extra sexcombs


EVOLUTIONARY HOMOLOGS

A yeast Esc homolog

The yeast transcriptional repressor Tup1, a potential homolog of Drosophila Extra sexcombs, contains seven WD repeats that interact with the DNA-binding protein alpha2. Mutations have been identified in Tup1 that disrupt this interaction. The positions of the amino acids changed by these mutations are consistent with Tup1 being folded into a seven-bladed propeller like that formed by another WD repeat-containing protein, the beta subunit of the heterotrimeric G protein used in signal transduction. These results also indicate that the interaction between Tup1 and alpha2 resembles the interaction between Gbeta and G alpha, suggesting that a similar structural interface is formed by WD repeat proteins used in both transcriptional regulation and signal transduction (Komachi, 1997).

Repression of a-cell specific gene expression in yeast alpha cells requires MAT alpha 2 and MCM1, as well as two global repressors, SSN6 and TUP1, the yeast homolog of ESC. Previous studies demonstrated that nucleosomes positioned adjacent to the alpha 2/MCM1 operator in alpha cells directly contribute to repression. To investigate the possibility that SSN6 and TUP1 provide a link between MAT alpha 2/MCM1 and neighboring histones, nucleosome locations were examined in mutant ssn6 and tup1 alpha cells. In both cases, nucleosome positions downstream of the operator were disrupted, and the severity of the disruption correlated with the degree of derepression. Nevertheless, the observed changes in chromatin structure were not dependent on transcription. These data strongly indicate that SSN6 and TUP1 directly organize repressive regions of chromatin (Cooper, 1994).

Invertebrate Esc homologs

Based on the results of mutational studies, the WD repeats appear to be essential for ESC protein to function as a repressor of homeotic genes. WD repeats may mediate interactions between ESC and other Polycomb Group proteins, recruiting still other proteins to their target genes, perhaps by additional interactions with transiently expressed repressors such as hunchback. The ESC protein is highly conserved between D. melanogaster and D. virulis, particularly its WD motifs. The high degree of conservation suggests that each of the WD repeats in the ESC protein is functionally specialized and that this specialization has been highly conserved during evolution. The highly charged N-terminus of ESC exhibits the greatest divergence, but even these differences are conservative of its predicted physical properties. These observations suggest that the ESC protein is functionally compact, nearly every residue making an important contribution to its function (Sathe, 1995b).

Four Caenorhabditis elegans genes, mes-2, mes-3, mes-4 and mes-6, are essential for the normal proliferation and viability of the germline. Mutations in these genes cause a maternal-effect sterile (i.e. mes) or grandchildless phenotype. The mes-6 gene is in an unusual operon, the second example of this type of operon found in C. elegans: mes-6 encodes the nematode homolog of Extra sex combs, a WD-40 protein in the Polycomb group in Drosophila. mes-2 encodes another Polycomb group protein, Enhancer of zeste. Consistent with the known role of Polycomb group proteins in regulating gene expression, MES-6 is a nuclear protein. It is enriched in the germline of larvae and adults and is present in all nuclei of early embryos. Molecular epistasis results predict that the MES proteins, like Polycomb group proteins in Drosophila, function as a complex to regulate gene expression. Database searches reveal that there are considerably fewer Polycomb group genes in C. elegans than in Drosophila or vertebrates. MES-6 and MES-2 are the only recognizable Pc-G products in the C. elegans genome. These studies suggest that their primary function is in controlling gene expression in the germline and ensuring the survival and proliferation of that tissue (Korf, 1998).

Sequence analysis of the mes-6 cDNA has revealed that it encodes a 459 amino acid protein similar to Drosophila Extra sex combs (Esc) and the murine homolog of Esc, termed Eed for Embryonic ectodermal development. Similarity searches involving ~90% of the entire worm genomic sequence indicates that Esc is more similar to MES-6 than to any other sequence in the worm genome, and therefore, MES-6 likely represents the worm ortholog of Esc. MES-6, Esc and Eed share regions of sequence similarity that line up in register along the entire length of the proteins. These regions contain WD-40 repeats, which are thought to be involved in protein-protein interactions. WD-40 repeats within a protein are often very dissimilar. In contrast, positionally equivalent repeats in homologous proteins are more highly conserved. Indeed, positionally equivalent WD-40 repeats in MES-6, Esc and Eed are more similar to each other than to any non-equivalent repeats compared within or between proteins. MES-6 also appears to contain seven WD-40 repeats, so it also may adopt a propeller-like tertiary stucture. Interestingly, the gly-to-glu change found in the bn66 allele of mes-6 maps to one of the loops that is predicted to project from the top of the Esc propeller. The this region is likely to be important for protein-protein interactions based on sequence conservation among insect esc genes. (Korf, 1998).

As predicted by the similarity of MES-6 to Esc, MES-6 is localized in nuclei. In wild-type adults, MES-6 staining is most prominent in the germline, but is also detectable in intestinal nuclei. A maternal load of protein is seen in the nuclei of oocytes. In early embryos, MES-6 is present in the nuclei of all cells. As embryogenesis proceeds, staining gradually fades in somatic cells. In late embryos and L1 larvae, MES-6 remains faintly visible in a number of cell types, including the intestine, but is most prominent in Z2 and Z3, the primordial germ cells. Nuclear staining is diminished or reduced below detectability in worms bearing any of the four mutant alleles of mes-6 (Korf, 1998).

Do Pc-G genes serve an essential role in germline development in Drosophila, in addition to their well-known roles in somatic development? Since Pc-G mutations are generally lethal, addressing this question has required generating mutant germline clones by pole cell transplantation or by induction of mitotic recombination in the germline. Most of the Pc-G genes tested appear not to be essential for female germline development. E(z) does appear to be essential in the germline, since certain temperature-sensitive alleles of E(z) are sterile: transplanted pole cells mutant for a null allele of E(z) generate germlines indistinguishable from those in the temperature-sensitive alleles, indicating that the defect is germline autonomous (A. Shearn, personal communication to Korf, 1998). The strict maternal-effect sterility caused by mes mutations reveals that a maternal supply of wild-type mes gene product is both necessary and sufficient for normal germline development in the next generation. With this in mind, it has been hypothesized that the MES proteins are required to maintain a germline-specific organization of chromatin from one generation to the next, and that this chromatin state is essential to initiate the correct pattern of gene expression in the germline. This hypothesis integrates both the maintenance function of Pc-G proteins and the initiation function and germline specificity suggested by the maternal-effect sterile phenotype of mes mutants (Korf, 1998).

Recent results from Kelly and Fire (1998) provide strong support for this hypothesis. Kelly and Fire observed that ‘housekeeping’ genes introduced into worms as transgenes and present in many tandem copies in extrachromosomal arrays are efficiently expressed in somatic cells but are specifically silenced in the germline of wild-type worms. Remarkably, gene expression is desilenced in the germline of animals mutant for any of the four mes genes. Gene expression also can be desilenced in the germline of wild-type worms by placing the test genes in the context of complex DNA in the array. These findings indicate that gene expression in the germline is dependent on chromatin context and wild-type MES function, supporting the notion that the MES proteins repress gene expression through an influence on chromatin state. The following model of germline establishment and protection of immortality is presented (Korf, 1998).

