repressive methyl-lysine marks is a feature, limited among WD40 proteins, to EED-related molecules (Margueron, 2009).

This methyl-lysine interaction provides an extra contribution to nucleosome binding that is mainly driven by a combination of contacts from other subunits of PRC2; RbAp48 binds to histone H4 and the N-terminal domain of EED binds to H3, and it may well be that these different interactions act cooperatively. In Drosophila, recruitment of PRC2 may also be facilitated by certain DNA-binding factors. The Drosophila experiments show that when the Drosophila EED orthologue ESC bears mutations in the aromatic cage, the recruitment of PRC2 to the PRE is less effective, as shown by the drop in E(Z) binding to the bxd PRE, the massive reduction in the global level of H3K27me2/3 and by the phenotype of the Phe77Ala and Phe345Ala mutants. The chromatin modification assays suggest that a major effect of EED binding to repressive methyl-lysine marks is the stimulation of PRC2 methyltransferase activity, thus providing a mechanism for the propagation of this mark. Thus, when PRC2 is recruited to appropriate chromatin domains, the presence of pre-existing H3K27me3 marks on neighbouring nucleosomes activates the complex to carry out further methylation of unmodified H3K27. Accordingly, a polycomb group target gene that had been repressed in one cell cycle will tend to be repressed again in the next cell cycle, and previously active genes will be left unmodified at H3K27. It is proposed that the ability to recognize a previously established mark that triggers its renewal is a feature that will be found in other epigenetic mechanisms mediated by histone modifications (Margueron, 2009).

EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair

Polycomb protein group (PcG)-dependent trimethylation on H3K27 (H3K27me3) regulates identity of embryonic stem cells (ESCs). How H3K27me3 governs adult SCs and tissue development is unclear. This study conditionally target H3K27 methyltransferases Ezh2 and Ezh1 to address their roles in mouse skin homeostasis. Postnatal phenotypes appear only in doubly targeted skin, where H3K27me3 is abolished, revealing functional redundancy in EZH1/2 proteins. Surprisingly, while Ezh1/2-null hair follicles (HFs) arrest morphogenesis and degenerate due to defective proliferation and increased apoptosis, epidermis hyperproliferates and survives engraftment. mRNA microarray studies reveal that, despite these striking phenotypic differences, similar genes are up-regulated in HF and epidermal Ezh1/2-null progenitors. Featured prominently are (1) PcG-controlled nonskin lineage genes, whose expression is still significantly lower than in native tissues, and (2) the PcG-regulated Ink4a/Inkb/Arf locus. Interestingly, when EZH1/2 are absent, even though Ink4a/Arf/Ink4b genes are fully activated in HF cells, they are only partially so in epidermal progenitors. Importantly, transduction of Ink4b/Ink4a/Arf shRNAs restores proliferation/survival of Ezh1/2-null HF progenitors in vitro, pointing toward the relevance of this locus to the observed HF phenotypes. These findings reveal new insights into Polycomb-dependent tissue control, and provide a new twist to how different progenitors within one tissue respond to loss of H3K27me3 (Ezhkova, 2011).

Histone H3 K36 methylation is mediated by a trans-histone methylation pathway involving an interaction between Set2 and histone H4

Set2-mediated H3 K36 methylation is an important histone modification on chromatin during transcription elongation. Although Set2 associates with the phosphorylated C-terminal domain (CTD) of RNA polymerase II (RNAPII), the mechanism of Set2 binding to chromatin and subsequent exertion of its methyltransferase activity is relatively uncharacterized. This study identified a critical lysine residue in histone H4 that is needed for interaction with Set2 and proper H3 K36 di- and trimethylation. It was also determined that the N terminus of Set2 contains a histone H4 interaction motif that allows Set2 to bind histone H4 and nucleosomes. A Set2 mutant lacking the histone H4 interaction motif is able to bind to the phosphorylated CTD of RNAPII and associate with gene-specific loci but is defective for H3 K36 di- and trimethylation. In addition, this Set2 mutant shows increased H4 acetylation and resistance to 6-Azauracil. Overall, this study defines a new interaction between Set2 and histone H4 that mediates trans-histone regulation of H3 K36 methylation, which is needed for the preventative maintenance and integrity of the genome (Du, 2008).

