Enhancer of zeste


EVOLUTIONARY HOMOLOGS

Enhancer of zeste homologs in plants and yeast

The CURLEY LEAF gene of Arabidopsis is necessary for stable repression of a floral homeotic gene (AGAMOUS) required to specify stamen and carpel identity in whorls 3 and 4 respectively. AGAMOUS is a MADS box protein (see Drosophila Serum response factor and MEF2). The CURLEY LEAF protein shows extensive homology to Enhancer of zeste, with three regions conserved between the two proteins. First, the C-terminus contains a 115 amino-acid region, the SET domain, previously recognized as a conserved region in the products of E(z), Trithorax and Suppressor of variegation 3-9. Second, residues 655-720 of CLF show 46% identity with the region of E(Z) (residues 538-603) that are rich in cysteine residues, but with an arrangement unlike that of zinc fingers. Third, residues 270-317 of CLF contain seven cysteines with a similar spacing to a region of E(Z) (residues 321-367) containing five cysteines, and there is a small region of sequence similarity at the N termini of these regions (Goodrich, 1997).

The Polycomb-group (PcG) proteins MEDEA (MEA; encodes a Polycomb-group (PcG) protein for which the closest Drosophila homolog is Enhancer of Zeste), FERTILIZATION INDEPENDENT ENDOSPERM, and FERTILIZATION INDEPENDENT SEED2 regulate seed development in Arabidopsis by controlling embryo and endosperm proliferation. All three of these FIS-class proteins are likely subunits of a multiprotein PcG omplex, which epigenetically regulates downstream target genes. The MADS-box gene PHERES1 (PHE1) is commonly deregulated in the fis-class mutants. PHE1 belongs to the evolutionarily ancient type I class of MADS-box proteins that have not yet been assigned any function in plants. Both MEDEA and FIE directly associate with the promoter region of PHE1, suggesting that PHE1 expression is epigenetically regulated by PcG proteins. PHE1 is expressed transiently after fertilization in both the embryo and the endosperm; however, it remains up-regulated in the fis mutants, consistent with the proposed function of the FIS genes as transcriptional repressors. Reduced expression levels of PHE1 in medea mutant seeds can suppress medea seed abortion, indicating a key role of PHE1 repression in seed development. PHE1 expression in a hypomethylated medea mutant background resembles the wild-type expression pattern and is associated with rescue of the medea seed-abortion phenotype. In summary, these results demonstrate that seed abortion in the medea mutant is largely mediated by deregulated expression of the type I MADS-box gene PHE1 (K&oml;hler, 2003).

A feature shared by some Trithorax family members (in Drosophila Trithorax and Enhancer of zeste), and by other proteins that function in chromatin-mediated transcriptional regulation, is the presence of a 130- to 140-amino acid motif dubbed the SET or Tromo domain. Analysis is presented of SET1, a yeast member of the trithorax gene family that encodes a 1080-amino acid protein with a C-terminal SET domain. In addition to its SET domain, which is 40-50% identical to those previously characterized, SET1 also shares dispersed but significant similarity to Drosophila and human trithorax homologs. To understand SET1 function(s), a null mutant was created. Mutant strains, although viable, are defective in transcriptional silencing of the silent mating-type loci and telomeres. The telomeric silencing defect is rescued not only by full-length episomal SET1 but also by the conserved SET domain of SET1. set1 mutant strains display other phenotypes, including morphological abnormalities, stationary phase defects, and growth and sporulation defects. Candidate genes that may interact with SET1 include those with functions in transcription, growth, and cell cycle control. These data suggest that yeast SET1, like its SET domain counterparts in other organisms, functions in diverse biological processes including transcription and chromatin structure (Nislow, 1997).

A Caenorhabditis elegans Polycomb protein complex

A unique and essential feature of germ cells is their immortality. In Caenorhabditis elegans, germline immortality requires the maternal contribution from four genes, mes-2, mes-3, mes-4 and mes-6. mes-2 encodes a protein similar to the Drosophila Polycomb group protein, Enhancer of zeste. Sequence analysis of the mes-2 cDNA revealed that it encodes a 773 amino acid protein. Most of the similarity between MES-2 and E(z) lies in two domains found in the carboxy-terminal half of each protein. MES-2 and E(z) are 57% identical at the amino acid level in a motif of ~115 amino acids, named the SET domain for three Drosophila proteins: Suppressor of variegation 3-9 [Su(var)3-9]. The fact that SET domains have been found in proteins that are not otherwise similar and in organisms as diverse as plants, yeast and mammals suggests that the SET domain represents an important functional motif. While the function of the SET domain is not known, all characterized SET domain proteins associate with chromatin. Currently there is no evidence that this domain binds to DNA directly; instead it has been suggested that SET-domain-containing proteins associate with their targets through protein-protein interactions. Consistent with the SET domain being an important functional motif, molecular lesions have been identified in highly conserved SET domain residues in two alleles of mes-2 (Holdeman, 1998).

It is thought that Polycomb group proteins form heteromeric complexes and control gene expression by altering chromatin conformation of target genes. As predicted from its similarity to a Polycomb group protein, MES-2 localizes to nuclei. MES-2 is found in germline nuclei in larval and adult worms and in all nuclei in early embryos. By the end of embryogenesis, MES-2 is detected primarily in the two primordial germ cells. The correct distribution of MES-2 requires the wild-type functions of mes-3 and mes-6. It is hypothesized that mes-2 encodes a maternal regulator of gene expression in the early germline; its function is essential for normal early development and the viability of germ cells (Holdeman, 1998).

The current working model for MES protein function is that a maternal supply of wild-type MES proteins is required for proper chromatin structure and gene expression in the nascent germline. Germline development is known to require maternal PIE-1, a transcriptional repressor that keeps somatically expressed genes turned off in the germline of early embryos. Several somatically expressed genes were tested for ectopic expression in the germlines of mes mutants and no evidence is seen for ectopic expression. This argues that the mes gene products act neither in concert with the PIE-1- mediated system, nor as a continuation of it. It is hypothesized that after PIE-mediated repression is lifted, the mes genes participate in establishing or maintaining germline chromatin in a conformation that ensures the proper pattern of gene expression when the germline starts its developmental program in early larvae. The chromosomes in the primordial germ cells of embryos and young larvae display a unique morphology, which is retained in the germ cells of mes mutants. This suggests that the MES proteins do not operate at the level of controlling gross chromosome morphology. Kelly and Fire (1998) find support for an involvement of the MES proteins in controlling some aspect of germline chromatin organization and gene expression: expression of transgenes present in many copies in extrachromosomal arrays is silenced in the germlines of wild-type worms, but desilenced in the germlines of mes mutants. Desilencing can also be achieved in wild-type germlines by placing the transgenes in the context of additional, complex DNA in the array. These findings suggest that the MES+ system participates in keeping at least some genes silenced in the germline, by having an effect on the state of chromatin. It is speculated that defects in the MES system result in aberrant patterns or levels of gene expression, leading to death of the germline (Holdeman, 1998).

Evolutionary conservation of the Extra sex combs-E(z) partner relationship is supported by recent studies of two Caenorhabditis elegans maternal-effect sterile genes, mes-2 and mes-6. Mutant alleles of either mes-2 or mes-6 produce grandchildless phenotypes that result from the limited proliferation and death of germ cells. MES-2 and MES-6 share sequence similarity with E(z) and Esc, respectively, across extensive portions of these proteins. However, unlike E(z) and Esc functions, mes-2 and mes-6 functions appear to be restricted to the germ line; thus far, there is no evidence for their involvement in Hox gene control. Thus, the developmental roles of MES-2 and MES-6 in worms are distinct from those of their E(z) and Esc homologs in flies and mammals. Nevertheless, there is evidence that the basic biochemical partnership between these proteins has been conserved. The spatial and temporal patterns of MES-2 and MES-6 accumulation in nuclei are identical, and mutations in either gene disrupt the spatial distribution or stability of the other protein. One of these mutations, mes-6bn66, substitutes a residue at a position that aligns with the predicted loop region of Esc that is required for binding to E(z) protein. Taken together, these results are consistent with a direct physical interaction between MES-2 and MES-6 in vivo. The conservation of these worm homologs, together with the evolutionary divergence of their developmental functions, may reflect a common biochemical role in chromatin that has been adapted for use in different cell lineages in worms and flies (Jones, 1998 and references therein).

The C. elegans maternal-effect sterile genes, mes-2, mes-3, mes-4, and mes-6, encode nuclear proteins that are essential for germ-line development. They are thought to be involved in a common process because their mutant phenotypes are similar. MES-2 and MES-6 are homologs of Enhancer of zeste and extra sex combs, both members of the Polycomb group of chromatin regulators in insects and vertebrates. MES-3 is a novel protein, and MES-4 is a SET-domain protein. To investigate whether the MES proteins interact and likely function as a complex, biochemical analyses were performed on C. elegans embryo extracts. Results of immunoprecipitation experiments indicate that MES-2, MES-3, and MES-6 are associated in a complex and that MES-4 is not associated with this complex. Based on in vitro binding assays, MES-2 and MES-6 interact directly, via the amino terminal portion of MES-2. Sucrose density gradient fractionation and gel filtration chromatography were performed to determine the Stokes radius and sedimentation coefficient of the MES-2/MES-3/MES-6 complex. Based on those two values, it is estimated that the molecular mass of the complex is ~255 kDa, close to the sum of the three known components. These results suggest that the two C. elegans Polycomb group homologs (MES-2 and MES-6) associate with a novel partner (MES-3) to regulate germ-line development in C. elegans (Xu, 2001).

Twelve PcG proteins have been cloned so far in Drosophila, but only two of them, E(Z) and ESC, are conserved in worms and also in plants. These data suggest that these two proteins might be distinctive PcG members, which function independently of the other PcG proteins. In Drosophila, E(Z) and ESC are associated in a complex in vivo and interact with each other directly in vitro. This association is conserved for their mammalian homologs, ENX and EED, and also for their worm homologs, MES-2 and MES-6 (Xu, 2001).

The portion of MES-2 that is important for its interaction with MES-6 in vitro is the N-terminal 194 aa. Similarly, sequences within the N-terminal region of fly E(Z) (amino acids 34-66) and the mouse E(Z) homolog (amino acids 132-160) are responsible for their interactions with ESC homologs. Although there is very little sequence similarity between the N termini of MES-2 and E(Z) homologs, their tertiary structure and the nature of the interaction between the partners might be conserved. The ESC-binding region of E(Z) is predicted to include a long stretch of helix, and the N-terminal 194 aa of MES-2 is predicted to form multiple long helices (Xu, 2001).

The conservation of the interaction between E(Z) and ESC among worms, flies, and mammals suggests that the molecular mechanism by which these protein partners function has been maintained throughout evolution. The MES-2/MES-6 complex therefore might regulate gene expression in C. elegans by the same mechanism used by the E(Z)/ESC complex in Drosophila. Indeed, MES-2 and MES-6 participate in repressing gene expression in C. elegans, as E(Z) and ESC are known to do in Drosophila (Xu, 2001).

The polycomb complex protein mes-2/E(z) promotes the transition from developmental plasticity to differentiation in C. elegans embryos

Expression profiling and in vivo imaging were used to characterize C. elegans embryos as they transit from a developmentally plastic state to the onset of differentiation. Normally, this transition is accompanied by activation of developmental regulators and differentiation genes, downregulation of early-expressed genes, and large-scale reorganization of chromatin. This study found that loss of plasticity and differentiation onset depends on the Polycomb complex protein mes-2/E(Z). mes-2 mutants display prolonged developmental plasticity in response to heterologous developmental regulators. Early-expressed genes remain active, differentiation genes fail to reach wild-type levels, and chromatin retains a decompacted morphology in mes-2 mutants. By contrast, loss of the developmental regulators pha-4/FoxA or end-1/GATA does not prolong plasticity. This study establishes a model by which to analyze developmental plasticity within an intact embryo. mes-2 orchestrates large-scale changes in chromatin organization and gene expression to promote the timely loss of developmental plasticity. These findings indicate that loss of plasticity can be uncoupled from cell fate specification (Yuzyuk, 2009).

This study has made three contributions toward understanding developmental plasticity in the early C. elegans embryo. (1) The molecular and morphological features of developmentally plastic cells were characterized and compared to cells that have undergone cell fate restriction. In wild-type embryos, large-scale changes were observed in gene expression and global alterations in chromatin morphology during the transition to cell fate restriction. (2) The PRC2 component mes-2/E(z) was examined and found to be required for the timely onset of differentiation, and not to maintain developmental plasticity. (3) Two possible roles for mes-2 in differentiating cells were considered: silencing of developmental regulators versus global reorganization of chromatin. Neither end-1 nor pha-4 was required to terminate plasticity, indicating that cell fate specification can be uncoupled from plasticity. The findings implicate large-scale restructuring of chromatin and gene expression by MES-2 as an important facet of differentiation onset (Yuzyuk, 2009).