The early germline requires the silencing of gene expression that takes two forms. As the germline is being set apart from the soma in the early embryo, maternally supplied PIE-1, a transcriptional repressor that keeps somatically expressed genes turned off in the germline of early PIE-1 embryos keeps the germline blastomeres transcriptionally quiescent, while the surrounding somatic cells respond to differentiation factors and become transcriptionally active. During this period, maternally supplied MES proteins provide an underlying 'memory' of the germline state of chromatin, by maintaining nucleosomes or higher order chromatin in a particular conformation. When PIE-1 decays and the primordial germ cells become transcriptionally active, the MES-regulated chromatin conformation maintains repression of certain genes or regions of the genome and selectively allows initiation of expression of germline-required genes. In the absence of a functional MES system, death of the germline could be due to ectopic expression of genes that are normally kept silent in the germline, or alternatively, could be due to altered levels or timing of expression of genes that are normally expressed in the germline. The chromosomal binding sites for the predicted MES complex are currently unknown. However, several observations suggest that a key feature of these sites may be the number of copies of target sequences. MES+ function participates in keeping repetitive arrays of transgenes silenced in the germline, while non-repetitive arrays escape this silencing (Kelly, 1998). Somewhat analogously, Drosophila Pc-G proteins repress expression of transgenes that are present in multiple copies (Pal-Bhadra, 1997). Interestingly, the requirement for MES+ function is sensitive to chromosome dosage: animals with three X chromosomes absolutely require MES+ function for fertility, while animals with one X show a reduced requirement. This sensitivity to X-chromosome dosage may indicate participation of the MES proteins in dosage compensation in the germline. Future identification of MES targets will enhance understanding of the mechanism of Pc-G/MES control of gene expression and how this control contributes to the germline/soma dichotomy in C. elegans (Korf, 1998 and references).

Evolutionary conservation of the Esc-Enhancer of zeste 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).

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).

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).

ESC in fish: The ESC-E(Z) complex participates in the hedgehog signaling pathway

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).

EED mutation

A classic mouse mutation, eed (embryonic ectoderm development) is a highly conserved homolog of esc. Mutants for a null allele of eed display disrupted anterior-posterior patterning of the primitive streak during gastrulation. Mutant embryos lack a node, notochord and somites, and there is no neural induction. In contrast to absence of anterior structures, extra-embryonic mesoderm is abundant. Mice carrying a hypomorphic eed mutation exhibit posterior transformations along the axial skeleton. EED consists of 441 amino acids and contains five tandemly repeated WD motifs. Sequence comparison with ESC reveals a 55% identity, with conservation between WD motifs almost as high as that within the WD motifs. The high evolutionary conservation spans 83% of the EED sequence, and is not interrupted by a single gap or insertion. Mutation in eed causes spatial derepression of Hox genes. Disruption of the unsegmented primitive streak during gastrulation reflects eed involvement in regulating anterior-posterior patterning before segmentation. Homeodomain transcription factor Evx1 (the mouse homolog of Even-skipped) is downstream of eed. It is conceivable the eed may be required to establish and/or maintain an Evx1 expression gradient, which in turn may impart positional information to mesodermal cells leaving the primitive streak (Schumacher, 1996)

Similar to Drosophila, murine Polycomb-group (PcG) genes regulate anterior-posterior patterning of segmented axial structures by transcriptional repression of homeotic gene expression. The murine PcG gene eed (embryonic ectoderm development) encodes a 441-amino-acid protein with five WD motifs which, except for the amino terminus, is highly homologous to Drosophila Extra Sex Combs (Esc). Sequence and expression analysis as well as chromosomal mapping of the human ortholog of eed are described in this study. Absolute conservation of the human eed protein along with significant divergence at the nucleotide level reveals functional constraints operating on all residues. The human orthologue appears to be ubiquitously expressed and maps to chromsome 11q14.2-q22.3. Using the first WD motif of the beta-subunit of the bovine G protein as a structural reference, the predicted locations of two previously identified eed point mutations are reported in this study. The proline substitution (L196P) in the second WD motif of the l7Rn5(3354SB) null allele maps to the internal core of the inner end of the beta-propeller blade and is likely to disrupt protein folding. In contrast, the asparagine substitution (I193N) in the second WD motif of the hypomorphic l7Rn5(1989SB) allele maps onto the surface of the beta-propeller blade near the central cavity and may affect surface interactions without compromising propeller packing. These results illustrate the critical importance of all residues for eed function in mammals and support a model whereby the amino terminus might implement function(s) related to embryonic development in higher organisms (Schumacher, 1998).

An induced mutation, embryonic ectoderm development or eed, has been characterized: it disrupts A-P patterning of the mouse embryo during gastrulation. Positional cloning of this gene reveals it to be the highly conserved homolog of the Drosophila gene extra sex combs, which is required for maintenance of long-term transcriptional repression of homeotic gene expression. Mouse embryos homozygous for loss-of-function alleles of eed initiate gastrulation but display abnormal mesoderm production. Very little embryonic mesoderm is produced; in contrast, extraembryonic mesoderm is relatively abundant. These observations, along with mRNA in situ hybridization analyses, suggest a defect in the anterior primitive streak, from which much of the embryonic mesoderm of the wild-type embryo is derived. Clonal analysis of the pre-streak epiblast was initiated in eed mutant embryos, using the lineage tracer horseradish peroxidase (HRP). The results of these studies indicate that epiblast cells ingress through the anterior streak, but the newly formed mesoderm does not migrate to the anterior and is mislocalized to the extraembryonic compartment. Abnormal localization of mesoderm to the extraembryonic region does not appear to be due to a restriction and alteration of distal epiblast cell fate, since the majority of clones produced from regions fated to ingress through the anterior streak are mixed, displaying descendants in both embryonic and extraembryonic derivatives. eed mutant embryos also fail to display proper epiblast expansion, particularly with respect to the A-P axis. Based on patterns of clonal spread and calculated clone doubling times for the epiblast, this does not appear to be due to decreased epiblast growth. Rather, epiblast, which is normally fated to make a substantial contribution to the axial midline, appears to make mesoderm preferentially. The data are discussed in terms of global morphogenetic movements in the mouse gastrula and a disruption of signalling activity in the anterior primitive streak (Faust, 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).