Trimethylation of Lys36 on H3 restricts gene expression change during aging and impacts life span

Functional data indicate that specific histone modification enzymes can be key to longevity in Caenorhabditis elegans, but the molecular basis of how chromatin structure modulates longevity is not well understood. In this study, the genome-wide pattern of trimethylation of Lys36 on histone 3 (H3K36me3) was profiled in the somatic cells of young and old Caenorhabditis elegans. A new role of H3K36me3 was revealed in maintaining gene expression stability through aging with important consequences on longevity. Genes with dramatic expression change during aging are marked with low or even undetectable levels of H3K36me3 in their gene bodies irrespective of their corresponding mRNA abundance. Interestingly, 3' untranslated region (UTR) length strongly correlates with H3K36me3 levels and age-dependent mRNA expression stability. A similar negative correlation between H3K36me3 marking and mRNA expression change during aging was also observed in Drosophila melanogaster, suggesting a conserved mechanism for H3K36me3 in suppressing age-dependent mRNA expression change. Importantly, inactivation of the methyltransferase met-1 resulted in a decrease in global H3K36me3 marks, an increase in mRNA expression change with age, and a shortened life span, suggesting a causative role of the H3K36me3 marking in modulating age-dependent gene expression stability and longevity (Pu, 2014).

Mouse polycomb proteins bind differentially to methylated histone H3 and RNA and are enriched in facultative heterochromatin

The chromodomain (CD) of the Drosophila Polycomb protein exhibits preferential binding affinity for histone H3 when trimethylated at lysine 27. The five mouse Polycomb homologs known as Cbx2, Cbx4, Cbx6, Cbx7, and Cbx8 have been investigated. Despite a high degree of conservation, the Cbx chromodomains display significant differences in binding preferences. Not all CDs bind preferentially to K27me3; rather, some display affinity towards both histone H3 trimethylated at K9 and H3K27me3, and one CD prefers K9me3. Cbx7, in particular, displays strong affinity for both H3K9me3 and H3K27me3 and is developmentally regulated in its association with chromatin. Cbx7 associates with facultative heterochromatin and, more specifically, is enriched on the inactive X chromosome. Finally, it was found that, in vitro, the chromodomain of Cbx7 can bind RNA and that, in vivo, the interaction of Cbx7 with chromatin, and the inactive X chromosome in particular, depends partly on its association with RNA. It is proposed that the capacity of this mouse Polycomb homolog to associate with the inactive X chromosome, or any other region of chromatin, depends not only on its chromodomain but also on the combination of histone modifications and RNA molecules present at its target sites (Bernstein, 2005).

The founding member of the PcG genes is Drosophila melanogaster Polycomb (Pc) -- mutations in Pc result in body segment transformations. Pc is encoded by a single gene in Drosophila, while the mouse homologs have expanded into five family members known as Chromobox 2 (Cbx2) (mPc1 or M33), Cbx4 (mPc2), Cbx6, Cbx7, and Cbx8 (mPc3) (34). Importantly, these proteins contain a highly conserved N-terminal chromodomain (CD), a module first identified in the Drosophila proteins heterochromatin protein 1 (HP1) and Pc. The CD is found in a wide range of chromatin-associated proteins, most with transcriptionally repressive functions. The CD binds to methylated histones: the CD of Drosophila HP1 binds histone H3K9me2 and me3, while that of Pc specifically binds K27me3 on H3. Besides methyl-lysine binding, several reports have also suggested that certain CDs bind nucleic acids (Bernstein, 2005).