Studies of wild-type and perturbed C. elegans embryos have revealed that somatic blastomeres from pregastrula embryos are developmentally plastic. The present study used gene expression profiling and chromatin analysis to characterize the plastic state, and to track the changes that accompany differentiation onset. The expression arrays revealed dramatic shifts in transcript pools from the 2E->4E stages to the 8E stage. This observation agrees well with previous studies that noted a transition from maternal to embryonically expressed genes at the onset of gastrulation. The switch from maternal to zygotic control suggests that this stage may resemble a mid-blastula transition (MBT) similar to other animals. Consistent with this idea, C. elegans embryos undergo cell cycle lengthening and cell movements at the 2E stage, akin to the MBT of other animals. These cellular behaviors and gene expression profiles suggest that the 2E->8E period of development constitutes a major transition during embryogenesis (Yuzyuk, 2009).

At least five classes of transcription factors contribute to pluripotency in mammals: Oct4/Pou, Sox2, Nanog, Klf/Kruppel-like, and c-myc. C. elegans embryos possess homologs of several of these factors, but their functions are not obviously linked to developmental plasticity. For example, C. elegans mep-1/Kruppel-like represses germline transcription in somatic cells, indicating that Kruppel-like factors promote a somatic or differentiated state in C. elegans. mml-1 is homologous to vertebrate c-myc, but mml-1 mutants have no known embryonic phenotype (Yuzyuk, 2009).

Genes expressed at the 2E stage were searched for homologs of mammalian pluripotency genes, but no obvious candidates were identified. For example, the three Pou proteins unc-6, unc-86, and ceh-18 are not expressed in the early embryo. Promoters of 2E-stage-expressed genes were examined to determine if any were enriched for the known binding sites of these factors, but they were not. Thus, it is unclear if developmentally plastic cells in C. elegans depend on the same constellation of sequence-specific transcription factors as do mammalian embryos (Yuzyuk, 2009).

In addition to gene expression changes, large-scale reorganization of chromatin was observed between the 2E and 8E stages. Developmentally plastic cells contained decompacted florets, and this configuration was lost during the transition toward differentiation. Florets were associated with a marker of elongating RNA pol II and lacked repressive histone marks, suggesting an open chromatin configuration. Ellipsoids and crescents were detected at the 4E and 8E stages. These conformations were associated with a repressive histone mark and reduced elongating RNA pol II, consistent with a more closed or silenced configuration. Changes in arrays were mirrored by morphological changes near the myo-2 and pax-1 loci, indicating that chromatin reorganization is a feature of endogenous loci as well as arrays. In embryos from other species, there may also be a transition from open to compacted chromatin, based on changes in nuclear size, but this has not been investigated directly. In culture, chromatin from pluripotent ES is decondensed and becomes condensed when cells are induced to differentiate. Thus, many types of cells undergo a transition in chromatin conformation as they lose developmental plasticity (Yuzyuk, 2009).

What is the underlying organization that establishes different chromatin morphologies? The florets appear relatively unstructured and diffuse within the nucleus. Ellipsoids and crescents may contain compacted and/or looped DNA that associates with the nuclear periphery. Electron microscopy studies have revealed that C. elegans nuclei at both the 2E and 8E stages lack electron-dense material, which typically characterizes heterochromatin. Thus, the nature of the compacted chromatin at the 4E and 8E stages is unclear (Yuzyuk, 2009).

Studies with other animals have shown a strong correlation between decompaction and the onset of transcription. What is the relationship of transcriptional activity with the chromatin configurations in early versus late embryonic cells? The foregut promoters included in the arrays are active many hours after the 2E stage. Moreover, arrays bearing mutated promoters or no added promoter formed florets at the 2E stage, similar to those bearing wild-type promoters. These observations indicate that decompaction is not dependent on productive transcription that generates mature mRNAs. However, the H5 staining suggests that arrays at the 2E stage are transcriptionally active to some extent. One possibility is that early C. elegans embryos, like ES cells, are transcriptionally hyperactive, meaning that DNA is transcribed promiscuously. A speculative idea is that open chromatin may be an important attribute of developmentally plastic cells that provides accessibility to the genome (Yuzyuk, 2009).

Chromatin reorganization during the transition toward differentiation may restrict transcriptional access or help partition unneeded DNA within the nucleus. This effect is unlikely to depend on the activity of specific promoters. One reason for thinking so is that arrays with and without foregut promoters underwent analogous conformational changes at the 4E and 8E stages. At endogenous loci, three genes within the myo-2 region were active in 4E- to 8E-stage embryos, whereas at least four were silent. Two genes from the pax-1 region were active in 2E- to 4E-stage embryos, whereas at least five were not. Thus, changes in chromatin conformation from the 2E to 8E developmental stages are likely distinct from the classic examples of decompaction by promoter firing. These data suggest that chromatin reorganization can reflect different developmental states (Yuzyuk, 2009).

Two models are considered regarding how developmental plasticity is lost. One appealing idea is that developmental regulators inhibit plasticity as well as promote cell fate. Conversely, silencing of developmental regulators by repressors such as the Polycomb complex might maintain developmental plasticity. This idea was tested four ways and no evidence was found for inhibition of plasticity by developmental regulators or promotion of plasticity by Polycomb. (1) Inactivation of the developmental regulator pha-4/FoxA failed to prolong plasticity, and loss of mes-2 failed to promote differentiation, as measured by the Cell Fate Challenge Assay. (2) Expression profiling of mes-2 and mes-3 mutants revealed an inability to downregulate Early genes or activate Differentiation genes at the 8E stage, suggesting a delayed onset of differentiation. (3) Inactivation of end-1 or pha-4 in mes-2 mutants did not suppress the mes-2 phenotype. (4) Chromatin from mes-2 mutants failed to compact at the 8E stage and resembled chromatin at earlier stages. These data suggest that developmental regulators like pha-4 and end-1 are not key terminators of plasticity, and that Polycomb does not maintain plasticity. Instead, the findings indicate that cell fate restriction can be uncoupled from cell fate specification. This separation may explain how early embryos can retain developmental plasticity even as they undergo rapid changes in gene expression at the onset of embryogenesis. This feature of developing embryos is different from pluripotent cell lines in which the pluripotent state is associated with arrested development and a static expression landscape (Yuzyuk, 2009).

What activity of mes-2 is required for the timely transition to differentiation? One possibility is that derepression of multiple developmental regulators in mutant embryos activates diverse developmental programs within single cells. The resulting confusion might interfere with the ability of cells to terminate plasticity and differentiate in a timely fashion. This scenario seems unlikely since most developmental regulators were activated normally in mes-2 mutants. Moreover, the target genes of these regulators were also expressed normally. Thus, widespread expression of end-1 in mes-2 mutants did not generate widespread end-1 activity, as detected by elt-7 expression. It is noted that although derepression of developmental regulators cannot explain the mes-2 phenotype, it is still possible that other critical genes are targeted by the MES factors (Yuzyuk, 2009).

A second appealing hypothesis is that MES factors modulate large-scale chromatin organization. In mes-2 mutants, Polycomb has been implicated in the reorganization of chromatin in other contexts, including mammalian X inactivation and genomic imprinting. Moreover, PRC2-Ezh1 can compact nucleosomes in vitro. It will be of interest to learn whether C. elegans PRC2 shares mechanistic features with mammalian PRC2 complexes, and how these activities contribute to the loss of plasticity and the transition toward differentiation (Yuzyuk, 2009).

Enhancer of zeste homologs in fish and Xenopus

The Xenopus homolog of Drosophila Enhancer of Zeste has been identified using a differential display strategy designed to identify genes involved in early anterior neural differentiation. XEZ codes for a protein of 748 amino acids that is very highly conserved in evolution and is 96% identical to both human and mouse EZ(H)2. In common with most other Xenopus Pc-G genes and unlike mammalian Pc-G genes, XEZ is anteriorly restricted. Zygotic expression of XEZ commences during gastrulation, much earlier than other anteriorly localized Pc-G genes; expression is restricted to the anterior neural plate and is confined later to the forebrain, eyes and branchial arches. XEZ is induced in animal caps overexpressing noggin; up-regulation of XEZ therefore represents a response to inhibition of BMP signaling in ectodermal cells. The midbrain/hindbrain junction marker En-2, and hindbrain marker Krox-20, are target genes of XEZ and that XEZ functions to repress these anteroposterior marker genes. Conversely, XEZ does not repress the forebrain marker Otx-2. XEZ overexpression results in a greatly thickened floor of the forebrain. These results implicate an important role for XEZ in the patterning of the nervous system (Barnett, 2001).

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).

Interactions of mammalian Enhancer of zeste homologs

Drosophila Extra sex combs (Esc) and its mammalian homolog, embryonic ectoderm development (eed), are special Pc-G members; they are required early during development when Pc-G repression is initiated, a process that is still poorly understood. To gain insight into the molecular function of Eed, the yeast two-hybrid method was used to find Eed-interacting proteins. Described is the specific in vivo binding of Eed to Enx1 and Enx2, two mammalian homologs of the essential Drosophila Pc-G gene Enhancer-of-zeste [E(z)]. No direct biochemical interactions are found between Eed/Enx and a previously characterized mouse Pc-G protein complex containing several mouse Pc-G proteins, including mouse polyhomeotic (Mph1). This suggests that different Pc-G complexes with distinct functions may exist. However, and in support of a bridging role for Enx1, partial colocalization of Enx1 and Mph1 to subnuclear domains may point to more transient interactions between these complexes (van Lohuizen, 1998).

If the observed interactions between Enx1, Enx2, and Eed are relevant for their in vivo function, one would expect the expression patterns to coincide during development. Since the onset of Eed expression possibly occurs earlier than for most other Pc-G genes, a sensitive, semiquantitative RT-PCR assay was used to monitor the expression patterns of Enx1 and Enx2 during development and in adult mice. Whereas no expression is detected in blastocysts, Enx1 is already highly expressed in day 7.3 mouse embryos and becomes gradually more restricted to specific organs during development, with the highest expression being in the testis. In contrast, Enx2 expression was first detected on day 9 and continues to be expressed at moderate levels in most tissues. These results indicate that there is indeed extensive overlap in onset and tissue distribution between Enx1, Enx2, and Eed: the onset of Enx1 expression parallels that of Eed, whereas in adult mice Enx2 and Eed are expressed in most tissues (van Lohuizen, 1998).

The Polycomb group proteins are involved in maintenance of the silenced state of several developmentally regulated genes. These proteins form large aggregates with different subunit compositions. To explore the nature of these complexes and their function(s), the full-length Eed (embryonic ectoderm development) protein, a mammalian homolog of the Drosophila Polycomb group protein Esc, was used as a bait in a yeast two-hybrid screen. Several strongly interacting cDNA clones were isolated. The cloned cDNAs all encode the 150- to 200-amino-acid N-terminal fragment of the mammalian homolog of the Drosophila Enhancer of zeste [E(z)] protein, Ezh2. The full-length Ezh2 binds strongly to Eed in vitro, and Eed coimmunoprecipitates with Ezh2 from murine 70Z/3 cell extracts, confirming the interaction between these proteins that was observed in yeast. Mutations T1031A and T1040C in one of the WD40 repeats of Eed, which account for the hypomorphic and lethal phenotype of eed in mouse development, block binding of Ezh2 to Eed in a two-hybrid interaction in both yeast and mammalian cells. These mutations also blocked the interaction between these proteins in vitro. In mammalian cells, the Gal4-Eed fusion protein represses the activity of a promoter bearing Gal4 DNA elements. The N-terminal fragment of the Ezh2 protein abolishes the transcriptional repressor activity of Gal4-Eed protein when these proteins are coexpressed in mammalian cells. Eed and Ezh2 are also found to bind RNA in vitro, and RNA alters the interaction between these proteins. These findings suggest that Polycomb group proteins Eed and Ezh2 functionally interact in mammalian cells, an interaction that is mediated by the WD40-containing domain of Eed protein (Denisenko, 1998).

X inactivation in female mammals is one of the best studied examples of heritable gene silencing and provides an important model for studying maintenance of patterns of gene expression during differentiation and development. The process is initiated by a cis-acting RNA, the X inactive specific transcript (Xist). Xist RNA is thought to recruit silencing complexes to the inactive X, that then serve to establish and maintain the inactive state in all subsequent cell divisions. Most lineages undergo random X inactivation, there being an equal probability of either the maternally (Xm) or paternally (Xp) inherited X chromosome being inactivated in a given cell. In the extraembryonic trophectoderm and primitive endoderm lineages of mouse embryos, however, there is imprinted X inactivation of Xp. This process is also Xist dependent. A recent study has shown that imprinted X inactivation in trophectoderm is not maintained in embryonic ectoderm development (eed) mutant mice. Eed, the mammalian homolog of Drosophila Extra sex combs, and Enx1, the mammalian homolog of enhancer of Zeste, are directly localized to the inactive X chromosome in XX trophoblast stem (TS) cells. The association of Eed/Enx1 complexes is mitotically stable, suggesting a mechanism for the maintenance of imprinted X inactivation in these cells (Mak, 2002).