The murine Polycomb-Group (PcG) proteins Bmi1 (B cell-specific Mo-MLV integration site 1) and eed (embryonic ectoderm development) govern axial patterning during embryonic development by segment-specific repression of Hox gene expression. The two proteins engage in distinct multimeric complexes that are thought to use a common molecular mechanism to render the regulatory regions of Hox and other downstream target genes inaccessible to transcriptional activators. Beyond axial patterning, Bmi1 is also involved in hemopoiesis because a loss-of-function allele causes a profound decrease in bone marrow progenitor cells. Here, evidence is presented that is consistent with an antagonistic function of eed and Bmi1 in hemopoietic cell proliferation. Heterozygosity for an eed null allele causes marked myelo- and lympho-proliferative defects, indicating that eed is involved in the negative regulation of the pool size of lymphoid and myeloid progenitor cells. This antiproliferative function of eed does not appear to be mediated by Hox genes or the tumor suppressor locus p16INK4a/p19ARF because expression of these genes was not altered in eed mutants. Intercross experiments between eed and Bmi1 mutant mice reveal that Bmi1 is epistatic to eed in the control of primitive bone marrow cell proliferation. However, the genetic interaction between the two genes is cell-type specific, because the presence of one or two mutant alleles of eed trans-complements the Bmi1-deficiency in pre-B bone marrow cells. Thus, these studies suggest that hemopoietic cell proliferation is regulated by the relative contribution of repressive (Eed-containing) and enhancing (Bmi1-containing) PcG gene complexes. Biochemical studies have indicated that the PcG proteins Bmi1, Mel18, M33, and Mph1/Rae28 are constituents of a multimeric protein complex A, which localizes to discrete nuclear foci in U-2 OS osteosarcoma cells. Importantly, Eed neither interacts physically with Bmi1 nor engages in this protein complex. Instead, Eed forms a complex B with the PcG proteins Enx1/EzH2 and Enx2/EzH1, which lack signs of a discrete subnuclear distribution and are found rather uniformly throughout the nucleoplasm of U-2 OS osteosarcoma cells (Lessard, 1999).

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).

EED and X chromosome inactivation

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).

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).

EED protein interactions

The heterogeneous nuclear ribonucleoprotein K protein represents a novel class of proteins that may act as docking platforms that orchestrate cross-talk among molecules involved in signal transduction and gene expression. Using a fragment of K protein as bait in the yeast two-hybrid screen, a cDNA was isolated that encodes a protein whose primary structure has extensive similarity to the Drosophila melanogaster extra sexcombs (esc) gene product, Esc, a putative silencer of homeotic genes. The cDNA that was isolated is identical to the cDNA of the recently positionally cloned mouse embryonic ectoderm development gene, eed. Like Esc, Eed contains six WD-40 repeats in the C-terminal half of the protein and is thought to repress homeotic gene expression during mouse embryogenesis. Eed binds to K protein through a domain in its N terminus, but interestingly, this domain is not found in the Drosophila Esc. Gal4-Eed fusion protein represses transcription of a reporter gene driven by a promoter that contains Gal4-binding DNA elements. Eed also represses transcription when recruited to a target promoter by Gal4-K protein. Point mutations within the eed gene, responsible for severe embryonic development abnormalities, abolish the transcriptional repressor activity of Eed. Results of this study suggest that Eed-restricted homeotic gene expression during embryogenesis reflects the action of Eed as a transcriptional repressor. The Eed-mediated transcriptional effects are likely to reflect the interaction of Eed with multiple molecular partners, including K protein (Denisenko, 1997).

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).

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).

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).

Polycomb-group (PcG) proteins form multimeric protein complexes, which are involved in maintaining the transcriptional repressive state of genes over successive cell generations. Components of PcG complexes and their mutual interactions have been identified and analysed through extensive genetic and biochemical analyses. However, molecular mechanisms underlying PcG-mediated repression of gene activity have remained largely unknown. There are two distinct human PcG protein complexes. The EED/EZH protein complex contains the embryonic ectoderm development (EED) and enhancer of zeste 2 (EZH2) PcG proteins. The HPC/HPH PcG complex contains the human polycomb 2 (HPC2), human polyhomeotic (HPH), BMI1 and RING1 proteins. In this study, EED has been shown to interact, both in vitro and in vivo, with histone deacetylase (HDAC) proteins. This interaction is highly specific because the HDAC proteins do not interact with other vertebrate PcG proteins. Histone deacetylation activity co-immunoprecipitates with the EED protein. The histone deacetylase inhibitor trichostatin A relieves transcriptional repression mediated by EED, but not by HPC2, a human homolog of polycomb. These data indicate that PcG-mediated repression of gene activity involves histone deacetylation. This mechanistic link between two distinct, global gene repression systems is accomplished through the interaction of HDAC proteins with a particular PcG protein, EED (van der Vlag, 1999).

Polycomb group (PcG) proteins form multimeric protein complexes that are involved in the heritable stable repression of genes. Two distinct human PcG protein complexes have been identified. The EED-EZH protein complex contains the EED and EZH2 PcG proteins, and the HPC-HPH PcG complex contains the HPC, HPH, BMI1, and RING1 PcG proteins. YY1, a homolog of the Drosophila PcG protein Pleiohomeotic (Pho), interacts specificially with the human PcG protein EED (Drosophila homolog: Extra sexcombs) but not with proteins of the HPC-HPH PcG complex. Since YY1 and Pho are DNA-binding proteins, the interaction between YY1 and EED provides a direct link between the chromatin-associated EED-EZH PcG complex and the DNA of target genes. To study the functional significance of the interaction, the Xenopus homologs of EED and YY1 were expressed in Xenopus embryos. Both Xeed and XYY1 induce an ectopic neural axis but do not induce mesodermal tissues. In contrast, members of the HPC-HPH PcG complex do not induce neural tissue. The exclusive, direct neuralizing activity of both the Xeed and XYY1 proteins underlines the significance of the interaction between the two proteins. These data also indicate a role for chromatin-associated proteins, such as PcG proteins, in Xenopus neural induction (Satijn, 2001).

Esc, the Drosophila homolog of EED, is distinguished from other PcG proteins in Drosophila in that it is primarily required only during embryogenesis. It has been speculated that deacetylation of histones by HDACs and the recruitment of EED to the HDAC proteins may be among the initial repressive events during embryogenesis that eventually lead to stable and heritable PcG-mediated repression of target genes. Now it has been found that YY1 is part of or associated with the EED-EZH PcG complex, which displays HDAC activity. Since Pho and YY1 display specific DNA-binding properties, this finding suggests a model in which YY1 directs the EED-EZH PcG complex to target genes. This first step is consistent with the early developmental role of the EED-EZH complex, as has been defined genetically. It is also consistent with a role for histone deacetylation, mediated by the HDACs, which are associated with both EED and YY1, as an early event by which PcG proteins set up stable repression of target genes (Satijn, 2001).

The existence of two distinct PcG protein complexes has also been observed in Drosophila. Using a two-hybrid analysis, the Esc and E(z) proteins have been found to interact. Furthermore, two distinct Drosophila PcG protein complexes have been characterized biochemically. One complex contains the Pc, Psc, and Ph proteins; the other contains the Esc and E(z) proteins. These findings are very similar to the observations in the human system. There are, however, significant differences between the two developmental systems. For instance, the Drosophila Pho protein lacks a domain that mediates histone deacetylation activity. This domain is present in the YY1 protein. It is possible that this constitutes a fundamental difference between the Drosophila and the vertebrate systems, indicating that histone deacetylation plays a less significant role in the Drosophila system. Further, the Pho protein has not been detected in the Pc-Ph complex. However, the Pho protein could not be detected in the biochemically purified ESC-E(Z) complex either. These puzzling findings may reflect a more transient nature of interactions between Pho and other proteins, which precludes biochemical purification as part of a stable protein complex. The observation that even an in vitro interaction between EED and the Drosophila Pho protein exists at least suggests a highly conserved nature of the interaction between EED and YY1. Also, the virtually identical phenotypes that are induced by Xeed, XYY1, and Pho in Xenopus embryos suggest that YY1 or Pho is either a stable component of or at least transiently associated with the EED-EZH PcG complex and not the HPC-HPH PcG complex (Satijn, 2001).