Based on findings with Cbx7, it is concluded that although the H3K27me3 mark seems to be important for the recruitment of Cbx7 via its CD, other components, including RNAs, must also be required for its recruitment and maintenance on the Xi. Indeed, recent reports have described the recruitment of several PRC1 proteins to the Xi, which may be part of one or multiple complexes. It will be interesting to identify Cbx7-interacting proteins at different stages of development in order to understand the mechanism(s) by which Cbx7 associates with the inactive X (Xi) chromosome, and with chromatin in general, during ES cell differentiation. For example, in addition to representing a potential mechanism by which Ring1 is able to ubiquitylate H2A on the Xi, the association of Cbx7 with chromatin during ES cell differentiation may be linked to the rather sudden appearance of macroH2A on the Xi. Finally, the role of RNA in Cbx7 chromatin association is of particular interest in light of recent evidence in fission yeast, flies, and mammals suggesting that noncoding RNAs can impact the chromatin template. Moreover, TAP-tag purification of Cbx7 from human cells has demonstrated that this Pc protein interacts with an RNA-helicase, suggesting the involvement of RNA in Cbx7-mediated repression. The ability of particular CDs to potentially bind both methylated histone tails and RNA suggests that a cooperative binding mechanism may mediate the enrichment of particular CD-containing proteins in chromatin. Future functional and structural studies will be required to determine the nature of this potential synergy, particularly in the case of Cbx7 and Xist RNA in the context of the inactive X chromosome (Bernstein, 2005).

HOTAIR interacts with Polycomb Repressive Complex 2 (PRC2) and is required for PRC2 occupancy and histone H3 lysine-27 trimethylation of HOXD locus

Noncoding RNAs (ncRNA) participate in epigenetic regulation but are poorly understood. This study characterized the transcriptional landscape of the four human HOX loci at five base pair resolution in eleven anatomic sites, and identified 231 HOX ncRNAs that extend known transcribed regions by more than 30 kilobases. HOX ncRNAs are spatially expressed along developmental axes, possess unique sequence motifs, and their expression demarcate broad chromosomal domains of differential histone methylation and RNA polymerase accessibility. A 2.2 kilobase ncRNA was identified residing in the HOXC locus, termed HOTAIR, which represses transcription in trans across 40 kilobases of the HOXD locus. HOTAIR interacts with Polycomb Repressive Complex 2 (PRC2) and is required for PRC2 occupancy and histone H3 lysine-27 trimethylation of HOXD locus. Thus, transcription of ncRNA may demarcate chromosomal domains of gene silencing at a distance; these results have broad implications for gene regulation in development and disease states (Rinn, 2007).

By analyzing the transcriptional and epigenetic landscape of the HOX loci at high resolution in cells with many distinct positional identities, a panoramic view was obtained of multiple layers of regulation involved in maintenance of site-specific gene expression. The HOX loci are demarcated by broad chromosomal domains of transcriptional accessibility, marked by extensive occupancy of RNA polymerase II and H3K4 dimethylation and, in a mutually exclusive fashion, by occupancy of PRC2 and H3K27me3. The active, PolII-occupied chromosomal domains are further punctuated by discrete regions of transcription of protein-coding HOX genes and a large number of long ncRNAs. These results confirm the existence of broad chromosomal domains of histone modifications and occupancy of HMTases over the Hox loci, and extend on those observation in several important ways (Rinn, 2007).

First, by comparing the epigenetic landscape of cells with distinct positional identities, it was showm that the broad chromatin domains can be programmed with precisely the same boundary but with diametrically opposite histone modifications and consequences on gene expression. The data thus functionally pinpoint the locations of chromatin boundary elements in the HOX loci, the existence of some of which have been predicted by genetic experiments. One such boundary element appears to reside between HOXA7 and HOXA9. This genomic location is also the switching point in the expression of HOXA genes between anatomically proximal versus distal patterns and is the boundary of different ancestral origins of HOX genes, raising the possibility that boundary elements are features demarcating the ends of ancient transcribed regions. Second, the ability to monitor 11 different HOX transcriptomes in the context of the same cell type conferred the unique ability to characterize changes in ncRNA regulation that reflect their position in the human body. This unbiased analysis identified more than 30 kb of new transcriptional activity, revealed ncRNAs conserved in evolution, mapped their anatomic patterns of expression, and uncovered enriched ncRNA sequence motifs correlated with their expression pattern -- insights which could not be gleamed from examination of EST sequences alone. The finding of a long ncRNA that acts in trans to repress HOX genes in a distant locus is mainly due to the ability afforded by the tiling array to comprehensively examine the consequence of any perturbation over all HOX loci. The expansion of a handful of Hox-encoded ncRNAs in Drosophila to hundreds of ncRNAs in human HOX loci suggests increasingly important and diverse roles for these regulatory RNAs (Rinn, 2007).