Drosophila suppressor of zeste 12 (Su(z)12) is a Polycomb group (PcG) transcriptional repressor and is present in E(z)-ESC, a multiprotein complex with methylation activity specific for lysine 9 and 27 of histone H3. Although PcG- and heterochromatin-mediated gene silencing have been considered distinct, mutant flies of Su(z)12 show not only homeotic transformation but also position effect variegation. The mammalian SU(Z)12 directly interacts with heterochromatin protein 1alpha (HP1alpha) and PcG enhancer of zeste 2 (EZH2), the mammalian counterpart of E(z), in vitro and in vivo. Two distinct domains in SU(Z)12 are involved in these interactions, the region between the zinc finger motif and the VEFS (VRN2-EMF2-FIS2-Su(z)12) box for HP1alpha (amino acid residues 479-536) and the VEFS box for EZH2 (amino acid residues 600-639), which are not mutually exclusive. Interestingly this region of the VEFS box has been shown to be critical for the phenotype of the Su(z)12 mutant fly. In addition SU(Z)12 represses transcription activity in the presence of HP1alpha in a reporter assay. These results provide a molecular explanation for the functional link of these epigenetic silencing processes mediated by Su(z)12 (Yamamoto, 2004).

Recent studies have revealed the intrinsic histone methyltransferase (HMTase) activity of the EED-EZH2 complex and its role in Hox gene silencing, X inactivation, and cancer metastasis. This study focuses on the function of individual components. It was found that the HMTase activity requires a minimum of three components -- EZH2, EED, and SUZ12 -- while AEBP2 is required for optimal enzymatic activity. Using a stable SUZ12 knockdown cell line, it has been shown that SUZ12 knockdown results in cell growth defects, which correlate with genome-wide alteration on H3-K27 methylation as well as upregulation of a number of Hox genes. Chromatin immunoprecipitation (ChIP) assay identified a 500 bp region located 4 kb upstream of the HoxA9 transcription initiation site as a SUZ12 binding site, which responds to SUZ12 knockdown and might play an important role in regulating HoxA9 expression. Thus, this study establishes a critical role for SUZ12 in H3-lysine 27 methylation and Hox gene silencing (Cao, 2004).

The importance of histone methylation by the polycomb group proteins was examined in the mouse circadian clock mechanism. Endogenous EZH2, a polycomb group enzyme that methylates lysine 27 on histone H3, co-immunoprecipitates with CLOCK and BMAL1 throughout the circadian cycle in liver nuclear extracts. Chromatin immunoprecipitation revealed EZH2 binding and di- and tri-methylation of H3-K27 on both the Period 1 and Period 2 promoters. A role of EZH2 in cryptochrome-mediated transcriptional repression of the clockwork was supported by overexpression and RNA interference studies. Serum-induced circadian rhythms in NIH 3T3 cells in culture were disrupted by transfection of an RNA interfering sequence targeting EZH2. These results indicate that EZH2 is important for the maintenance of circadian rhythms and extend the activity of the polycomb group proteins to the core clockwork mechanism of mammals (Etchegaray, 2006).

The Polycomb group (PcG) protein, enhancer of zeste homologue 2 (EZH2), has an essential role in promoting histone H3 lysine 27 trimethylation (H3K27me3) and epigenetic gene silencing. This function of EZH2 is important for cell proliferation and inhibition of cell differentiation, and is implicated in cancer progression. This study demonstrates that under physiological conditions, cyclin-dependent kinase 1 (CDK1) and cyclin-dependent kinase 2 (CDK2) phosphorylate EZH2 at Thr 350 in an evolutionarily conserved motif. Phosphorylation of Thr 350 is important for recruitment of EZH2 and maintenance of H3K27me3 levels at EZH2-target loci. Blockage of Thr 350 phosphorylation not only diminishes the global effect of EZH2 on gene silencing, it also mitigates EZH2-mediated cell proliferation and migration. These results demonstrate that CDK-mediated phosphorylation is a key mechanism governing EZH2 function and that there is a link between the cell-cycle machinery and epigenetic gene silencing (Chen, 2010).

Enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of Polycomb repressive complex 2 (PRC2) and catalyses the trimethylation of histone H3 on Lys 27 (H3K27), which represses gene transcription. EZH2 enhances cancer-cell invasiveness and regulates stem cell differentiation. This study demonstrates that EZH2 can be phosphorylated at Thr 487 through activation of cyclin-dependent kinase 1 (CDK1). The phosphorylation of EZH2 at Thr 487 disrupts EZH2 binding with the other PRC2 components SUZ12 and EED, and thereby inhibits EZH2 methyltransferase activity, resulting in inhibition of cancer-cell invasion. In human mesenchymal stem cells, activation of CDK1 promotes mesenchymal stem cell differentiation into osteoblasts through phosphorylation of EZH2 at Thr 487. These findings define a signalling link between CDK1 and EZH2 that may have an important role in diverse biological processes, including cancer-cell invasion and osteogenic differentiation of mesenchymal stem cells (Wei, 2011).

Hira-dependent Histone H3.3 deposition facilitates PRC2 recruitment at developmental loci in ES cells

Polycomb repressive complex 2 (PRC2) regulates gene expression during lineage specification through trimethylation of lysine 27 on histone H3. In Drosophila, polycomb binding sites are dynamic chromatin regions enriched with the histone variant H3.3. This study shows that, in mouse embryonic stem cells (ESCs), H3.3 is required for proper establishment of H3K27me3 at the promoters of developmentally regulated genes. Upon H3.3 depletion, these promoters show reduced nucleosome turnover measured by deposition of de novo synthesized histones and reduced PRC2 occupancy. Further, this study shows H3.3-dependent interaction of PRC2 with the histone chaperone, Hira, and that Hira localization to chromatin requires H3.3. The data demonstrate the importance of H3.3 in maintaining a chromatin landscape in ESCs that is important for proper gene regulation during differentiation. Moreover, these findings support the emerging notion that H3.3 has multiple functions in distinct genomic locations that are not always correlated with an 'active' chromatin state (Banaszynski, 2013).

Nucleosome-binding activities within JARID2 and EZH1 regulate the function of PRC2 on chromatin

Polycomb-repressive complex 2 (PRC2) comprises specific members of the Polycomb group of epigenetic modulators. PRC2 catalyzes methylation of histone H3 at Lys 27 (H3K27me3) through its Enhancer of zeste (Ezh) constituent, of which there are two mammalian homologs: Ezh1 and Ezh2. Several ancillary factors, including Jarid2, modulate PRC2 function, with Jarid2 facilitating its recruitment to target genes. Jarid2, like Ezh2, is present in poorly differentiated and actively dividing cells, while Ezh1 associates with PRC2 in all cells, including resting cells. Jarid2 was found to exhibit nucleosome-binding activity that contributes to PRC2 stimulation. Moreover, such nucleosome-binding activity is exhibited by PRC2 comprising Ezh1 (PRC2-Ezh1), in contrast to PRC2-Ezh2. The presence of Ezh1 helps to maintain PRC2 occupancy on its target genes in myoblasts where Jarid2 is not expressed. These findings lead to a model in which PRC2-Ezh2 is important for the de novo establishment of H3K27me3 in dividing cells, whereas PRC2-Ezh1 is required for its maintenance in resting cells (Son, 2013).

A dimeric state for PRC2

Polycomb repressive complex-2 (PRC2) is a histone methyltransferase required for epigenetic silencing during development and cancer. EZH2 is the catalytic subunit of PRC2, and SUZ12 is an essential regulatory subunit. EED is a histone-binding subunit that binds H3K27me3-modified histone tails, resulting in increased affinity to nucleosomes and stimulation of the catalytic activity of PRC2. Long non-coding RNAs (lncRNAs) can recruit PRC2 to chromatin. Previous studies identified PRC2 subunits in a complex with the apparent molecular weight of a dimer, which might be accounted for by the incorporation of additional protein subunits or RNA rather than PRC2 dimerization. This study shows that reconstituted human PRC2 is in fact a dimer, using multiple independent approaches including analytical size exclusion chromatography (SEC), SEC combined with multi-angle light scattering and co-immunoprecipitation of differentially tagged subunits. Even though it contains at least two RNA-binding subunits, each PRC2 dimer binds only one RNA molecule. Yet, multiple PRC2 dimers bind a single RNA molecule cooperatively. These observations suggest a model in which the first RNA binding event promotes the recruitment of multiple PRC2 complexes to chromatin, thereby nucleating repression (Davidovich, 2014).

Mutation in mammalian Enhancer of zeste homologs

Polycomb-group (Pc-G) genes are required for the stable repression of the homeotic selector genes and other developmentally regulated genes, presumably through the modulation of chromatin domains. Among the Drosophila Pc-G genes, Enhancer of zeste [E(z)] merits special consideration since it represents one of the Pc-G genes most conserved through evolution. In addition, the E(Z) protein family contains the SET domain, which has recently been linked with histone methyltransferase (HMTase) activity. Although E(Z)-related proteins have not (yet) been directly associated with HMTase activity, mammalian Ezh2 is a member of a histone deacetylase complex. To investigate its in vivo function, mice deficient for Ezh2 were generated. The Ezh2 null mutation results in lethality at early stages of mouse development. Ezh2 mutant mice either cease developing after implantation or initiate but fail to complete gastrulation. Moreover, Ezh2-deficient blastocysts display an impaired potential for outgrowth, preventing the establishment of Ezh2-null embryonic stem cells. Interestingly, Ezh2 is up-regulated upon fertilization and remains highly expressed at the preimplantation stages of mouse development. Together, these data suggest an essential role for Ezh2 during early mouse development and genetically link Ezh2 with eed and YY1, the only other early-acting Pc-G genes (O'Carroll, 2001).

Enhancer of zeste 2 (Ezh2), a SET domain-containing protein, is crucial for development in many model organisms, including early mouse development. In mice, Ezh2 is detected as a maternally inherited protein in the oocyte but its function at the onset of development is unknown. A conditional allele of Ezh2 was used to deplete the oocyte of this maternal inheritance. The loss of maternal Ezh2 has a long-term effect causing severe growth retardation of neonates despite 'rescue' through embryonic transcription from the paternal allele. This phenotypic effect on growth could be attributed to the asymmetric localization of the Ezh2/Eed complex and the associated histone methylation pattern to the maternal genome, which is disrupted in Ezh2 mutant zygotes. During subsequent development, distinct histone methylation patterns were detected in the trophectoderm and the pluripotent epiblast. In the latter, where Oct4 expression continues from the zygote onwards, the Ezh2/Eed complex apparently establishes a unique epigenetic state and plasticity, which probably explains why loss of Ezh2 is early embryonic lethal and obligatory for the derivation of pluripotent embryonic stem cells. By contrast, in the differentiating trophectoderm cells where Oct4 expression is progressively downregulated, Ezh2/Eed complex is recruited transiently to one X chromosome in female embryos at the onset of X-inactivation. This accumulation and the associated histone methylation are also lost in Ezh2 mutants, suggesting a role in X inactivation. Thus, Ezh2 plays significant and diverse roles during early development, as well as during the establishment of the first differentiated cells, the trophectoderm, and of the pluripotent epiblast cells (Erhardt, 2003).

Polycomb group protein Ezh2 is an essential epigenetic regulator of embryonic development in mice, but its role in the adult organism is unknown. High expression of Ezh2 in developing murine lymphocytes suggests Ezh2 involvement in lymphopoiesis. Using Cre-mediated conditional mutagenesis, a critical role has been demonstrated for Ezh2 in early B cell development and rearrangement of the immunoglobulin heavy chain gene (Igh). Ezh2 is a key regulator of histone H3 methylation in early B cell progenitors. These data suggest Ezh2-dependent histone H3 methylation as a novel regulatory mechanism controlling Igh rearrangement during early murine B cell development (Su, 2003).

Enhancer of zeste homologs function downstream of E2F

E2F is a family of transcription factors that regulate both cellular proliferation and differentiation. To establish the role of E2F3 in vivo, an E2f3 mutant mouse strain was generated. E2F3-deficient mice arise at one-quarter of the expected frequency, demonstrating that E2F3 is important for normal development. To determine the molecular consequences of E2F3 deficiency, the properties of embryonic fibroblasts derived from E2f3 mutant mice were analyzed. Mutation of E2f3 dramatically impairs the mitogen-induced, transcriptional activation of numerous E2F-responsive genes. A number of genes, including B-myb, cyclin A, cdc2, cdc6, and DHFR, could be identified whose expression is dependent on the presence of E2F3 but not E2F1. The E2Fs regulate the expression of several proteins that are involved in early development, including homeobox proteins, transcription factors involved in cell fate decisions, a number of proteins that determine homeotic gene transcription, and signaling pathways such as the TGFbeta and Wnt pathways that are essential for early development. As an example of the relevance of these findings, it has been reported that position-effect variegation (PEV) in Drosophila depends on E2F activity. Loss of E2F activity enhances PEV, whereas overexpression of E2F activity suppresses PEV in Drosophila. These data suggested that the E2Fs themselves have an epigenetic effect by regulating chromatin structure or, more likely, that the E2Fs control PEV by regulating genes of the Polycomb group (PcG) family. In this screen, several PcG genes have been identified, like Enhancer of Zeste 2 (EZH2), Embryonic Ectoderm Development protein (EED) and Homolog of Polyhomeotic (EDR2/HPH2). The E2F-induced expression of these genes may provide an explanation for the role of E2F in the regulation of PEV and, more importantly, in development (Muller, 2001).