To study the functional significance of the interaction between EED and YY1, the expression levels of the Xenopus homologs of these proteins, Xeed and XYY1, were manipulated. Both proteins, but no other PcG proteins, induce an ectopic neural axis in Xenopus embryos, but neither Xeed nor XYY1 is able to induce mesodermal tissue, such as muscle or notochord. Importantly, the Drosophila Pho protein induces the same phenotype. The similarity of effects underlines the significance of the EED-YY1 interaction. The fact that Pho induces the same phenotype and neural tissue in ectoderm explants also substantiates the notion that YY1 is indeed a functional homolog of the Drosophila Pho protein (Satijn, 2001).

These data point towards an early developmental role for the EED-EZH complex. Also, in homozygous eed minus mice the earliest developmental decisions are affected, pointing towards an early role for EED in setting up vertebrate PcG-mediated repression. It may be significant that homozygous eed minus mice lack a node and, probably as a consequence of this, also neural tissue. Whereas the homozygous YY1 minus mutation is embryonic lethal, in heterozygote YY1/+ mice the formation of a proper neural tube is seriously hampered. Both phenotypes are complementary to the phenotypes observed after overexpression of both Xeed and XYY1 proteins in Xenopus embryos. Although a detailed comparison between the loss-of-function data in mice and overexpression of proteins in Xenopus is not possible, the opposing effects on neural tissue are compatible with each other. The results reinforce one another and both point towards an early role of these PcG proteins in developmental decisions, such as the induction of embryonic tissues (Satijn, 2001).

The following questions remain: which are the target genes of Xeed and XYY1, and how does the modulation of the activity of these target genes result in the induction of neural tissue? Since EED is a repressor of gene activity, it is likely that Xeed is also a repressor of gene activity. Furthermore, both XYY1 and XYY1-EnR directly induce neural tissue, and by virtue of the EnR domain, XYY1-EnR is a transcriptional repressor. It is, therefore, likely that the target genes of Xeed and XYY1 are repressed by these proteins and that this repression results in the induction of neural tissue. It will be of considerable interest to identify these target genes. Since the effects of Xeed and XYY1 occur early in development, these target genes may well represent a class of PcG target genes other than the known PcG target genes in Drosophila that are affected relatively late during development. Also, identification of such target genes may reveal pathways, distinct from the known ones, that are involved in mediating neural induction in Xenopus (Satijn, 2001).

heed, the human homolog of mouse eed and Drosophila esc, two members of the trithorax (trx) and Polycomb group (Pc-G) of genes, were isolated by screening an activated lymphocyte cDNA library versus the immunodeficiency virus type 1 (HIV-1) MA protein used as a bait in a two-hybrid system in yeast. The human EED protein (HEED) has 99.5% identity with the mouse EED protein and contains seven WD repeats. Two heed gene transcripts were identified, with a putative 407-nucleotide-long intron, giving rise to two HEED protein isoforms of 535 and 494 residues in length, respectively. The shorter HEED isoform, originating from the unspliced message, lacks the seventh WD repeat. HEED binds to MA protein in vitro, as efficiently as in vivo in yeast cells. Site-directed mutagenesis and phage biopanning suggest that the interaction between HEED and MA involved the N-terminal region of the MA protein, including the first polybasic signal, in a MA conformation-dependent manner. In the HEED protein, however, two discrete linear MA-binding motifs were identified within residues 388-403, overlapping the origin of the fifth WD repeat. Deletion of the C-terminal 41 residues of HEED, spanning the seventh WD repeat, as in the 494-residue HEED protein, is detrimental to HEED-MA interaction in vivo, suggesting the existence of another C-terminal binding site and/or a conformational role of the HEED C-terminal domain in the MA-HEED interaction. MA and HEED proteins co-localize within the nucleus of co-transfected human cells and of recombinant baculovirus co-infected insect cells. This and the failure of HEED to bind to uncleaved GAG precursor suggests a role of HEED at the early stages of virus infection, rather than late in the virus life cycle (Peytavi, 1999).

The mouse eed locus encodes the highly conserved ortholog of the Drosophila ESC protein. To test the functional conservation between the two genes, eed was introduced into the fly to determine whether it could rescue the esc mutant phenotype. eed exerts a dominant negative effect on the leg transformation phenotype associated with the esc mutation. This result is interpreted in light of in vitro protein-protein binding data and in vivo polytene chromosome staining indicating the lack of significant interaction between Eed and fly E(Z), a molecular partner of ESC (Wang, 2000).

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).

Role of Histone H3 lysine 27 methylation in Polycomb-Group silencing

Polycomb group (PcG) proteins play important roles in maintaining the silent state of HOX genes. Recent studies have implicated histone methylation in long-term gene silencing. However, a connection between PcG-mediated gene silencing and histone methylation has not been established. This study reports the purification and characterization of an EED-EZH2 complex, the human counterpart of the Drosophila ESC-E(Z) complex. The complex specifically methylates nucleosomal histone H3 at lysine 27 (H3-K27). Using chromatin immunoprecipitation assays, it is shown that H3-K27 methylation colocalizes with, and is dependent on, E(z) binding at an Ultrabithorax (Ubx) Polycomb response element (PRE), and that this methylation correlates with Ubx repression. Methylation on H3-K27 facilitates binding of Polycomb (Pc), a component of the PRC1 complex, to histone H3 amino-terminal tail. Thus, these studies establish a link between histone methylation and PcG-mediated gene silencing (Cao, 2002).

To understand the function of histone methylation, attempts were made to identify histone methyltransferase (HMTase) using a systematic biochemical approach. Certain fractions derived from HeLa cell nuclear pellet contained high levels of HMTase activity toward nucleosomal histone H3. To identify the enzyme(s) present in these fractions, the proteins were further fractionated in a DEAE5PW column, which separated the HMTase activities into two peaks. The present study focuses on the second peak. After fractionation on phenyl sepharose and hydroxyapatite columns, the active fractions were further purified through a gel filtration Superose 6 column. Analysis of the fractions derived from this column indicates that the HMTase activity elutes between fraction 47 and 50 with an estimated mass of about 500 kD. Silver staining of an SDS-polyacrylamide gel containing these fractions revealed that six major polypeptides copurify with the enzymatic activity. Because the largest protein band neither cofractionates with the HMTase activity in the hydroxyapatite column, nor coimmunoprecipitates with the other components, it is concluded that this largest band is not a part of the HMTase protein complex (Cao, 2002).