An important limitation of the tiling array approach is that while improved identification of transcribed regions is obtained, the data does not address the connectivity of these regions. The precise start, end, patterns of splicing, and regions of double-stranded overlap between ncRNAs will need to be addressed by detailed molecular studies in the future (Rinn, 2007).

The results uncovered a new mechanism whereby transcription of ncRNA dictates transcriptional silencing of a distant chromosomal domain. The four HOX loci demonstrate complex cross regulation and compensation during development. For instance, deletion of the entire HOXC locus exhibits a milder phenotype than deletion of individual HOXC genes, suggesting that there is negative feedback within the locus. Multiple 5' HOX genes, including HOXC genes, are expressed in developing limbs, and deletion of multiple HOXA and HOXD genes are required to unveil limb patterning defects. The results suggest that deletion of the 5' HOXC locus, which encompass HOTAIR, may lead to transcriptional induction of the homologous 5' HOXD genes, thereby restoring the total dosage of HOX transcription factors. How HOX ncRNAs may contribute to cross-regulation among HOX genes should be addressed in future studies (Rinn, 2007).

HOTAIR ncRNA is involved in Polycomb Repressive Complex 2-mediated silencing of chromatin. Because many HMTase complexes lack DNA binding domains but possess RNA binding motifs, it has been postulated that ncRNAs may guide specific histone modification activities to discrete chromatin loci. This study has shown that HOTAIR ncRNA binds PRC2 and is required for robust H3K27 trimethylation and transcriptional silencing of the HOXD locus. HOTAIR may therefore be one of the long sought after RNAs that interface the Polycomb complex with target chromatin. A potentially attractive model of epigenetic control is the programming of active or silencing histone modifications by specific noncoding RNAs. Just as transcription of certain ncRNA can facilitate H3K4 methylation and activate transcription of the downstream Hox genes (Sanchez-Elsner, 2006; Schmitt, 2005), distant transcription of other ncRNAs may target the H3K27 HMTase PRC2 to specific genomic sites, leading to silencing of transcription and establishment of facultative heterochromatin. In this view, extensive transcription of ncRNAs is both functionally involved in the demarcation of active and silent domains of chromatin as well as being a consequence of such chromatin domains (Rinn, 2007).

Several lines of evidence suggest that HOTAIR functions as a bona fide long ncRNA to mediate transcriptional silencing. First, full length HOTAIR is detected in vivo and in primary cells, but not small RNAs derived from HOTAIR indicative of miRNA or siRNA production. Second, depletion of full length HOTAIR led to loss of HOXD silencing and H3K27 trimethyation by PRC2, and third, endogenous or in vitro transcribed full length HOTAIR ncRNA physically associated with PRC2. While these results do not rule out the possibility that RNA interference pathways may be subsequently involved in PcG function, they support the notion that the long ncRNA form of HOTAIR is functional. The role of HOTAIR is reminiscent of XIST, another long ncRNA shown to be involved in transcriptional silencing of the inactive X chromosome. An important difference between HOTAIR and XIST is the strictly cis-acting nature of XIST. HOTAIR is the first example of a long ncRNA that can act in trans to regulate a chromatin domain. While a trans repressive role for HOTAIR was observed, the data do not permit ruling out a cis-repressive role in the HOXC locus. siRNA-mediated depletion of HOTAIR was substantial but incomplete; further, the proximity between the site of HOTAIR transcription and the neighboring HOXC locus may ensure significant exposure to HOTAIR even if the total pool of HOTAIR in the cell were depleted. The precise location of HOTAIR at the boundary of a silent chromatin domain in the HOXC locus makes a cis-repressive role a tantalizing possibility. Judicious gene targeting of HOTAIR may be required to address its role in cis-regulation of chromatin (Rinn, 2007).