EZH2, homolog of Drosophila E(z) is highly expressed in metastatic prostate cancer and in lymphomas. EZH2 is a component of the PRC2 histone methyltransferase complex, which also contains EED and SUZ12 and is required for the silencing of HOX gene expression during embryonic development. Both EZH2 and EED are essential for the proliferation of both transformed and non-transformed human cells. In addition, the pRB-E2F pathway tightly regulates their expression and, consistent with this, EZH2 is found to be highly expressed in a large set of human tumors. These results raise the question whether EZH2 is a marker of proliferation or if it is actually contributing to tumor formation. Significantly, it is proposed that EZH2 is a bona fide oncogene, since ectopic expression of EZH2 is found to be capable of providing a proliferative advantage to primary cells and, in addition, its gene locus is specifically amplified in several primary tumors (Bracken, 2003).

Mammalian homologues of the Polycomb-group gene Enhancer of zeste mediate gene silencing in Drosophila heterochromatin and at S. cerevisiae telomeres

Human (EZH2) and mouse (Ezh1) homologs of the Drosophila Polycomb-group (Pc-G) gene Enhancer of zeste (E[z]) have been isolated, a crucial regulator of homeotic gene expression implicated in the assembly of repressive protein complexes in chromatin. Mammalian homologs of E(z) are encoded by two distinct loci in mouse and man, and the two murine Ezh genes display complementary expression profiles during mouse development. Human EZH1 and murine Ezh1 (and probably also EZH2/Ezh2), are encoded by orthologous loci in mouse and man, and in both comparisons amino acid identities are 98%. EZH2/Ezh2 and E(z) appear to be most closely related, whereas EZH1/Ezh1 is the more divergent of the mammalian gene pairs. Human/mouse EZH2/Ezh2 is 61% and human/mouse EZH1/Ezh1 is 55% identical to E(z). There are four regions of high sequency identity. In addition to the most highly conserved SET domain (86% identity), a 115 amino acid, cysteine-rich region immediately precedes the SET domain (68% identity). Most of the cystein residues originally noted for E(z) are conserved in the mammalian proteins. In the amino-terminal half, two stretches, one of 66 amino acids (domain I; 66% identity) and one of 114 amino acids (domain II: 56% identity), are highly related between mammalian and fly E(z) proteins. Domain II also contains six conserved cysteine residues with unusual spacing and is separated from domain I by a stretch of charged amino acids. The position and sequence of the nuclear localization signal is also conserved (Laible, 1997).

In mice, Ezh1-specific transcripts are detected ubiquitously during embryogenesis, however their relative abundance is generally upregulated during development and in adults reaches 4- to 5-fold higher levels in kidney, brain and skeletal muscle. Ezh1 transcripts remain at a low level in liver, spleen and thymus. Ezh2 transcripts are downregulated as development progresses, to display a more thymus-restricted expression. Human EZH2 and murine Ezh2 are subject to alternative splicing that removes the amino-terminal half of the conserved cysteine-rich region or, in EZH1, part of the SET domain. The E(z) gene family reveals a striking functional conservation in mediating gene repression in eukaryotic chromatin: extra gene copies of human EZH2 or Drosophila E(z) in transgenic flies enhance position effect variegation of the heterochromatin-associated white gene, and expression of either human EZH2 or murine Ezh1 restores gene repression in Saccharomyces cerevisiae mutants that are impaired in telomeric silencing. Together, these data provide a functional link between Pc-G-dependent gene repression and inactive chromatin domains, indicating that silencing mechanism(s) may be broadly conserved in eukaryotes (Laible, 1997).

The finding of mammalian E(z) homologs allows the definition of a SET domain family, and suggests that the SET domain represents an ancient protein module that is intrinsically involved in chromatin-controlled gene regulation. Four subgroups are described. The SET domain of E(z) related proteins (subgroup I) appears to be most similar to that of subgroup II genes, which include the Trithorax homologs and S. cerevisiae SET1. The SET domain of S. cerevisiae Ezl-1 and Drosophila Ash-1 appear to be unique, because in both of these genes the SET domain is not located at the carboxy terminus (subgroup III). Finally, SET domains for subgroup IV genes comprose those present in Drosophila Su(var)3-9 related proteins and human G9a and KG-1 (Laible, 1997).

Polycomb group regulation of Hox gene expression in C. elegans.

Polycomb group (PcG) chromatin proteins regulate homeotic genes in both animals and plants. In Drosophila and vertebrates, PcG proteins form complexes and maintain early patterns of Hox gene repression, ensuring fidelity of developmental patterning. PcG proteins in C. elegans form a complex and mediate transcriptional silencing in the germline, but no role for the C. elegans PcG homologs in somatic Hox gene regulation has been demonstrated. Surprisingly, it is found that the PcG homologs MES-2 [E(Z)] and MES-6 (ESC), along with MES-3, a protein without known homologs, do repress Hox expression in C. elegans. mes mutations cause anteroposterior transformations and disrupt Hox-dependent neuroblast migration. Thus, as in Drosophila, vertebrates, and plants, C. elegans PcG proteins regulate key developmental patterning genes to establish positional identity (Ross, 2003).

The three mes genes act upstream of the Hox genes mab-5 and egl-5 during V ray differentiation, and loss of mes activity can restore normal ray development and mating ability to males mutant in the mab-5 activator pal-1. Males lacking mes activity display anterior expansions of tail structures and ectopic expression of the Hox reporter egl-5::gfp and the Hox target lin-32::gfp. This regulation is not restricted to the male tail: mes-2, -3, and -6 also repress lin-39::lacZ expression in the midbody and head and mab-5 activity in a migrating neuroblast. Consistent with a general somatic regulatory function, MES protein expression is widespread in larvae, particularly males. These findings suggest that the regulatory relationship between PcG chromatin proteins and the Hox genes has been conserved in nematodes (Ross, 2003).

Characterization of interactions between the mammalian polycomb-group proteins Enx1/EZH2 and EED suggests the existence of different mammalian polycomb-group protein complexes

Several lines of evidence suggest a functional interaction between the PcG and trxG proteins. For example, genetic evidence indicates that the Enhancer of zeste [E(z)] gene can be considered both a PcG and a trxG gene. To better understand the molecular interactions in which the E(z) protein is involved, a two-hybrid screen was performed with Enx1/EZH2, a mammalian homolog of E(z), as the target. The human EED protein is shown to interact with Enx1/EZH2. EED is the human homolog of eed, a murine PcG gene that has extensive homology with the Drosophila PcG gene extra sex combs (esc). Enx1/EZH2 and EED coimmunoprecipitate, indicating that they also interact in vivo. However, Enx1/EZH2 and EED do not coimmunoprecipitate with other human PcG proteins, such as HPC2 and BMI1. Furthermore, unlike HPC2 and BMI1, which colocalize in nuclear domains of U-2 OS osteosarcoma cells, Enx1/EZH2 and EED do not colocalize with HPC2 or BMI1. These findings indicate that Enx1/EZH2 and EED are members of a class of PcG proteins that is distinct from previously described human PcG proteins (Sewalt, 1998).

To define the domains that are responsible for the interaction between Enx1 and EED, different parts of Enx1 and EED were subcloned in frame with the GAL4-DBD and to see whether these proteins could still interact with full-length EED or full-length Enx1. Enx1 comprises two N-terminal domains that show strong homology between Drosophila E(z) and its mammalian homologs. These domains have been designated domains I and II. Furthermore, Enx1 contains a C-terminal cysteine-rich domain and a SET domain. This last domain is found in a number of different proteins such as the Trithorax protein. The region encompassing both the cysteine-rich domain and the SET domain (aa 498 to 746) does not interact with EED. A region extended toward the N terminus (aa 285 to 746) also does not interact with EED. In contrast, the region encompassing domain I and a part of domain II (aa 1 to 285) does interact with EED. To analyze this region in more detail, two constructs were made, one containing homology domain I (aa 1 to 195) and the other containing homology domain II (aa 172 to 335). Only the region of Enx1 that contains homology domain I interacts with the EED protein. It is concluded that EED binds to the N-terminal region of Enx1 that encompasses homology domain I (Sewalt, 1998).

EED contains five WD-40 domains which are thought to be involved in protein-protein interactions. The importance of these WD-40 domains for the interaction between Enx1 and EED was tested. Truncated EED protein constructs were made that contain an increasing number of WD-40 domains. None of the truncated EED proteins, which contain up to four WD-40 domains, interact with Enx1. Only when all five WD-40 domains are present is this truncated EED protein (aa 184 to 535) able to interact with Enx1. The most N-terminal region of EED, which does not contain WD-40 domains, is not important for mediating the interaction between Enx1 and EED (Sewalt, 1998).

It has been proposed that Drosophila Esc interacts with the transcriptional machinery through the WD-40 domains. This model is based on the homology that is found between Esc and Tup1, a yeast protein that also contains seven WD-40 domains. These WD-40 domains are important for the involvement of the Tup1 protein in the repression of gene activity and in its binding to the DNA-binding homeodomain protein 2. Point mutations in the WD-40 domains have also been found in several esc mutants. Either one of two point mutations in the second WD-40 domain completely abolishes the interaction in the two-hybrid system between Enx1 and EED. Precisely these two point mutations are responsible for the severe developmental defects in eed mutant mice. It is significant that the ability of the eed535 protein to repress gene activity is also completely abolished by these point mutations. It is therefore tempting to speculate that both the interference with the binding capacity and the repressing abilities of the eed/EED protein through these point mutations contribute to the developmental defects in eed mutant mice. One immediate consequence of these point mutations can be that the Enx1 protein is no longer able to bind to eed; this leads to a subsequent loss of integrity for the protein complex formed in part by both Enx1/EZH2 and eed (Sewalt, 1998).

Histone methyltransferase activity associated with a human multiprotein complex containing the Enhancer of Zeste protein

Enhancer of Zeste [E(z)] is a Polycomb-group transcriptional repressor and one of the founding members of the family of SET domain-containing proteins. Several SET-domain proteins possess intrinsic histone methyltransferase (HMT) activity. However, recombinant E(z) protein was found to be inactive in a HMT assay. A multiprotein E(z) complex has been isolated from humans that contains extra sex combs, suppressor of zeste-12 [Su(z)12], and the histone binding proteins RbAp46/RbAp48 (see Caf1). This complex, which has been termed Polycomb repressive complex (PRC) 2, possesses HMT activity with specificity for Lys 9 (K9) and Lys 27 (K27) of histone H3. The HMT activity of PRC2 is dependent on an intact SET domain in the E(z) protein. It is hypothesized that transcriptional repression by the E(z) protein involves methylation-dependent recruitment of PRC1. The presence of Su(z)12, a strong suppressor of position effect variegation, in PRC2 suggests that PRC2 may play a widespread role in heterochromatin-mediated silencing (Kuzmichev, 2002).

The polypeptide composition of the PRC2, specifically the presence of Su(z)12, suggests that PRC2 plays a more general role in transcriptional silencing outside of the repression of HOX genes. Su(z)12 is a protein with dual PcG and Su(var) functions, and this, therefore, suggests that PRC2 has functions other than homeotic gene repression and, in fact, may play a more general role in heterochromatin-mediated silencing. The observation that human E(z) can function as an inducer of silencing in yeast and as an enhancer of PEV in Drosophila supports this notion. It is speculated that the requirement for E(z) and ESC during early embryonic development reflects its function in general transcriptional silencing. The multifunctional nature of both the E(z) and Su(z)12 proteins suggests that they may also display biochemical heterogeneity. For example, the heterogeneous elution profile of E(z) on various columns suggests that E(z) exists in several distinct complexes (Kuzmichev, 2002).

Purified PRC2 displayed specificity for K9 and K27 of the histone H3 tail. The complex, under the conditions of the assays used, displayed a strong preference for K27. However, when the H3-tail was used as a GST-fusion protein, PRC2 displayed apparently equal specificity for K9 and K27. Analyses of the amino acid sequence in which these lysines are embedded shows a great deal of conservation. K9 is present within the sequence QTARK9STG, whereas K27 is present within the sequence KAARK27SAP. Therefore, at least two different possibilities can be postulated to account for the specificity observed. In one case, the specificity of PRC2 is relaxed in vitro, under the assay conditions used, and the methylation of K9 is nonspecific because of the sequence similarity of the residues within which K9 resides. An apparently similar situation was observed in studies analyzing the specificity of the histone methyltransferase G9a (see Drosophila G9a), which biochemically behaves as a H3-histone methyltransferase that preferentially targets K9 and, to much lower levels, K27. In vivo, however, G9a clearly targets H3-K9: whether or not the extent of H3-K27 methylation is decreased in G9a-null cells is unknown. A second possibility is that E(z) targets both K9 and K27, but that this is a regulated process such that methylation of K9 and/or K27 is modulated by factors that associate with E(z) and/or by other modifications existing in the nucleosome. This second possibility is favored based on the following observations. First, the E(z) protein can be considered to be a PcG as well as a TrxG. Not surprisingly, the analyses demonstrate that E(z) is present in distinct complexes. One of the complexes containing E(z) is PRC2; however; this complex also includes Su(z)12. Su(z)12 is a polypeptide that has been found in genetic analyses to regulate the expression of the HOX genes, but loss of function of Su(z)12 suppresses PEV. Therefore, the presence of Su(z)12 in PRC2 may regulate the methylation sites within the histone H3 tail (Kuzmichev, 2002).