To identify the proteins that copurify with the HMTase activity, the protein bands were excised and analyzed by a combination of peptide mass fingerprinting and mass spectrometric sequencing. In addition to RbAp48, a polypeptide present in many protein complexes involved in histone metabolism, several human PcG proteins, including EZH2, SUZ12, and EED, were identified in the HMTase complex. A zinc finger transcriptional repressor named AEBP2 was also identified. Whether this protein is involved in targeting the complex remains to be determined. EZH2 contains a SET domain, a signature motif for all known histone lysine methyltransferases, except the H3-K79 methyltransferase DOT1, and is therefore likely to be the catalytic subunit. However, recombinant EZH2 made in Escherichia coli or baculovirus-infected SF9 cells has no detectable HMTase activity, indicating that either a posttranslational modification or other components in the complex are required for the HMTase activity. This is consistent with previous results in which a partial EZH2 protein containing the SET domain was used (Cao, 2002).

Although mammalian EZH2 and EED, and their respective homologs in Drosophila and Caenorhabditis elegans, are known to interact directly, the presence of SUZ12 in such a complex has not been previously reported. To verify that these proteins are components of the same protein complex, antibodies against each of these proteins were generated. Western blot analysis of the column fractions derived from the last two columns indicates that these proteins copurify with the HMTase activity. To further confirm that the copurified proteins exist as a single protein complex, the last column fractions were immunoprecipitated with an antibody to SUZ12. All five proteins coimmunoprecipitate. Because a protein complex containing Drosophila Esc and E(z), respective homologs of EED and EZH2, has been previously named the ESC-E(Z) complex, the human counterpart is referred to as the EED-EZH2 complex. Although both EED-EZH2 and Esc-E(z) complexes physically associate with HDACs, the purified complex neither contains any HDAC polypeptide nor possesses detectable HDAC activity. It is possible that a different protein complex containing EED, EZH2, and HDAC may exist. Alternatively, HDACs may be recruited to target sites through direct interaction with EED, yet may not exist as a stable subunit of EED-EZH2 complexes. Further work is needed to differentiate these possibilities (Cao, 2002).

To characterize the substrate specificity of the EED-EZH2 complex, equivalent amounts of histone H3 that exist alone, in complex with other core histones, and in mono- or oligo-nucleosome forms were subjected to methylation by equal amounts of the enzyme. The EED-EZH2 complex is capable of methylating all forms of histone H3, but shows a strong preference for H3 in oligonucleosome forms (Cao, 2002).

Attempts were made to identify the residue methylated by the EED-EZH2 complex. Because oligonucleosomes are preferred substrates, they were subjected to methylation by the EED-EZH2 complex in the presence of S-adenosyl-L-[methyl-3H]methionine (3H-SAM). After purification, the labeled H3 was subjected to microsequencing followed by liquid scintillation counting. Neither K4 nor K9 released numbers of counts clearly greater than background. However, a small radioactive peak was detected in cycle 27. Given that the recovery efficiency decreases with each microsequencing cycle, the detection of a small peak on cycle 27 indicates that K27 is likely to be the site targeted by the EED-EZH2 complex. To confirm this possibility, each of the five potential methylation sites on H3 were mutated and the effect of the mutation on the ability of H3 to serve as a substrate for the enzyme was compared. As a control, the ability of these H3 mutants to be methylated by SUV39H1 was also analyzed. Mutation of K27 completely abolishes the ability of H3 to serve as a substrate, whereas mutations of other sites have little effect. As expected, only mutation of K9 affects the SUV39H1-mediated H3 methylation. These data, led to the conclusion that K27 is the predominant site, if not the only site, that is targeted for methylation by the EED-EZH2 complex (Cao, 2002).

To gain insight into the function of H3-K27 methylation in vivo, a polyclonal antibody was generated against a dimethyl-K27 H3 peptide. This antibody is highly specific for mK27 when evaluated by peptide competition and enzyme-linked immunosorbent assay. Western blot analysis with the H3-mK27-specific antibody demonstrates that H3-K27 methylation occurs in a variety of multicellular organisms, including human, chicken, and Drosophila . However, it does not appear to occur in the budding yeast Saccharomyces cerevisiae (Cao, 2002).

Given that both H3-K27 methylation as well as the EED-EZH2 counterpart exist in Drosophila , whether the ESC-E(Z) complex is responsible for H3-K27 methylation was examined in this organism. Several E(z) temperature-sensitive mutant alleles have been characterized, one of which, E(z)61, contains a Cys-to-Tyr substitution (C603Y) in the cysteine-rich region immediately preceding the SET domain. When reared continuously at 18°C (permissive temperature), E(z)61 homozygotes exhibit no detectable mutant phenotype and maintain wild-type expression patterns of HOX genes, such as Ubx. However, at 29°C (restrictive temperature), E(z)61 produces multiple homeotic phenotypes due to derepression of HOX genes, which correlates with loss of polytene chromosome binding by the E(Z)61 protein and disruption of chromosome binding by Polycomb (PC) and other PRC1 components. Given that chromosome binding by E(Z)61 protein is abolished at 29°C, H3-K27 methylation should be correspondingly reduced in the mutants at 29°C, if E(Z) is responsible for H3-K27 methylation. Western blot analysis of the histones from wild-type and E(z)61 fly embryos at 18° and 29°C demonstrate that the H3-K27 methylation is abolished in the E(z)61 embryos at 29°C. However, these conditions do not have a detectable effect on H3-K9 methylation. It is therefore concluded that functional E(Z) protein is required for H3-K27 methylation in vivo (Cao, 2002).

To understand the functional relation between E(z)-mediated H3-K27 methylation and HOX gene silencing, a study was carried out of E(z) binding, H3-K27 methylation, and recruitment of PC, a core component of the PRC1 complex, to the major PRE of the Ubx gene in S2 tissue culture cells by chromatin immunoprecipitation (ChIP). Consistent with the involvement of E(z) in H3-K27 methylation, ChIP analysis of a 4.4-kb region that includes this PRE showed precise colocalization of E(z) binding and H3-K27 methylation. In contrast, similar colocalization was not observed for mK9, indicating that H3-K9 methylation, or at least K9-dimethylation, is independent of E(z) binding. To further verify the importance of E(z) binding for H3-K27 methylation, attempts were made to disrupt Esc-E(z) complex activity using RNA interference (RNAi). It was reasoned that depletion of the Esc protein, a direct binding partner of E(z) and a component of the Esc-E(z) complex, would result in disruption of PRE binding by E(z). Depletion of Esc with RNAi results in greatly reduced PRE binding by E(z), loss of H3-K27 methylation, and concomitant loss of PC binding. Depletion of PC in S2 cells has been shown to result in derepression of Ubx. Therefore, these data collectively suggest that the Esc-E(z) complex is critical not only for H3-K27 methylation, but also for PC binding to the PRE region, and that H3-K27 methylation is associated with Ubx repression (Cao, 2002).