The discovery of a long ncRNA that can mediate epigenetic silencing of a chromosomal domain in trans has several important implications. First, ncRNA guidance of PRC2-mediated epigenetic silencing may operate more globally than just in the HOX loci, and it is possible that other ncRNAs may interact with chromatin modification enzymes to regulate gene expression in trans. Second, PcG proteins are important for stem cell pluripotency and cancer development; these PcG activities may also be guided by stem cell or cancer-specific ncRNAs. Third, Suz12 contains a zinc finger domain, a structural motif that can bind RNA, and EZH2 and EED both have in vitro RNA binding activity. The interaction between HOTAIR and PRC2 may also be indirect and mediated by additional factors. Detailed studies of HOTAIR and PRC2 subunits are required to elucidate the structural features that establish the PRC2 interaction with HOTAIR. As is illustrated in this study, high throughput approaches for the discovery and characterization of ncRNAs may aid in dissecting the functional roles of ncRNAs in these diverse and important biological processes (Rinn, 2007).

H3K27me3 regulates BMP activity in developing spinal cord

During spinal cord development, the combination of secreted signaling proteins and transcription factors provides information for each neural type differentiation. Studies using embryonic stem cells show that trimethylation of lysine 27 of histone H3 (H3K27me3) contributes to repression of many genes key for neural development. However, it remains unclear how H3K27me3-mediated mechanisms control neurogenesis in developing spinal cord. This study demonstrates that H3K27me3 controls dorsal interneuron generation by regulation of BMP activity. Expression of Noggin, a BMP extracellular inhibitor, is repressed by H3K27me3. Moreover, Noggin expression is induced by BMP pathway signaling, generating a negative-feedback regulatory loop. In response to BMP pathway activation, JMJD3 histone demethylase interacts with the Smad1/Smad4 complex to demethylate and activate the Noggin promoter. Together, these data reveal how the BMP signaling pathway restricts its own activity in developing spinal cord by modulating H3K27me3 levels at the Noggin promoter (Alizu, 2010).

Polycomb eviction as a new distant enhancer function

Remote distal enhancers may be located tens or thousands of kilobases away from their promoters. How they control gene expression is still poorly understood. This study analyzed the influence of a remote enhancer on the balance between repression (Polycomb-PcG) and activation (Trithorax-TrxG) of a developmentally regulated gene associated with a CpG island. Its essential, nonredundant role in clearing the PcG complex and H3K27me3 from the CpG island is revealed. In the absence of the enhancer, the H3K27me3 demethylase (JMJD3) is not recruited to the CpG island. A new role is proposed for long-range regulatory elements in removing repressive PcG complexes (Vernimmen, 2011).

There is increasing evidence that CpG islands, such as those associated with the α-globin promoter, constitute at least one element that can mediate recruitment of PcG and TrxG complexes to mammalian promoters (Mendenhall et al. 2010). As previously noted, PcG-binding sites are dynamic, are nucleosome-depleted, and have a rapid histone turnover (the residency time of PcG is in the order of a few minutes). PcG binding is therefore thought to be dynamic and sensitive to the antagonistic action of TrxG proteins together with positive and negative input from other TFs and cofactors. However, it is not known whether the eviction of PcG silencing complex from its targets, seen during development and differentiation, depends on the presence of distal regulatory elements or only on (co)factors acting at proximal cis elements. This study used a mouse experimental model to analyze the CpG island associated with the human α-globin promoter in two states: without and with its interacting distant enhancer, both in terminally differentiated erythroid cells. The recruitment was compared of CGBP in nonexpressing versus expressing human cells. In nonerythroid cells, the unmethylated, nuclease-insensitive CpG island associated with the α-globin gene is bound by PcG and is transcriptionally silenced (referred to as the 'silent state'). In erythroid cells without MCS-R2 (referred to as 'basal state'), and in contrast to nonerythroid cells, the promoter becomes accessible to some TFs and is associated with some active chromatin modifications (e.g., H3K4me3) with relatively low levels of transcription (~2% of normal). Nevertheless, the PcG complex with its associated modification (H3K27me3) is still prominent at the α-globin CpG island. It was also demonstrates that PcG and CGBP binding are mutually exclusive. In erythroid cells with MCS-R2 (referred to as 'active state'), PcG complexes are completely removed from the CpG island. Furthermore, the histone H3K27 demethylase JMJD3, which may remove H3K27me3 and thereby facilitate transcription, is also recruited at high levels. Recruitment of the SAGA complex (e.g., PCAF and GCN5) becomes prominent and the downstream effects (e.g., deposition of H2Bub and H3K79 methylation) are established. At this stage, high levels of transcription are associated with binding of CGBP. This study thus shows that the recruitment of the demethylase JMJD3 and full clearance of the PcG-repressive complex (including PRC2 and HDAC1) at the α-globin CpG island depend on one or more activities mediated by the remote regulatory element and are associated with the transition between basal and fully activated transcription (Vernimmen, 2011).