Methylation of histone H3-K9 was shown to be an essential step in the establishment of inactive X chromosome. H3-Lys 9 methylation of the inactive X chromosome is not mediated by Suv39 or by G9a. Studies have demonstrated that the imprinted inactivation of the X chromosome in females is lost in mutant mice lacking eed (the mammalian homolog of ESC). Moreover, studies have also demonstrated that during imprinted X inactivation, the mammalian ESC-E(z) complex is localized to the inactive X chromosome in a mitotically stable manner. It is speculated, in light of the accumulated data, that H3-K9 methylation of the inactive X chromosome might be mediated by E(z) within PRC2 or a PRC2-like complex. Importantly, however, the function of methylation of histone H3 at K27 has not been analyzed in the establishment and/or maintenance of the inactive X chromosome. In light of the results discussed above, it is postulated that methylation of H3-K27 may also be important in the process of X inactivation (Kuzmichev, 2002).

It is proposed that the role of E(z) HMT activity in the repression of homeotic gene expression is to establish a binding site for other PcG proteins. It is suggested that PRC2 is recruited to the HOX gene cluster by a transiently acting repressor, for example, through an EED-YY1/Pho interaction or an RbAp46/48-HDAC/dMi2/Hb interaction. Once recruited, PRC2 methylates K27 on histone H3, and this mark recruits PC1. The PC1 protein can convert this mark into a permanently repressed state through methylation of K9 through the recruitment of the Su(var)3-9 H3-K9-specific histone methyltransferase and/or the recruitment of PRC1. Alternatively and/or additionally, PC1 may stimulate the H3-K9 HMT activity of PRC2. This hypothesis is supported by studies demonstrating that trimethylation of K27 is necessary for binding of PC1 to an H3 tail peptide. These findings are in full agreement with studies demonstrating loss of chromosome binding for several PRC1 components upon inactivation of E(z). Interestingly, immunolocalization experiments using antibodies specific for methylated H3-K9 suggest that almost all of the H3-K9 methylation is concentrated in the chromocenter of Drosophila polytene chromosomes, with almost no staining detectable on the chromosomal arms. In contrast, E(z) and other PcG proteins, with subnuclear localization that is regulated by E(z), bind only to discrete bands along the arms of polytene chromosomes. These observations suggest that methylation at K27, rather than methylation at K9, is more likely to establish a binding site for the PC1 protein. This may explain why methylation of K9 alone was not sufficient to allow PC1 to recognize specifically the H3 tail in vitro. The observed PC1 binding was independent of DNA. However, repression of HOX genes in vivo is dependent on PRE. From these results, it must be concluded that although methylation of the H3 tail is important in creating a recognition site for PC1 binding, stable and specific binding must require additional factors and or modifications. A likely candidate is a nucleosome on the PRE with the histone H3-tail methylated at position 27 (Kuzmichev, 2002).

The presence of the RbAp46 and RbAp48 proteins in the ESC-E(z) complex may be important for several reasons. First, these histone-binding proteins are often found in complexes with enzymes involved in the covalent modification of histones. For example, RbAp46 is essential for substrate recognition by, and enzymatic activity of, the histone acetyltransferase enzyme Hat1. Therefore, it is speculated that the inability to detect HMT activity in preparations of recombinant E(z) protein is owing, in part, to the lack of the RbAp46/RbAp48. Another implication of the presence of RbAp proteins in the E(z) complex is that they might facilitate interaction with HDACs. During development, GAP proteins facilitate repression of the HOX genes. GAP proteins, such as Hunchback, are short-lived. Hunchback represses HOX genes by recruiting the Drosophila homolog of human Mi-2 protein, a constituent of the NuRD complex which also contains HDACs 1 and 2 and RbAp46/RpAp48. An interesting possibility is that HDACs or RbAp proteins initially recruited by Hunchback can later recruit PRC2 containing HMT activity via interaction with E(z). This may constitute a switch from short-term to long-term repression (Kuzmichev, 2002).

In support of this hypothesis, PRC2 contains E(z) and RbAp proteins. In addition, there is strong experimental evidence for an interaction between HDACs and E(z). One function of the E(z)-HDAC interaction is to deacetylate histones so that the E(z)-containing complex can methylate them. A similar mechanism was found to operate in yeast, in which methylation of H3-K9 by Clr4 requires deacetylation of H3-K9 and Lys 14 (K14) by Clr6 and Clr3, respectively. A similar mechanism is likely to operate in higher eukaryotes because acetylation and methylation are mutually exclusive marks, and methylation of H3-K9 by Suv39h1 requires deacetylation of this residue. The findings demonstrating two distinct ESC-E(z) complexes, one of which coelutes with HDAC1, raises the possibility that the PRC2 can transiently associate with an HDAC complex. This observation raises the possibility that PRC2 HDAC1 may be a highly-specialized complex dedicated to the methylation of H3-K27, which apparently is not acetylated in vivo in higher eukaryotes. Therefore, it is possible that these two different ESC-E(z) multiprotein complexes establish different marks on the histone H3 tail (Kuzmichev, 2002).

Establishment of Histone H3 methylation on the inactive X chromosome requires transient recruitment of Eed-Enx1 polycomb group complexes

The Eed-Enx1 Polycomb group complex has been implicated in the maintenance of imprinted X inactivation in the trophectoderm lineage in mouse. Recruitment of Eed-Enx1 to the inactive X chromosome (Xi) also occurs in random X inactivation in the embryo proper. Localization of Eed-Enx1 complexes to Xi occurs very early, at the onset of Xist expression, but then disappears as differentiation and development progress. This transient localization correlates with the presence of high levels of the complex in totipotent cells and during early differentiation stages. Functional analysis demonstrates that Eed-Enx1 is required to establish methylation of histone H3 at lysine 9 and/or lysine 27 on Xi and that this, in turn, is required to stabilize the Xi chromatin structure (Silva, 2003).

In eed-/- XX embryos Enx1 protein does not localize to Xi. This result is consistent with in vitro analysis demonstrating that the eed3354SB mutation disrupts a WD40 domain required for the interaction of Eed with Enx1. Thus, in the absence of functional Eed protein, the Enx1 HMTase cannot be directed toward specific targets. Eed/Enx1 complexes methylate H3-K9 and K27 in vitro, with a strong preference for K27. Failure to localize Enx1 clearly accounts for the absence of H3-K9/K27 methylation on Xi in eed-/- embryos (Silva, 2003).

It should be noted that, while previous studies have identified H3-K9 methylation as an early mark of silent chromatin on the inactive X chromosome, more recent data indicates that this could be attributable to cross-reactivity of di/tri-meH3-K9 antisera toward di/tri-meH3-K27. New antisera highly specific for di/tri-meH3-K9 detect pericentromeric heterochromatin, but not Xi, while the tri-meH3-K27 antibody used in this study detects Xi, but not pericentromeric heterochromatin. It is also possible that Xi is di/trimethylated both at K9 and K27 and that this configuration is not recognized by the novel di/trimethylH3-K9 antisera (Silva, 2003).

H3-K9/K27 methylation is shown to serve to stabilize the Xi chromatin structure. Thus, in eed-/- embryos, H3-K9 hypoacetylation and loss of H3-K4 methylation on Xi are not seen in a significant number of cells. Moreover, both the Xa and Xi alleles of two X-linked genes were seen to be expressed in a similar proportion of cells. These observations provide a basis for explaining reactivation of the X-linked GFP transgene in trophectoderm cells of eed-/- embryos. However, other studies have not observe reactivation of the GFP transgene in cells of the embryo proper, leading to the conclusion that Eed is required for the maintenance of imprinted, but not random, X inactivation. This difference can be accounted for by two factors. First, it is probable that reactivation of any given locus on Xi is sporadic and progressive. Since embryos exhibit mosaic expression of the XGFP transgene because of random X inactivation, a relatively small increase in the proportion of cells expressing the transgene, as observed for endogenous X-linked genes in this study, would be difficult to quantify. A second factor, is that additional levels of epigenetic silencing, for example, DNA methylation, play a more significant role in maintenance of X inactivation in cells of the embryo proper compared with the trophectoderm, potentially masking the effects of failure to establish H3-K9/K27 methylation (Silva, 2003 and references therein).

Initiation and propagation of X inactivation occurs coincident with cellular differentiation and involves a large nonprotein-coding RNA, the X inactive-specific transcript (Xist). Xist RNA spreads over the X chromosome and is thought to induce chromosome inactivation through recruitment of as yet unidentified silencing factors. The silencing function of Xist RNA has been shown to map to a short tandemly repeated element at the 5' end of the transcript (Silva, 2003 and references therein).

The banded localization of Eed-Enx1 complexes on Xi parallels that observed for Xist RNA. There could be an interaction, either direct or indirect, between Xist and the Eed-Enx1 complex. This view is further supported by observations reported in this study. (1) Recruitment of Eed-Enx1 complexes occurs extremely rapidly after the onset of stable Xist RNA accumulation in differentiating XX ES cells. (2) Eed-Enx1 recruitment occurs in response to expression of ectopic Xist RNA transgenes, both in undifferentiated XY ES cells and also in XYTg15 blastocysts. (3) Eed-Enx1 complexes are not recruited in response to expression of Xistinv mutant RNA, which fails to elicit X inactivation (Silva, 2003).

While the data support the idea that Eed-Enx1 complexes are recruited by Xist RNA, they do not prove that this interaction is direct. In fact, localization of Eed-Enx1 to Xi is first seen to occur at the morula stage, when imprinted X inactivation is initiated, whereas Xist RNA expression is detected earlier, in cleavage stage embryos. High levels of Eed-Enx1 complexes are available during the cleavage stages, suggesting that a factor(s) required for the interaction of the complex with Xist is absent (Silva, 2003).

Also relevant to the question of whether or not the Eed-Enx1 complex interacts directly with Xist RNA is the observation that eed-/- embryos are able to initiate X inactivation. This is, presumably, at least in part, attributable to recruitment of an HDAC complex that can deacetylate H3-K9. It is possible that components of the PcG complex other than Eed and Enx1 are still assembled in eed-/- embryos and that these confer H3-K9 HDAC activity. In the Drosophila ESC (Eed) protein, an equivalent mutation disrupts EZ (Enx1) recruitment but does not appear to ablate complex formation. An alternative scenario is that the complex responsible for H3-K9 deacetylation is a direct target of Xist RNA and that the Eed-Enx1 HMTase is then recruited by this complex. In support of this idea, heritable silencing at Drosophila homeotic genes is initiated by recruitment of the dMi2 protein, a component of an HDAC and chromatin remodeling complex, by Hunchback. Moreover, Eed protein can interact with type 1 HDACs, both in mammalian cells and in Drosophila. Thus, HDAC complexes may recruit Eed-Enx1 to target sites, including Xi, rather than vice versa (Silva, 2003).

It is interesting to note that the time during which high levels of Eed-Enx1 complex are available corresponds closely to the window of opportunity during which cells are responsive to Xist RNA. On the basis of this consideration, a model is proposed that speculates that Xist RNA recruits HDAC and Eed-Enx1 complexes, which lead to establishment of a primary level of chromatin silencing. Only during early differentiation stages would levels of these complexes be sufficient to establish chromosome-wide primary silencing. This would explain why expression of Xist in more differentiated cell types cannot induce X inactivation. It is further suggested that maintaining localization of the HDAC/Eed-Enx1 complexes is Xist RNA dependent. This would account for reversibility and Xist dependence of silencing in undifferentiated ES cells or during early differentiation stages. Extinguishing Xist expression would result in delocalization of HDAC/Eed-Enx1 complexes, loss of H3-K9/K27 methylation, increased H3-K9 acetylation, and, hence, reactivation of Xi (Silva, 2003).

To account for the fact that X inactivation does subsequently become stabilized and Xist independent, it is suggested that the chromatin modifications induced by the HDAC and Eed-Enx1 complex provide a template for recruitment of other silencing components. These could be responsible for further histone N-terminal modifications, for example, global H4 deacetylation, and also for DNA methylation at CpG islands and recruitment of macroH2A1.2. It should be noted that proteins of the PRC1 PcG group complex are not localized to Xi at any stage and are therefore unlikely to be involved in maintaining X inactivation in late development (Silva, 2003).