To examine the relation between E(z) binding, H3-K27 methylation, and Ubx gene repression in vivo, wing imaginal discs were dissected from homozygous E(z)61 larvae that had been either reared continuously at 18°C or shifted from 18° to 29°C ~48 hours before dissection, and analyzed E(z) binding and H3-K27 methylation in the same Ubx PRE region by ChIP. Consistent with previous studies demonstrating disruption of polytene chromosome binding by both E(z)61 and PC proteins at 29°C, ChIP analysis showed loss of E(z)61 and PC binding to this PRE at restrictive temperature. In addition, H3-K27 methylation colocalizes with E(z) binding at permissive temperature, but is lost along with E(z) binding at 29°C. In contrast, similar changes in H3-K9 methylation were not observed under the same conditions. Under normal conditions, Ubx is not expressed in wing discs due to PcG-mediated silencing. Similar inactivation of an E(z) temperature-sensitive allele during larval development has been shown to result in derepression of Ubx in wing discs. Thus, Ubx PRE-associated nucleosomes appear to be targeted by E(z)-mediated H3-K27 methylation, which correlates with PC binding and repression of Ubx. Collectively, these data suggest that H3-K27 methylation plays an important role in the maintenance of Ubx gene silencing (Cao, 2002).

The chromodomain of the heterochromatin protein HP1 specifically binds to H3 tails that are methylated at K9 by the HMTase SUV39H1. Given that PC contains a chromodomain and that loss of E(z) function abolishes H3-K27 methylation as well as Pc binding to the Ubx PRE, it is possible that methylation of H3-K27 by Esc-E(z) facilitates PRE binding by PC, analogous to the effect of H3-K9 methylation on nucleosome binding by HP1. To test this possibility, Drosophila PC was generated using the rabbit reticulocyte transcription/translation-coupled system and it was incubated with biotinylated H3 peptides with or without K27 methylation in the presence of streptavidin-conjugated Sepharose beads. Analysis by peptide pull-down assay indicated that methylation on K27 facilitates binding of Pc to the H3 peptide. Binding of Pc to the peptides is specific because the chromodomain-containing protein HP1 fails to bind to the same peptides under the same conditions (Cao, 2002).

Previous studies strongly suggest that the chromodomain of PC is necessary and sufficient for targeting PC to specific chromosomal locations in vivo because mutations in the PC chromodomain abolish the ability of PC to bind to chromatin in vivo. In addition, a chimeric PC/HP1 protein, in which the HP1 chromodomain is replaced by the PC chromodomain, binds to both heterochromatin and PcG target sites in euchromatin. To evaluate the contribution of the chromodomain in the preferential binding of PC to K27 methylated peptide, a PC mutant was generated in which two of the highly conserved amino acids Trp-47 and Trp-50 were changed to Ala. These two amino acids were chosen because the corresponding amino acids in the HP1 chromodomain have been shown to directly contact the methyl group of an H3-mK9 peptide. The mutant PC does not preferentially bind to the K27 methylated peptide, suggesting that the chromodomain of PC is responsible for the preferential binding to the H3-mK27 (Cao, 2002).

Collectively, these studies support a model in which Esc-E(z)-mediated H3-K27 methylation serves as a signal for the recruitment of the PRC1 complex by facilitating PC binding. Recruitment of PRC1 in turn prevents the access of nucleosome remodeling factors, such as SWI/SNF, leading to the formation of a repressive chromatin state. Although this model is attractive, it does not exclude the possibility that protein-protein interaction also contributes to the recruitment of PRC1 to PREs. Indeed, a recent study indicates that PC transiently associates with the Esc-E(z) complex during early embryogenesis. These studies established a correlation between H3-K27 methylation and PcG silencing. Further work is needed to establish the exact role of H3-K27 methylation in PcG silencing (Cao, 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).

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).

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).

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).

Juxtaposed Polycomb complexes co-regulate vertebral identity

Best known as epigenetic repressors of developmental Hox gene transcription, Polycomb complexes alter chromatin structure by means of post-translational modification of histone tails. Depending on the cellular context, Polycomb complexes of diverse composition and function exhibit cooperative interaction or hierarchical interdependency at target loci. The present study interrogated the genetic, biochemical and molecular interaction of BMI1 [Drosophila homologs Psc and Su(z)2] and EED (Drosophila homolog; Esc), pivotal constituents of heterologous Polycomb complexes, in the regulation of vertebral identity during mouse development. Despite a significant overlap in dosage-sensitive homeotic phenotypes and co-repression of a similar set of Hox genes, genetic analysis implicated eed and Bmi1 in parallel pathways, which converge at the level of Hox gene regulation. Whereas EED and BMI1 formed separate biochemical entities with EzH2 and Ring1B, respectively, in mid-gestation embryos, YY1 engaged in both Polycomb complexes. Strikingly, methylated lysine 27 of histone H3 (H3-K27), a mediator of Polycomb complex recruitment to target genes, stably associated with the EED complex during the maintenance phase of Hox gene repression. Juxtaposed EED and BMI1 complexes, along with YY1 and methylated H3-K27, were detected in upstream regulatory regions of Hoxc8 and Hoxa5. The combined data suggest a model wherein epigenetic and genetic elements cooperatively recruit and retain juxtaposed Polycomb complexes in mammalian Hox gene clusters toward co-regulation of vertebral identity (Kim, 2006).

At least two PcG complexes with diverse composition and function in chromatin remodeling have been identified in mammals. The Polycomb repressive complex 1 (PRC1) involves the paralogous PcG proteins BMI1/MEL18, M33/PC2, RAE28, and RING1A. Evidence for PRC1-mediated chromatin modification derived from ubiquitylation at lysine 119 of histone H2A (H2A-K119). A second PcG complex, PRC2, encompasses EED, the histone methyltransferase EZH2, the zinc finger protein SUZ12, the histone-binding proteins RBAP46/RBAP48, and the histone deacetylase HDAC1. Several EED isoforms, generated by alternate translation start site usage of eed mRNA, differentially engage in PRC2-related complexes (PRC2/3/4), targeting the histone methyltransferase activity of EZH2 to H3-K27 or H1-K26. PcG complexes bind to cis-acting Polycomb response elements (PREs), which encompass several hundred base pairs and are necessary and sufficient for PcG-mediated repression of target genes. Whereas the function of several PREs has been delineated in Drosophila, similar elements await characterization in mammals (Kim, 2006 and references therein).

An antibody raised against residues 123-140 of the EED amino terminus precipitated three distinct isoforms of approximately 50 and 75 kDA from E12.5 trunk, representing three of the four EED isoforms previously reported in 293 cells. In addition to EZH2 and YY1, dimethylated H3-K27 co-immunoprecipitated with EED. Immunoprecipitation identified three BMI1 isoforms of approximately 39-41 kDA. BMI1 was found in a complex with RING1B, but not dimethylated H3-K27. Similar to the EED complex, the BMI1 complex also contained YY1. It should be emphasized that all (co-)immunoprecipitating bands were detected by at least two antibodies against different epitopes. Strikingly, while dimethylated H3-K27 engaged in the EED complex, trimethylated H3-K27 did not appear to associate with either the EED or the BMI1 complex. Importantly, reciprocal co-immunoprecipitation detected EED and BMI1 in separate protein complexes (Kim, 2006).