A model is presented for long-range control of epigenetic regulation. In nonerythroid cells, the CpG island is entirely silenced by PcG and HDAC1, associated with the repressive histone mark H3K27me3. The promoter 'P' is not sensitive to DNaseI, and transcription does not occur. In erythroid cells lacking the enhancer, the gene remains repressed by PcG and marked by H3K27me3. At this basal level of expression, the promoter becomes accessible to some TFs and chromatin-modifying enzymes and is marked by moderate levels of H3K4me3, which reflect very low levels of transcription. In the presence of the enhancer, PcG is evicted and the H3K27me3 histone mark is erased by recruitment of demethylase JMJD3. Acetylation (H3ac and H4ac), H3K79me3, and H2Bub increases with spreading of HAT and Bre (SAGA) along the coding sequence. At this activated stage, the remaining TFs, including Pol II, are now fully recruited, and a high rate of transcription occurs. The CpG island at this stage is also bound by CGBP (Vernimmen, 2011).

These findings demonstrate for the first time that the pattern of PcG binding at a CpG island may be affected by cis-acting elements located far away from the associated promoter. In contrast, the chromatin modification associated with TrxG activity (H3K4me3) appears to be more dependent on local changes at the CpG island that occur in the context of basal transcription. Future studies will address how long-range enhancers exert these effects. It is possible that transcriptional activation per se competes with the competitive binding of PcG complexes and is responsible for the clearance of these complexes. The second is that upstream elements also deliver new proteins (e.g., JMJD3) or modify proteins (e.g., histones) that facilitate the removal of PcG. In the past, detailed analysis of the globin genes has established many of the general principles underlying mammalian gene regulation, and it therefore seems probable that this new role of distal regulatory elements in removing PcG from their target promoters will be of considerable general importance (Vernimmen, 2011).

Histone methylation by PRC2 is inhibited by active chromatin marks

The Polycomb repressive complex 2 (PRC2) confers transcriptional repression through histone H3 lysine 27 trimethylation (H3K27me3). This study examined how PRC2 is modulated by histone modifications associated with transcriptionally active chromatin by providing the molecular basis of histone H3 N terminus recognition by the PRC2 Nurf55-Su(z)12 submodule. Binding of H3 is lost if lysine 4 in H3 is trimethylated. It was found that H3K4me3 inhibits PRC2 activity in an allosteric fashion assisted by the Su(z)12 C terminus. In addition to H3K4me3, PRC2 is inhibited by H3K36me2/3 (i.e., both H3K36me2 and H3K36me3). Direct PRC2 inhibition by H3K4me3 and H3K36me2/3 active marks is conserved in humans, mouse, and fly, rendering transcriptionally active chromatin refractory to PRC2 H3K27 trimethylation. While inhibition is present in plant PRC2, it can be modulated through exchange of the Su(z)12 subunit. Inhibition by active chromatin marks, coupled to stimulation by transcriptionally repressive H3K27me3, enables PRC2 to autonomously template repressive H3K27me3 without overwriting active chromatin domains (Schmitges, 2011).