A key finding from these experiments is that recruitment of Eed-Enx1 to Xi is temporally regulated, rather than lineage specific, and that this, in turn, appears to relate to temporal regulation of overall levels of Eed and, to a lesser extent, Enx1 proteins. A similar expression profile has been reported for ESC (Eed) protein in Drosophila embryogenesis. It is striking that these factors are expressed at highest levels in totipotent or multipotent precursors and during early stages of differentiation. One interpretation of this observation is that Eed-Enx1 complexes are components of the machinery required to confer genome plasticity. Thus, like X inactivation during the window of opportunity, silent chromatin at other Eed-Enx1 targets may be reversible if the primary signal (for example Hunchback at homeotic loci in Drosophila) is removed. This would provide cells of the early embryo with the capacity to activate regions of the genome in response to specific differentiation signals, contrasting with the situation in differentiated cells, where heritable silencing is highly stable and is normally irreversible (Silva, 2003).

The Polycomb group (PcG) protein Eed is implicated in regulation of imprinted X-chromosome inactivation in extraembryonic cells but not of random X inactivation in embryonic cells. The Drosophila homolog of the Eed-Ezh2 PcG protein complex achieves gene silencing through methylation of histone H3 on lysine 27 (H3-K27), which suggests a role for H3-K27 methylation in imprinted X inactivation. This study demonstrates that transient recruitment of the Eed-Ezh2 complex to the inactive X chromosome (Xi) occurs during initiation of X inactivation in both extraembryonic and embryonic cells and is accompanied by H3-K27 methylation. Recruitment of the complex and methylation on the Xi depend on Xist RNA but are independent of its silencing function. Together, these results suggest a role for Eed-Ezh2-mediated H3-K27 methylation during initiation of both imprinted and random X inactivation and demonstrate that H3-K27 methylation is not sufficient for silencing of the Xi (Plath, 2003).

The Polycomb Ezh2 methyltransferase regulates muscle gene expression and skeletal muscle differentiation

The Ezh2 protein endows the Polycomb PRC2 and PRC3 complexes with histone lysine methyltransferase (HKMT) activity that is associated with transcriptional repression. Ezh2 expression is developmentally regulated in the myotome compartment of mouse somites and its down-regulation coincides with activation of muscle gene expression and differentiation of satellite-cell-derived myoblasts. Increased Ezh2 expression inhibits muscle differentiation, and this property is conferred by its SET domain, required for the HKMT activity. In undifferentiated myoblasts, endogenous Ezh2 is associated with the transcriptional regulator YY1. Both Ezh2 and YY1 are detected, with the deacetylase HDAC1, at genomic regions of silent muscle-specific genes: their presence correlates with methylation of K27 of histone H3. YY1 is required for Ezh2 binding because RNA interference of YY1 abrogates chromatin recruitment of Ezh2 and prevents H3-K27 methylation. Upon gene activation, Ezh2, HDAC1, and YY1 dissociate from muscle loci, H3-K27 becomes hypomethylated and MyoD and SRF are recruited to the chromatin. These findings suggest the existence of a two-step activation mechanism whereby removal of H3-K27 methylation, conferred by an active Ezh2-containing protein complex, followed by recruitment of positive transcriptional regulators at discrete genomic loci are required to promote muscle gene expression and cell differentiation (Cartetti, 2004).

These results indicate that Ezh2 is recruited at the chromatin of selected muscle regulatory regions by the transcriptional regulator YY1. Both can be coimmunoprecipitated from myoblast and not myotube cell extracts, and the proteins colocalize at the same muscle chromatin regions in a developmentally regulated manner. The interaction of endogenous YY1 and Ezh2 is likely to be mediated by the PcG EED protein because recombinant YY1 and Ezh2 do not directly associate. Previous reports have demonstrated a negative role for YY1 in regulating muscle gene expression through interaction with distinct nucleotides within the CarG-box [CC(A+T-rich)6GG], one of the DNA elements required for muscle-specific gene transcription. Transcriptional activation coincides with replacement of YY1 by the serum response factor (SRF), whose interaction with the CarG-box is required for muscle-specific transcription to proceed. These data suggest a two-step activation model of muscle gene expression. In the repressed state, YY1 recruits a complex containing both Ezh2 and HDAC1 that silences transcription through histone methylation (H3-K27) and deacetylation. Transcriptional activation entails the initial removal of the YY1-Ezh2-HDAC1 repressive complex and subsequent recruitment of the activators SRF (which replaces YY1) and the MyoD family of transcription factors and associated acetyltransferases. Since YY1 binding tolerates a substantial nucleotide heterogeneity in its DNA recognition sites, muscle and non-muscle-specific CarG-less regulatory regions may be also occupied and regulated in a similar manner. In contrast, Ezh2 does not appear to promiscuously regulate expression of all muscle-specific genes as indicated by the transient coexpression of Ezh2 and myogenin in the myotome of developing embryos and lack of Ezh2 recruitment and H3-K27 methylation at the myogenin promoter. Distinct histone methyltransferases and deacetylases have been shown to modify histones at the myogenin promoter (Cartetti, 2004 and references therein).

Polycomb EZH2 controls self-renewal and safeguards the transcriptional identity of skeletal muscle stem cells

Satellite cells (SCs) sustain muscle growth and empower adult skeletal muscle with vigorous regenerative abilities. This study reports that EZH2, the enzymatic subunit of the Polycomb-repressive complex 2 (PRC2), is expressed in both Pax7+/Myf5- stem cells and Pax7+/Myf5+ committed myogenic precursors and is required for homeostasis of the adult SC pool. Mice with conditional ablation of Ezh2 in SCs have fewer muscle postnatal Pax7+ cells and reduced muscle mass and fail to appropriately regenerate. These defects are associated with impaired SC proliferation and derepression of genes expressed in nonmuscle cell lineages. Thus, EZH2 controls self-renewal and proliferation, and maintains an appropriate transcriptional program in SCs (Juan, 2011).

Ezh2 is required for neural crest-derived cartilage and bone formation

The emergence of craniofacial skeletal elements, and of the jaw in particular, was a crucial step in the evolution of higher vertebrates. Most facial bones and cartilage are generated during embryonic development by cranial neural crest cells, while an osteochondrogenic fate is in more posterior neural crest cells. Key players in this process are Hox genes, which suppress osteochondrogenesis in posterior neural crest derivatives. How this specific pattern of osteochondrogenic competence is achieved remains to be elucidated. This study demonstrates that Hox gene expression and osteochondrogenesis are controlled by epigenetic mechanisms. Ezh2, which is a component of polycomb repressive complex 2 (PRC2), catalyzes trimethylation of lysine 27 in histone 3 (H3K27me3), thereby functioning as transcriptional repressor of target genes. Conditional inactivation of Ezh2 does not interfere with localization of neural crest cells to their target structures, neural development, cell cycle progression or cell survival. However, loss of Ezh2 results in massive derepression of Hox genes in neural crest cells that are usually devoid of Hox gene expression. Accordingly, craniofacial bone and cartilage formation is fully prevented in Ezh2 conditional knockout mice. The data indicate that craniofacial skeleton formation in higher vertebrates is crucially dependent on epigenetic regulation that keeps in check inhibitors of an osteochondrogenic differentiation program (Schwarz, 2014).

Silencing of human 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).

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 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).

Molecular architecture of human polycomb repressive complex 2

Polycomb Repressive Complex 2 (PRC2) is essential for gene silencing, establishing transcriptional repression of specific genes by tri-methylating Lysine 27 of histone H3, a process mediated by cofactors such as AEBP2 (Drosophila homolog: Jing). In spite of its biological importance, little is known about PRC2 architecture and subunit organization. This study presents the first three-dimensional electron microscopy structure of the human PRC2 complex bound to its cofactor AEBP2. Using a novel internal protein tagging-method, in combination with isotopic chemical cross-linking and mass spectrometry, all the PRC2 subunits and their functional domains have been localized and a detailed map of their interactions generated. The position and stabilization effect of AEBP2 suggests an allosteric role of this cofactor in regulating gene silencing. Regions in PRC2 that interact with modified histone tails are localized near the methyltransferase site, suggesting a molecular mechanism for the chromatin-based regulation of PRC2 activity (Ciferri, 2012).

Previous studies have shown that PRC2 favors di- and oligonucleosome substrates over mononucleosomes, octamers, or histone H3 peptides. Molecular explanations for this substrate preference have been largely hypothetical in the absence of any structural information. The positioning of the different subunits within the PRC2 structure suggests a model of how PRC2 could interact with a dinucleosome, by placing the regions interacting with histone tails in opposite sides of the complex, thus allowing interaction with two nucleosomes simultaneously, without any steric hindrance. A model of the structure suggests a possible arrangement illustrating this point that also agrees with the proposed binding of AEBP2 to nucleosomal DNA. In such arrangement, EED binding to one nucleosome would position the histone H3 tail from the second nucleosome in close proximity to the Ezh2 SET domain (see Mechanism and allosteric regulation of PRC2 during gene silencing). It is suggested that at loci of compact and repressed chromatin, H3K27-me3 marks are recognized by EED. This binding is signaled via the SANT domains to the SET domain increasing the methyl-transferase activity of Ezh2, strengthening the chromatin compaction. At loci of open and actively transcribed chromatin, H3K4me3 and H3K36me2,3 are recognized by the VEFS domain of Suz12 and transferred to Ezh2, with an allosteric regulation that blocks Ezh2's enzymatic activity (Ciferri, 2012).

In conclusion, the human PRC2 structure presented in this paper provides the first full picture of the molecular organization of this fundamental complex and offers an invaluable structural context to understand previous biochemical data. Furthermore, the functional mapping of different activities within the physical shape of the complex leads to novel, testable hypotheses on how PRC2 interacts with chromatin that should inspire future research of PRC2 function and regulation. Given the similarity in sequence between PRC2 components from different species, the molecular architecture that is seen for human PRC2 is expected to be conserved throughout higher eukaryotes (Ciferri, 2012).

Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs

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).

Polycomb group proteins Ezh2 and Rnf2 direct genomic contraction and imprinted repression in early mouse embryos

Genomic imprinting regulates parental-specific expression of particular genes and is required for normal mammalian development. How imprinting is established during development is, however, largely unknown. To address this question, the mouse Kcnq1 imprinted cluster, at which paternal-specific silencing depends on expression of the noncoding RNA Kcnq1ot1, was studied. Kcnq1ot1 was shown to be expressed from the zygote stage onward and to rapidly associates with chromatin marked by Polycomb group (PcG) proteins and repressive histone modifications, forming a discrete repressive nuclear compartment devoid of RNA polymerase II, a configuration also observed at the Igf2r imprinted cluster. In this compartment, the paternal Kcnq1 cluster exists in a three-dimensionally contracted state. In vivo the PcG proteins Ezh2 and Rnf2 are independently required for genomic contraction and imprinted silencing. It is proposed that the formation of a parental-specific higher-order chromatin organization renders imprint clusters competent for monoallelic silencing and assign a central role to PcG proteins in this process (Terranova, 2008).

Phosphorylation of the PRC2 component Ezh2 is cell cycle-regulated and up-regulates its binding to ncRNA

Ezh2 functions as a histone H3 Lys 27 (H3K27) methyltransferase when comprising the Polycomb-Repressive Complex 2 (PRC2). Trimethylation of H3K27 (H3K27me3) correlates with transcriptionally repressed chromatin. The means by which PRC2 targets specific chromatin regions is currently unclear, but noncoding RNAs (ncRNAs) have been shown to interact with PRC2 and may facilitate its recruitment to some target genes. This study shows that Ezh2 interacts with HOTAIR and Xist. Ezh2 is phosphorylated by cyclin-dependent kinase 1 (CDK1) at threonine residues 345 and 487 in a cell cycle-dependent manner. A phospho-mimic at residue 345 increased HOTAIR ncRNA binding to Ezh2, while the phospho-mimic at residue 487 was ineffectual. An Ezh2 domain comprising T345 was found to be important for binding to HOTAIR and the 5' end of Xist (Kaneko, 2010).

The results presented here demonstrate that PRC2 binding to HOTAIR (expressed from the HOXC cluster) and RepA (repeats found in Xist) ncRNAs is mediated through its Ezh2 component, and that phosphorylation of Ezh2-T345 up-regulates HOTAIR-binding activity. Given that phosphorylation at this site is cell cycle-regulated, it is speculated that PRC2 recruitment to chromatin, mediated through Ezh2 interaction with HOTAIR or RepA ncRNAs and presumably other ncRNAs, must be restricted to a tightly defined interval during the cell cycle (G2/M). Most importantly, the results establish that there are at least two populations of PRC2 complexes during the G2-M stages of the cell cycle. This is consistent with a model whereby PRC2 is recruited to specific genes to initiate repression as a function of its Ezh2 component being phosphorylated at T345, after which other PRC2 complexes then spread the repressing signature (H3K27me2/3). Of note, a recent study has also documented that human Ezh2 is phosphorylated at Thr 350 (murine T345) by CDK1, and, in agreement with the current findings, the report shows that this modification is ineffectual with respect to the integrity of the PRC2 complex and PRC2-mediated histone lysine methyltransferase activity. Instead, this study shows that mutant Ezh2 that is not subject to T350 phosphorylation results in down-regulated PRC2 recruitment, such that appropriate gene repression is thwarted. This report demonstrates that abrogation of this phosphorylation site within Ezh2 compromises Ezh2 interaction with ncRNAs, and this may bear directly on the mechanism by which PRC2 recruitment is impaired (Kaneko, 2010).