Ectopic expression in mutant embryos revealed Hoxc8 and Hoxa5 as downstream targets of EED and BMI1 function. ChIP detected EED and BMI1 binding immediately upstream of the Hoxc8 transcribed region near putative promoter elements. The binding sites could not be separated, indicating close proximity of the complexes. EED and BMI1 binding also clustered within a small fragment 1.5 kb upstream of the Hoxc8 transcription start site, suggesting long-range juxtaposition of heterologous PcG complexes. Similar to EED and BMI1, YY1 localized to both regions. In support of YY1 binding to Hox regulatory regions, inspection of the mouse genome sequence revealed clusters of putative YY1 binding sites in both regions a and b, including TGTCCATTAG and CCCCCATTCC (region a), as well as ACACCATGGC, TTTCCATTAG and TCCCCATAAA (region b). CCAT represents the core of the YY1 consensus binding site, while flanking sequences exhibited significant tolerance for multiple nucleotides. EED, BMI1 and YY1 also co-localized approximately 1.5 kb upstream of the transcription start site of Hoxa5. In addition to PcG binding, ChIP detected trimethylated H3-K27 throughout the regulatory regions of Hoxc8 and Hoxa5. Furthermore, dimethylated H3-K27 localized to region b of Hoxc8 (Kim, 2006).

Spatial regulation of EED and BMI1 binding to Hox regulatory regions was evident from ChIP analysis of dissected anterior and posterior regions of E12.5 trunk. In agreement with transcriptional silencing of Hoxc8 and Hoxa5, EED and BMI1 binding was detected upstream of these loci in anterior regions of the trunk. By contrast, EED and BMI1 binding was absent from posterior regions of the trunk, where Hoxc8 and Hoxa5 are transcribed. These findings implicate PcG complexes in Hox gene repression in anterior regions of the AP axis (Kim, 2006).

The combined interpretation of the co-immunoprecipitation and ChiP results indicates that trimethylated H3-K27 did not form a complex with EED or BMI1, despite co-localization of the three proteins in Hox regulatory regions. By contrast, co-immunoprecipitation demonstrated physical association of the EED complex with dimethylated H3-K27. In aggregate, the results support a model in which EED- and BMI1-containing chromatin remodeling complexes exist as separate, but juxtaposed, biochemical entities at Hox target loci (Kim, 2006).

Polycomb complexes repress developmental regulators in murine embryonic stem cells

The mechanisms by which embryonic stem (ES) cells self-renew while maintaining the ability to differentiate into virtually all adult cell types are not well understood. Polycomb group (PcG) proteins are transcriptional repressors that help to maintain cellular identity during metazoan development by epigenetic modification of chromatin structure. PcG proteins have essential roles in early embryonic development and have been implicated in ES cell pluripotency, but few of their target genes are known in mammals. This study shows that PcG proteins directly repress a large cohort of developmental regulators in murine ES cells, the expression of which would otherwise promote differentiation. Using genome-wide location analysis in murine ES cells, it was found that the Polycomb repressive complexes PRC1 and PRC2 co-occupied 512 genes, many of which encode transcription factors with important roles in development. All of the co-occupied genes contained modified nucleosomes (trimethylated Lys 27 on histone H3). Consistent with a causal role in gene silencing in ES cells, PcG target genes were de-repressed in cells deficient for the PRC2 component Eed (homolog of Drosophila Extra sexcombs), and were preferentially activated on induction of differentiation. These results indicate that dynamic repression of developmental pathways by Polycomb complexes may be required for maintaining ES cell pluripotency and plasticity during embryonic development (Boyer, 2006).

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).

Erk1/2 activity promotes chromatin features and RNAPII phosphorylation at developmental promoters in mouse embryonic stem cells

Erk1/2 activation contributes to mouse ES cell pluripotency. This study found a direct role of Erk1/2 in modulating chromatin features required for regulated developmental gene expression. Erk2 binds to specific DNA sequence motifs typically accessed by Jarid2 and PRC2. Negating Erk1/2 activation leads to increased nucleosome occupancy and decreased occupancy of PRC2 and poised RNAPII at Erk2-PRC2-targeted developmental genes. Surprisingly, Erk2-PRC2-targeted genes are specifically devoid of TFIIH, known to phosphorylate RNA polymerase II (RNAPII) at serine-5, giving rise to its initiated form. Erk2 interacts with and phosphorylates RNAPII at its serine 5 residue, which is consistent with the presence of poised RNAPII as a function of Erk1/2 activation. These findings underscore a key role for Erk1/2 activation in promoting the primed status of developmental genes in mouse ES cells and suggest that the transcription complex at developmental genes is different than the complexes formed at other genes, offering alternative pathways of regulation (Tee, 2014).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

A region of the human HOXD cluster that confers polycomb-group responsiveness

Polycomb group (PcG) proteins are essential for accurate axial body patterning during embryonic development. PcG-mediated repression is conserved in metazoans and is targeted in Drosophila by Polycomb response elements (PREs). However, targeting sequences in humans have not been described. While analyzing chromatin architecture in the context of human embryonic stem cell (hESC) differentiation, a 1.8kb region between HOXD11 and HOXD12 (D11.12) was deciphered that is associated with PcG proteins, becomes nuclease hypersensitive, and then shows alteration in nuclease sensitivity as hESCs differentiate. The D11.12 element repressed luciferase expression from a reporter construct and full repression required a highly conserved region and YY1 binding sites. Furthermore, repression was dependent on the PcG proteins BMI1 and EED and a YY1-interacting partner, RYBP. It is concluded that D11.12 is a Polycomb-dependent regulatory region with similarities to Drosophila PREs, indicating conservation in the mechanisms that target PcG function in mammals and flies (Woo, 2010).

The D11.12 element has several characteristics of a Drosophila PRE, indicating that there is conservation of the mechanisms that target PcG function. The multiple components that combine to make a functional PRE in Drosophila are diverse and still not fully understood. While the study of mammalian PREs is in its infancy, there is reason to think that, like Drosophila, multiple components might contribute to function. Roles in D11.12 were observed for a hyperconserved region, for YY1 and the interacting protein RYBP, and it is suggested that nucleosome free region is also central to function (Woo, 2010).

Focused was placed on D11.12 as playing a potential regulatory role due to its depletion in nucleosome occupancy in mesenchymal stem cells, a level of depletion that changes during differentiation. It is intriguing and somewhat counter-intuitive that sequences associated with recruiting the PcG system are nucleosome depleted. Most characterized activities of the PRC1 and PRC2 families in vitro, including histone methylation, histone ubiquitylation, and chromatin compaction, involve nucleosomes. However, several studies have directly examined depletion of nucleosomes on Drosophila PREs and their association with PcG proteins. Dynamic accessibility of protein-binding sequences might be important for recruiting PcG complexes in vivo (Woo, 2010).

Recent studies suggest that in addition to nucleosome depletion, high levels of histone replacement could be observed where PcG and trxG binding sites exist. This suggests that PRE sequences in flies might be open and dynamic, consistent also with proposals that RNA production from these regions might be important for function. It was found that D11.12 is nuclease-sensitive and associated with the PcG proteins BMI1 and SUZ12. Nucleosome depletion might therefore play a key role mechanistically in establishing the ability to recruit PcG function to a region of the genome, explaining the apparent conservation of this feature between Drosophila and humans (Woo, 2010).