Understanding how histone modification patterns are propagated during cell division is essential for understanding the molecular basis of epigenetic inheritance. Trimethylation of H3K27 by PRC2 has emerged as a key step in generating transcriptionally repressed chromatin in animals and plants. This study investigates how PRC2 recognizes the H3 tail and responds to H3-associated marks of active chromatin. Crystallographic analyses reveal the molecular basis for H31-14 recognition by the Nurf55-Su(z)12 module of PRC2 and demonstrate that H3 tails carrying K4me3 are no longer recognized by Nurf55-Su(z)12. In the context of the whole PRC2 complex, H3K4me3 triggers allosteric inhibition of PRC2, a process that requires H3K4me3 to be present on the same histone molecule containing the substrate Lys27. PRC2 inhibition by H3K36me2/3 was also observed. PRC2 inhibition by active chromatin marks (H3K4me3 and H3K36me2/3) is conserved in PRC2 complexes reconstituted from humans, mouse, flies, and plants (Schmitges, 2011).

Minimal PRC2 complexes lacking Nurf55 retain partial catalytic activity and are inhibited by H3K4me3. H3K4me3, once free of Nurf55, is thereby able to trigger PRC2 inhibition. A model is favored where Nurf55-Su(z)12 serves in sequestration and release of histone H3. It is proposed that the release of the H3 tail from Nurf55-Su(z)12 is required, but not sufficient, to induce H3K4me3 inhibition as it needs to trigger allosteric inhibition in conjunction with Su(z)12 and the E(z) SET domain. Unmodified H3, H3K9me3, or H3R2me-modified tails, on the other hand, remain sequestered and are shielded from other chromatin factors. These sequestered marks are also not expected to interfere with PRC2 regulation. In line with this prediction, it is observed that H3K9me3, which remained bound to Nurf55-Su(z)12, also did not interfere with PRC2 activity in vitro. In vitro, binding of Nurf55 to the N terminus of H3 was not critical for the overall nucleosome binding affinity of PRC2 under the assay conditions. However, small differences in PRC2 affinity amplified by large chromatin arrays could skew PRC2 recruitment toward sites of unmodified H3K4. Additionally, the Nurf55 interaction might play a more subtle role in positioning the complex correctly on nucleosomes (Schmitges, 2011).

The in vitro findings suggest that active chromatin mark inhibition by PRC2 is largely governed through allosteric inhibition of the PRC2 HMTase activity thereby limiting processivity of the enzyme. A minimal trimeric PRC2 subcomplex that retains both activity and H3K4me3/H3K36me2/3 inhibition was defined. This minimal complex consists of ESC, an E(z) fragment that comprises the ESC binding region at the N terminus, the Su(z)12 binding domain in the middle, and the C-terminal catalytic domain, and the Su(z)12 C terminus harboring the VEFS domain. The importance of Su(z)12 is underlined by findings on the Arabidopsis PRC2 complexes that revealed that active mark inhibition is determined by the choice of Su(z)12 subunit (i.e., inhibition with EMF2, but not with VRN2). As the extent of methylation inhibition and the domains required for inhibition were similar for H3K4me3 and H3K36me2/3, it is hypothesized that both peptides function through a related mechanism allosterically affecting E(z) SET domain processivity with the help of Su(z)12. Further structural studies are required to reveal how these active marks are recognized and how this recognition is linked to inhibition of the E(z) SET domain (Schmitges, 2011).

H3K27me3 recognition by PRC2 has been reported to recruit and stimulate PRC2, a mechanism implicated in creating and maintaining the extended H3K27me3 domains at target genes in vivo. Such positive feedback, however, necessitates a boundary element curtailing the expansion of H3K27me3. The results suggest that actively transcribed genes (i.e., marked with H3K4me3 and H3K36me2/3) that flank domains of H3K27me3 chromatin may represent such boundary elements. In conjunction with H3K27me3-mediated stimulation, this provides a model how PRC2 could template domains of H3K27me3 chromatin during replication without expanding H3K27me3 domains into the chromatin of active genes. The inhibitory circuitry present in PRC2, however, does not function as a binary ON/OFF switch. PRC2 is able to integrate opposing H3K4me3 and H3K27me3 modifications into an intermediary H3K27 methylation activity (Schmitges, 2011).