It has been demonstrated previously that the Eed component of PRC2 binds to trimethylated histone-repressive marks, but its binding to H3K27me3 in particular results in an allosteric effect that markedly increases the histone methyltransferase activity of its partner, Ezh2. Thus, PRC2 binding to the product of its activity increases its production of this mark. It is postulated that HOTAIR and RepA ncRNAs (and other ncRNAs) recruit PRC2 to initiate repression of target genes, and that this recruitment is enhanced by Ezh2 phosphorylation at T345. This proposed mechanism is consistent with only a small percentage of Ezh2 being phosphorylated at T345. If ncRNA species are responsible for targeting PRC2 to chromatin during G2/M, it is postulated that the recruited PRC2 would set the initial H3K27me3 mark. A larger number of PRC2 complexes, independent of their Ezh2 component being phosphorylated, would then propagate this mark upon their Eed component binding to the initial H3K27me3, with resultant allosteric activation of their Ezh2 activity (Kaneko, 2010).

An important question remaining is whether Ezh2 is the only component of PRC2 that binds to ncRNAs. These studies established that a 30-amino-acid domain of the PRC2-associated protein Jarid2 (see Drosophila Jarid2) also binds to ncRNAs. Additionally, a recent study suggested that the Suz12 subunit of PRC2 binds to nascent transcripts. It was postulated that this binding results in the halting of RNA polymerase II. Interestingly, it was postulated that Suz12 binding to nascent transcripts requires a unique stem-loop structure on the RNA. This structure is similar to the structure of RepA, and computer analysis of the amino acid sequence of Suz12 revealed a putative domain at its N terminus with a predicted RNA-binding domain. These observations collectively suggest that PRC2 recruitment to its target genes is mediated by ncRNA, different species of which likely bind to different PRC2 subunits. Whether specificity or affinity of PRC2 for its target genes is regulated by PRC2 binding through its component(s) to one family of ncRNA (specificity), or whether multiple subunits of PRC2 simultaneously bind different ncRNAs or domains within a ncRNA (affinity), remains to be established. Regardless, the studies described here are beginning to shed light on the role of ncRNAs in mediating the recruitment of mammalian PRC2 to its target genes (Kaneko, 2010).

Jarid2 and PRC2, partners in regulating gene expression.

The Polycomb group proteins foster gene repression profiles required for proper development and unimpaired adulthood, and comprise the components of the Polycomb-Repressive Complex 2 (PRC2) including the histone H3 Lys 27 (H3K27) methyltransferase Ezh2. How mammalian PRC2 accesses chromatin is unclear. This study found Jarid2 associates with PRC2 and stimulates its enzymatic activity in vitro. Jarid2 contains a Jumonji C domain, but is devoid of detectable histone demethylase activity. Instead, its artificial recruitment to a promoter in vivo resulted in corecruitment of PRC2 with resultant increased levels of di- and trimethylation of H3K27 (H3K27me2/3). Jarid2 colocalizes with Ezh2 and MTF2, a homolog of Drosophila Pcl, at endogenous genes in embryonic stem (ES) cells. Jarid2 can bind DNA and its recruitment in ES cells is interdependent with that of PRC2, as Jarid2 knockdown reduced PRC2 at its target promoters, and ES cells devoid of the PRC2 component EED are deficient in Jarid2 promoter access. In addition to the well-documented defects in embryonic viability upon down-regulation of Jarid2, ES cell differentiation is impaired, as is Oct4 silencing (Li, 2010).

Since the first characterization of the PRC2 core complex, the subsequent, persuasive evidence supports that PRC2 is actually a family of complexes whose composition varies during development, as a function of cell type, or even from one promoter to another. This study identified two new components that interact with PRC2: MTF2 and Jarid2. These analyses of the proteins that interact with the PRC2 complex initiated with transformed cells. Yet it has become clear that interactions observed using transformed cells might be specific to such cells, and not a determinant to the integrity of a normal organism. Thus, studies of a developmentally relevant process was incorporated and it was confirmed that the interactions observed between PRC2 and Jarid2 were of consequence to the developmental program (Li, 2010).

MTF2 is a paralog of Drosophila Pcl. PHF1, another mammalian paralog of Pcl, is required for efficient H3K27me3 and gene silencing in HeLa cells. Although PHF1 appears dispensable for PRC2 recruitment in HeLa cells, work in Drosophila has suggested that the absence of Pcl could impair PRC2 gene targeting. It is possible that the other paralogs of Pcl (MTF2 and PHF19) exhibit a role that is partially redundant with PHF1 function and thereby maintain PRC2 recruitment upon its knockdown. Pcl and its mammalian paralogs contain two PHD domains and a tudor domain, domains reported to potentially recognize methylated histones. Although the ability of Pcl to specifically bind modified histone has not been elucidated to date, it is tempting to speculate that the PHD and tudor domains could target Pcl to specific chromatin regions. Its presence would then stabilize PRC2 recruitment and promote its enzymatic activity. In support of this hypothesis, it was observed that, whereas Ezh2 targeting is severely impaired in Eed-/- ES cells, MTF2 recruitment is affected in a promoter-dependent manner and to a lesser extent than that of Ezh2. This observation suggests that MTF2 gene targeting could be partially independent of PRC2 (Li, 2010).

The exact function of Jarid2 is more enigmatic. Indeed, Jarid2 is a member of a family of enzymes capable of demethylating histones. However, Jarid2 is devoid of the amino acids required for iron and αKG binding, and consequently is unable to catalyze this reaction. It is considered that Jarid2 could act as a dominant negative and inhibit the activity of other histone demethylases; however, coexpression of Jarid2 with, for instance, SMCX did not affect H3K4me3 demethylation. Jarid2 has two domains that could potentially bind DNA: the ARID domain and a zinc finger. Although the ARID domain of Jarid2 was reported to bind DNA, band shift assay suggests that other parts of the Jarid2 C terminus (potentially a zinc finger) are also important for binding to DNA. The SELEX experiment performed with the full-length Jarid2 did not allow identification of any sequence-specific DNA binding, but did result in a slight enrichment of GC-rich DNA sequences. Importantly, it was found that the N-terminal part of Jarid2 could robustly stimulate PRC2-Ezh2 enzymatic activity on nucleosomes. A knockdown of Jarid2 decreased the enrichment of PRC2 at its target genes. Conversely, overexpression of a Gal4-Jarid2 chimera recruited PRC2 at a stably integrated reporter and increased PRC2 enrichment at its target genes, supporting the hypothesis that Jarid2 contributes to PRC2 recruitment (Li, 2010).

In the case of Drosophila, PRE (Polycomb group response element) sequences have been described, and PRC2 access to chromatin is expected to involve the concerted action of several distinct and specific DNA-binding proteins that interact directly or indirectly with PRC2. However, these same DNA-binding factors, or even a combination thereof, are also found at active genes devoid of PRC2. What distinguishes PRE sequences harboring PRC2 from active genes is still not clear. During the evolution from Drosophila to mammals, only a few of the DNA-binding factors that bind PREs (Dsp1 and Pho) are conserved. Either PRC2 recruitment in mammals involves other mechanisms, or distinct transcription factors have emerged to stabilize PRC2 at its target genes. A recent study has identified a presumed mammalian PRE; however, the role of this putative PRE at the endogenous locus that is enriched for PRC2 is not reproduced when the element is integrated upstream of a transgene, as PRC2 is absent. Of note, whereas DNA-binding proteins are likely to play an important role for PRC2 recruitment in mammals, some studies have now suggested that long noncoding RNA could also be involved in this process. These observations together suggest that the recruitment of PRC2 to target genes is complex and requires more than one factor. These findings suggest that the DNA-binding activity of Jarid2 is one such factor, but its affinity for DNA is low and likely requires the help of other factors (Li, 2010).

A critical issue at this juncture is whether or not the composition of PRC2 changes during development. This study reports that Jarid2 interacts with PRC2, but its expression, unlike the PRC2 core components, seems to be restricted to some cell lines. In agreement with previous gene expression profiles that monitored mRNA levels during the reprogramming of mouse embryonic fibroblast cells into ES cells, it is observed that Jarid2 expression is higher in undifferentiated ES cells and decreases upon differentiation. Polycomb target genes are enriched with the H2A variant H2A.Z in undifferentiated ES cells; furthermore, H2A.Z and PRC2 targeting are interdependent in these cells. This result suggests that PRC2 recruitment might involve distinct mechanisms in ES cells and differentiated cells. It is possible that Jarid2 somehow contributes to this specificity (Li, 2010).

Knockdown of Jarid2 in undifferentiated ES cells does not give rise to an obvious phenotype; gene expression patterns appear to be only moderately affected, and cell proliferation is unchanged. In contrast, when cells are induced to differentiate, a process that entails dramatic changes in gene expression, impairments were observed as a function of Jarid2 knockdown. Interference with Jarid2 resulted in a failure to accurately coordinate the expression of genes required for the differentiation process, consistent with the previous report on Suz12 knockout cells. Instead of the requisite silencing of OCT4 and Nanog loci that occurs upon normal differentiation, each of which become enriched in H3K27me3, Jarid2 knockdown prevented such H3K27 methylation at these genes, and this correlated with their delayed repression. Thus, the Jumonji family of proteins that usually exhibits demethylase activity that might function in opposition to the role mediated by PRC2 contains the member Jarid2 that is devoid of such activity and instead facilitates the action of PRC2 through enabling its access to chromatin (Li, 2010).

pRB family proteins are required for H3K27 trimethylation and Polycomb repression complexes binding to and silencing p16INK4a tumor suppressor gene

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).

Polycomb limits the neurogenic competence of neural precursor cells to promote astrogenic fate transition

During neocortical development, neural precursor cells (NPCs, or neural stem cells) produce neurons first and astrocytes later. Although the timing of the fate switch from neurogenic to astrogenic is critical for determining the number of neurons, the mechanisms are not fully understood. This study shows that the polycomb group complex (PcG) restricts neurogenic competence of NPCs and promotes the transition of NPC fate from neurogenic to astrogenic. Inactivation of PcG by knockout of the Ring1B or Ezh2 gene or Eed knockdown prolonged the neurogenic phase of NPCs and delayed the onset of the astrogenic phase. Moreover, PcG was found to repress the promoter of the proneural gene neurogenin1 in a developmental-stage-dependent manner. These results demonstrate a role of PcG: the temporal regulation of NPC fate (Hirabayashi, 2009).

During neocortical development, the neurogenic phase normally persists for a limited time period (about 11 cell cycles on average in the mouse neocortex, and this restricted period may be a major parameter in determining the final number of neurons produced during development. PcG proteins contribute to the termination of the neurogenic phase, which normally takes place between E18.5 and E19.0 in the neocortex. Indeed, birthdating analysis showed that cells labeled by BrdU at E19.0 still contributed to upper-layer neurons at P6.5 in Ring1B- or Ezh2-deficient mice but not in control mice. Interestingly, the excess neurons produced at around the end of neurogenic phase appear to be eliminated (probably by cell death) later during postnatal development in both wild-type and Ring1B-deficient mice, suggesting that these late-born excess neurons fail to integrate into the appropriate neuronal networks and therefore cannot be supported by activity/target-dependent survival signals. In other words, the correct timing of the end of neurogenesis might help avoid production of excess (unnecessary, undesirable) neurons (Hirabayashi, 2009).

The roles of PcG in ES cells strikingly differ from those in NPCs. Components of the PcG are known to localize and repress a variety of target genes and play an essential role in the maintenance of pluripotency of ES cells by suppressing differentiation into multiple lineages. A previous report has shown that different arrays of genes are labeled with H3K27me3 in ES cells and ES-derived neuronal progenitors, suggesting that PcG targets are different between these cell types. Indeed, Ring1B deletion in late-stage neocortical NPCs preferentially increases the expression of genes associated with neuronal differentiation/development over those associated with other lineages based on microarray analyses, whereas developmental genes in multiple lineages are derepressed by Ring1B deletion in ES cells (Hirabayashi, 2009).

The fate restriction of ES cells during differentiation is accompanied by diminished occupancy of H3K27me3 at specific 'bivalent' gene promoters involved in the corresponding differentiation process, in contrast to the increased H3K27me3 at ngn loci during fate restriction of NPCs. Moreover, deletion of Ring1B or Suz12 in ES cells results in the loss of neurogenic capacity, whereas deletion of Ring1B in the late NPCs extended neurogenic capacity. These observations further support the difference of PcG functions between these cell types. Thus, this study has unveiled an in vivo role of PcG, namely, temporal (stage-dependent) fate conversion of multipotent progenitors during development (Hirabayashi, 2009).