The crosstalk between H3K4me3 and H3K36me2/3 versus H3K27me3 has been extensively studied in vivo. Specifically, HOX genes in developing Drosophila larvae, or in mouse embryos, show mutually exclusive H3K27me3 and H3K4me3 domains that correlate with transcriptional OFF and ON states, respectively. In Drosophila, maintenance of HOX genes in the ON state critically depends on the trxG regulators Trx and Ash1, which methylate H3K4 and H3K36, respectively. At the Ultrabithorax (Ubx) gene, lack of Ash1 results in PRC2-dependent H3K27me3 deposition in the coding region of the normally active gene and the concomitant loss of Ubx transcription. Similarly, in the Arabidopsis Flowering Locus C (FLC), CLF-dependent deposition of H3K27me3 reduces H3K4me3 levels, while deletion of the H3K4me3 demethylase FLD increases H3K4me3 levels and concomitantly diminishes H3K27me3 levels. The results provide a simple mechanistic explanation for these observations in plants and flies. It is proposed that H3K4 and H3K36 modifications in the coding region of active PcG target genes function as barriers that limit H3K27me3 deposition by PRC2 (Schmitges, 2011).

It is noted that a number of HMTase complexes contain histone mark recognition domains that bind the very same mark that is deposited by their catalytic domain. While this positive feedback loop guarantees the processivity of histone mark deposition, it also requires a control mechanism that avoids excessive spreading of marks. The direct inhibition of HMTases by histone marks, as seen for PRC2, may offer a paradigm of how excessive processivity can be counteracted in other HMTases (Schmitges, 2011).

Arabidopsis VRN2 is implicated in the control of the FLC locus after cold shock. FLC is a bivalent locus containing both repressive H3K27me3 and active H3K4me3 marks. In a VRN2-dependent fashion, H3K27me3 levels increase at FLC during vernalization. This study found that while EMF2-containing PRC2 complexes are sensitive to H3K4me3 and H3K36me3, their VRN2-containing counterparts are not. In response to environmental stimuli plant PRC2 H3K4me3/H3K36me3 inhibition can thus be switched OFF (or ON). This offers the possibility that inhibition in animal PRC2 could also be modulated either by posttranslational modification of SUZ12 or by association with accessory factors (Schmitges, 2011).

Quantitative mass spectrometry analyses of posttranslational modifications on the H3 N terminus in HeLa cells found no evidence for significant coexistence of H3K27me3 with H3K4me3 on the same H3 molecule. Similarly, the fraction of H3 carrying both H3K27me3 and H3K36me3 was reported to be extremely low (~0.078%), while H3K27me3 and H3K36me2 coexist on ~1.315% of H3 molecules. However, H3K27me3/H3K4me3 and H3K27me3/H3K36me2/3 bivalent domains have been reported to exist in embryonic stem cells, and they have been implicated to exist on the same nucleosome. Given that PRC2 is inhibited by active methylation marks, how then could such bivalent domains be generated? Two main possibilities are envisioned. First, PRC2 inhibition in vivo could be alleviated by specific posttranslational modifications on PRC2 in embryonic stem cells (see in plants, VRN2). Second, H3K27me3 could be deposited prior to modification of H3K4 or H3K36. According to this view, one would have to postulate that the HMTases depositing H3K4me3 or H3K36me2/3 can work on nucleosomes containing H3K27me3. In support of this view, it was found that H3K36 methylation by an NSD2 catalytic fragment is not inhibited by H3K27me3 marks on a peptide substrate (Schmitges, 2011).

In summary, this study found that mammalian and fly PRC2 complexes are not only activated by H3K27me3, but they are also inhibited by H3K4me3 and H3K36me2/3. PRC2, as a single biochemical entity, can thus integrate the information provided by histone modifications with antagonistic roles in gene regulation. While the biological network overseeing crosstalk between active and repressive chromatin marks in vivo probably extends beyond PRC2, including other chromatin modifiers such as histone demethylases, this identified a regulatory logic switch in PRC2 that intrinsically separates active and repressive chromatin domains. Given the dynamic nature of the nucleosome template that makes up eukaryotic chromosomes, this circuitry probably equips PRC2 with the necessary precision to heritably propagate a repressed chromatin state (Schmitges, 2011).

Evolutionary homologs: Table of contents

Histone H3: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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