This study found that PcG is responsible for ngn1suppression in late-stage NPCs. Since misexpression of ngn1 extends neurogenesis in late-stage NPCs, it is clear that suppression of ngn1 is a prerequisite for the neuronal-to-glial transition of NPC fate. Therefore, the suppression of ngn1 by PcG may partly account for the PcG restriction of neurogenic potential and transition to gliogenesis in the neocortex. However, it is unclear whether PcG also regulates other genes with similar functions. Ngn2 might be such a target, given that the level of H3K27me3 increases at the ngn2 locus in the late stage of neocortical NPCs. However, Ring1B deletion by itself did not cause much increase in ngn2 expression, suggesting that additional mechanisms might account for suppression of ngn2 at late stages of neocortical development (Hirabayashi, 2009).

Besides ngn1, no other proneural genes were found that were greatly upregulated by Ring1B deletion in neocortical NPCs. For instance, there was no elevation in neurogenic genes expressed in the neocortex such as Pax6, Math1, and Mash1. Among the basic helix-loop-helix or homeodomain-containing transcription factors expressed in brain, Dlx2 was significantly derepressed by Ring1B deletion. Although Dlx2 can contribute to neurogenesis in the ventral telencephalon in some contexts, it is not thought that this gene is responsible for the PcG suppression of neurogenesis in the neocortex, since Dlx2 is associated with differentiation of GABAergic interneurons rather than the glutamatergic neurons observed in the Ring1B-deficient mice. Nonetheless, a recent report has shown that the chromatin remodeling factor Mll1 suppresses the accumulation of H3K27me3 at the dlx2 locus and thus confers neurogenic potential in the adult neural stem cells. This implies a very interesting possibility that PcG participates in a common mechanism that suppresses neurogenic potential in both dorsal and ventral telencephalon in the late stages of development, and a small NPC population that escapes from this mechanism by Mll1 is selected to become adult neural stem cells that continue to produce neurons for lifetime (Hirabayashi, 2009).

Although knockdown of Bmi1, a component of PRC1, resulted in NPC loss and brain size reduction in a previous study, these phenotypes were not observed in the Ring1B-deficient mouse, implicating that Bmi1 and Ring1B may form distinct complexes that exert different functions. These functions of Bmi1 may not be related to PRC2, since it was found that brain size reduction was not seen in mice deficient for Ezh2 in the central nervous system, although H3K27me3 modification was barely seen in NPCs from these mice. Functional differences between Bmi1 and PRC2 have also been suggested in the hematopoietic system and tumors. For example, Bmi1 deletion reduced the numbers of myeloid and preB cells, whereas Eed deletion increased these cell types (Hirabayashi, 2009).

The levels of H3K27me3 gradually increase over time at the ngn1 promoter, and it is plausible that, at a certain threshold, their chromatin state becomes inactivated by PRC1, resulting in the suppression of ngn expression and the transition of NPC fate. It is proposed that the developmental-stage-dependent accumulation of H3K27me3 at specific gene loci functions as a timer to drive cell fate switching. Exactly how this accumulation occurs is not clear at present but might involve either a global increase in PcG activity or local recruitment of PcG to the ngn1 locus in late stages of neocortical NPCs. In either case, further analysis of this accumulation may shed light on the mechanism that underlies the developmental regulation of differentiation potential (Hirabayashi, 2009).

A genetic approach to the recruitment of PRC2 at the HoxD locus

Polycomb group (PcG) proteins are essential for the repression of key factors during early development. In Drosophila, the polycomb repressive complexes (PRC) associate with defined polycomb response DNA elements (PREs). In mammals, however, the mechanisms underlying polycomb recruitment at targeted loci are poorly understood. This study used an in vivo approach to identify DNA sequences of importance for the proper recruitment of polycomb proteins at the HoxD locus. Various genomic re-arrangements of the gene cluster do not strongly affect PRC2 recruitment, and relatively small polycomb interacting sequences appear necessary and sufficient to confer polycomb recognition and targeting to ectopic loci. In addition, a high GC content, while not sufficient to recruit PRC2, may help its local spreading. PRC2 recruitment over Hox gene clusters in embryonic stem cells is important for their subsequent coordinated transcriptional activation during development. It is concluded that a range of low affinity sequences synergize to recruit PRCs over the gene cluster, which makes this process very robust and resistant to genetic perturbations (Schorderet, 2013).

Mammalian Enhancer of zeste homologs and cancer

The Polycomb Group Protein EZH2 is a transcriptional repressor involved in controlling cellular memory and has been linked to aggressive prostate cancer. The functional role of EZH2 in cancer cell invasion and breast cancer progression has been investigated. EZH2 transcript and protein are consistently elevated in invasive breast carcinoma compared with normal breast epithelia. Tissue microarray analysis, which included 917 samples from 280 patients, demonstrate that EZH2 protein levels are strongly associated with breast cancer aggressiveness. Overexpression of EZH2 in immortalized human mammary epithelial cell lines promotes anchorage-independent growth and cell invasion. EZH2-mediated cell invasion requires an intact SET domain and histone deacetylase activity. This study provides compelling evidence for a functional link between dysregulated cellular memory, transcriptional repression, and neoplastic transformation (Kleer, 2003).

Prostate cancer is a leading cause of cancer-related death in males and is second only to lung cancer. Although effective surgical and radiation treatments exist for clinically localized prostate cancer, metastatic prostate cancer remains essentially incurable. Through gene expression profiling, the polycomb group protein enhancer of zeste homolog 2 (EZH2) is found to be overexpressed in hormone-refractory, metastatic prostate cancer. Small interfering RNA (siRNA) duplexes targeted against EZH2 reduce the amounts of EZH2 protein present in prostate cells and also inhibit cell proliferation in vitro. Ectopic expression of EZH2 in prostate cells induces transcriptional repression of a specific cohort of genes. Gene silencing mediated by EZH2 requires the SET domain and is attenuated by inhibiting histone deacetylase activity. Amounts of both EZH2 messenger RNA and EZH2 protein are increased in metastatic prostate cancer; in addition, clinically localized prostate cancers that express higher concentrations of EZH2 show a poorer prognosis. Thus, dysregulated expression of EZH2 may be involved in the progression of prostate cancer, as well as being a marker that distinguishes indolent prostate cancer from those at risk of lethal progression (Varambally, 2002).

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).

Control of developmental regulators by Polycomb in human embryonic stem cells; Targets of the Polycomb Repressive Complex 2 (PRC2) subunit SUZ12

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 has been mapped across the entire nonrepeat portion of the genome in human embryonic stem (ES) cells. 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).

It was striking that SUZ12 occupied many families of genes that control development and transcription. These included 39 of 40 of the homeotic genes found in the HOX clusters and the majority of homeodomain genes. SUZ12 bound homeodomain genes included almost all members of the DLX, IRX, LHX, and PAX gene families, which regulate early developmental steps in neurogenesis, hematopoiesis, axial patterning, tissue patterning, organogenesis, and cell-fate specification. SUZ12 also occupied promoters for large subsets of the FOX, SOX, and TBX gene families. The forkhead family of FOX genes is involved in axial patterning and tissue development from all three germ layers. Mutations in members of the SOX gene family alter cell-fate specification and differentiation and are linked to several developmental diseases. The TBX family of genes regulates a wide variety of developmental processes such as gastrulation, early pattern formation, organogenesis, and limb formation. Thus, the genes preferentially bound by SUZ12 have functions that, when expressed, promote differentiation. This is likely to explain, at least in part, why PRC2 is essential for early development and ES cell pluripotency (Lee, 2006).

A remarkable feature of PRC2 binding at most genes encoding developmental regulators was the extensive span over which the regulator occupied the locus. For the majority (72%) of bound sites across the genome, SUZ12 occupied a small region of the promoter similar in size to regions bound by RNA polymerase II. For the remaining bound regions, SUZ12 occupancy encompassed large domains spanning 2–35 kb and extending from the promoter into the gene. A large portion of genes encoding developmental regulators (72%) exhibited these extended regions of SUZ12 binding. In some cases, binding encompassed multiple contiguous genes. For instance, SUZ12 binding extended ~100 kb across the entire HOXA, HOXB, HOXC, and HOXD clusters but did not bind to adjacent genomic sequences, yielding a highly defined spatial pattern. In contrast, clusters of unrelated genes, such as the interleukin 1-β cluster, were not similarly bound by SUZ12. Thus, genes encoding developmental regulators showed an unusual tendency to be occupied by PRC2 over much or all of their transcribed regions (Lee, 2006).

Previous studies have noted that many highly conserved noncoding elements of vertebrate genomes are associated with genes encoding developmental regulators. Given SUZ12's strong association with this class of genes, the possibility that SUZ12 bound regions are associated with these highly conserved elements was investigated. Inspection of individual genes suggested that SUZ12 occupancy was associated with regions of sequence conservation. Eight percent of the approximately 1,400 highly conserved noncoding DNA elements were found to be associated with the SUZ12 bound developmental regulators. Using entries from the PhastCons database of conserved elements (Siepel, 2005), it was found that SUZ12 occupancy of highly conserved elements was highly significant. Since PRC2 has not been shown to directly bind DNA sequences, it is expected that specific DNA binding proteins occupy the highly conserved DNA sequences and may associate with PRC2, which spreads and occupies adjacent chromatin. Thus, the peaks of SUZ12 occupancy might not be expected to precisely colocate with the highly conserved elements, even if these elements are associated with PRC2 recruitment (Lee, 2006).

The observation that OCT4, SOX2, and NANOG are bound to a significant subset of developmental genes occupied by PRC2 supports a link between repression of developmental regulators and stem cell pluripotency. Like PRC2, OCT4 and NANOG have been shown to be important for early development and ES cell identity. It is possible, therefore, that inappropriate regulation of developmental regulators that are common targets of OCT4, NANOG, and PRC2 contributes to the inability to establish ES cell lines in OCT4, NANOG, and EZH2 mutants (Lee, 2006).

Polycomb group protein ezh2 controls actin polymerization and cell signaling

Polycomb group protein Ezh2, one of the key regulators of development in organisms from flies to mice, exerts its epigenetic function through regulation of histone methylation. This study reports the existence of the cytosolic Ezh2-containing methyltransferase complex, and the function of this complex has been tied to regulation of actin polymerization in various cell types. Genetic evidence supports the essential role of cytosolic Ezh2 in actin polymerization-dependent processes such as antigen receptor signaling in T cells and PDGF-induced dorsal circular ruffle formation in fibroblasts. Revealed function of Ezh2 points to a broader usage of lysine methylation in regulation of both nuclear and extra-nuclear signaling processes (Su, 2005; full text of article).

Polycomb protein Ezh2 regulates pancreatic beta-cell Ink4a/Arf expression and regeneration in diabetes mellitus

Proliferation of pancreatic islet beta cells is an important mechanism for self-renewal and for adaptive islet expansion. Increased expression of the Ink4a/Arf locus, which encodes the cyclin-dependent kinase inhibitor p16(INK4a) and tumor suppressor p19(Arf), limits beta-cell regeneration in aging mice, but the basis of beta-cell Ink4a/Arf regulation is poorly understood. This study shows that Enhancer of zeste homolog 2 (Ezh2), a histone methyltransferase and component of a Polycomb group (PcG) protein complex, represses Ink4a/Arf in islet beta cells. Ezh2 levels decline in aging islet beta cells, and this attrition coincides with reduced histone H3 trimethylation at Ink4a/Arf, and increased levels of p16(INK4a) and p19(Arf). Conditional deletion of beta-cell Ezh2 in juvenile mice also reduced H3 trimethylation at the Ink4a/Arf locus, leading to precocious increases of p16(INK4a) and p19(Arf). These mutant mice had reduced beta-cell proliferation and mass, hypoinsulinemia, and mild diabetes, phenotypes rescued by germline deletion of Ink4a/Arf. beta-Cell destruction with streptozotocin in controls led to increased Ezh2 expression that accompanied adaptive beta-cell proliferation and re-establishment of beta-cell mass; in contrast, mutant mice treated similarly failed to regenerate beta cells, resulting in lethal diabetes. The discovery of Ezh2-dependent beta-cell proliferation revealed unique epigenetic mechanisms underlying normal beta-cell expansion and beta-cell regenerative failure in diabetes pathogenesis (Chen, 2009).

Ezh2 regulates anteroposterior axis specification and proximodistal axis elongation in the developing limb

Specification and determination (commitment) of positional identities precedes overt pattern formation during development. In the limb bud, it is clear that the anteroposterior axis is specified at a very early stage and is prepatterned by the mutually antagonistic interaction between Gli3 and Hand2. There is also evidence that the proximodistal axis is specified early and determined progressively. Little is known about upstream regulators of these processes or how epigenetic modifiers influence axis formation. Using conditional mutagenesis at different time points, this study shows that the histone methyltransferase Ezh2 is an upstream regulator of anteroposterior prepattern at an early stage. Mutants exhibit posteriorised limb bud identity. During later limb bud stages, Ezh2 is essential for cell survival and proximodistal segment elongation. Ezh2 maintains the late phase of Hox gene expression and cell transposition experiments suggest that it regulates the plasticity with which cells respond to instructive positional cues (Wyngaarden, 2011).


Enhancer of zeste: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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