To date, there is only one known human DNA-binding protein, YY1, which has homology to one of the Drosophila proteins which functions to recruit PcG proteins at PREs. Several lines of evidence suggest that YY1 is important to D11.12 function, consistent with previous proposals based upon both functional studies and homology to PHO. It is important to note that while YY1 appears central to D11.12 function, it is unlikely that this protein (or any protein) is generally required for mammalian PRE function. In mice, the PRE-kr has a single YY1 binding site as determined by sequence analysis, however this YY1 binding site is not conserved in the homologous human sequence and no other apparent YY1 binding sites are present. The contribution of the YY1 binding site at the PRE-kr was not examined. It is noted note that in reporter constructs containing D11.12, mutation of the YY1 binding sites impacts binding of BMI1, a PRC1 component, but has little impact on binding of SUZ12, a PRC2 component. This is consistent with models in which PRC2 is recruited prior to PRC1, and suggests that different components of D11.12 might be involved differentially in recruitment of these two complexes. YY1 interacts with RYBP, which in turn interacts with three PRC1 proteins, RING1A, RING1B and CBX2. Thus, at D11.12, YY1 might be involved primarily in PRC1 recruitment (Woo, 2010).

A highly conserved region within D11.12, which shares sequence homologies to organisms as evolutionarily different as zebrafish, is essential for repressive function. This 237 bp conserved region was required for the recruitment of both PRC1 and PRC2 components and for full repression of the reporter gene. In a search for potential regulatory sequences in the Hoxd cluster, Duboule and colleagues made knockout mice deleted of highly conserved sequences, among them the conserved sequence in D11.12. Transgenic studies determined that deletion of this conserved region impacted hoxd11 and hoxd12 expression, however knockout mice with this region deleted displayed no gross phenotype. This lack of gross phenotype might reflect redundancy in either Hox protein function or in regulatory elements with the entire Hoxd cluster. These previous data are consistent with this conserved region having the potential to contribute to regulation in mice; further analysis is needed to determine whether there are contributions of the other nearby elements to function of D11.12 in the genomic context. The mouse PRE-kr element contains a conserved 450 bp sequence within the functionally defined 3kb fragment. Comparison of the conserved regions of D11.12 and PRE-kr using the TRANSFAC database revealed only conserved GAGA factor binding sites, a site defined in Drosophila that has no known binding protein in mammals. Interestingly both conserved region sequences were predicted to form NFRs when analyzed by the nucleosome occupancy feature at the UCSC Genome Browser (Woo, 2010).

The D11.12 element also contains a CpG island. It has not been tested whether this is important to D11.12 function, in part because it is surrounded by key functional elements (namely, the YY1 binding sites and the conserved element), making interpretation of any deletion effect problematic. This element might contribute to the nucleosome-free nature of D11.12, as CpG islands in other areas have been shown to form nucleosomes poorly thereby generating low nucleosome occupancy. It has previously been noted that there is a high correlation of PcG binding sites with CpG islands, leading to the proposal that these elements might be a key determinant of PRE function in mammals (Woo, 2010).

The D11.12 sequence behaves as a strong activating sequence in cells when PcG proteins are knocked down. These knockdowns therefore change the expression from the D11.12 reporter construct by several orders of magnitude in MSCs. A loss of association of the PcG proteins with the D11.12 construct in these cells might allow for the recruitment of activating factors. In Drosophila there is precedent for the same sequence being involved in repression and activation, as PRE elements overlap with Trithorax response elements involved in maintaining activation. It is possible that there is association of trxG components with D11.12 when PcG components have been removed (Woo, 2010).

A key aspect of PcG function is to maintain repression of genes as cells differentiate. It is not clear to what extent PRE sequences, as opposed to other aspects of PcG function, are required for this heritable repression. Repression of an integrated reporter is maintained when MSCs are differentiated into adipocytes. In its natural location, D11.12 remains associated with PcG proteins in adipocytes, although to a lesser degree than in MSCs. In Drosophila, it is known that PcG association can be plastic during differentiation and can be impacted by local activators. A test for whether D11.12 is required for embryonic development will require that the homologous mouse sequence function in this manner, as this type of experiment would require a genetically tractable model system (Woo, 2010).

Polycomb complexes act redundantly to repress genomic repeats and genes

Polycomb complexes establish chromatin modifications for maintaining gene repression and are essential for embryonic development in mice. This study used pluripotent embryonic stem (ES) cells to demonstrate an unexpected redundancy between Polycomb-repressive complex 1 (PRC1) and PRC2 during the formation of differentiated cells. ES cells lacking the function of either PRC1 or PRC2 can differentiate into cells of the three germ layers, whereas simultaneous loss of PRC1 and PRC2 abrogates differentiation. On the molecular level, the differentiation defect is caused by the derepression of a set of genes that is redundantly repressed by PRC1 and PRC2 in ES cells. Furthermore, it was found that genomic repeats are Polycomb targets, and it was shown that, in the absence of Polycomb complexes, endogenous murine leukemia virus elements can mobilize. This indicates a contribution of the Polycomb group system to the defense against parasitic DNA, and a potential role of genomic repeats in Polycomb-mediated gene regulation (Leeb, 2010).

The combined disruption of Ring1B and Eed results in an aberration of cell differentiation. Given the molecular differences of the activities of PRC1 and PRC2, the severity of this synthetic phenotype is unexpected. PRC1-catalyzed ubH2A has been reported to inhibit transcription elongation, suggesting a direct function in gene repression. Yet, the removal of ubH2A during mitosis is required for cell cycle progression, which precludes this modification as a heritable epigenetic mark. In contrast, H3K27me3 is transmitted through the cell cycle, but a direct function of H3K27me3 in gene repression has not been shown, and its only reported function is to recruit other Polycomb proteins such as Cbx7 or PRC2. The very different cell-biological properties of ubH2A and H3K27me3 indicate that the regulation of Polycomb-mediated silencing is a complex process. It is likely that considerable cross-talk exists between different Polycomb complexes, such as the reported binding of the PcG complex protein Rybp to ubH2A and Ring1B, and other chromatin-associated complexes. PRC1 has additional functions in replication, and it has been shown that PRC1 proteins remain bound to chromatin when replicated in an in vitro system (Leeb, 2010).

Ring1B recruitment to PcG target genes depends quantitatively on PRC2; most of the Ring1B signal on gene promoters is lost in Eed-deficient ES cells. Nevertheless, in the absence of PRC2, Ring1B remains detectable clearly over background level and is functionally relevant. In Ring1B-deficient ES cells, Suz12 and H3K27me3 signals are reduced on most nonredundantly silenced genes, but remain at wild-type levels on redundant gene promoters. These data could indicate that, to some extent, redundant gene silencing by PcG complexes reflects the recruitment of PRC1 and PRC2. However, it is believed that additional factors might also contribute to and determine gene repression of PcG target genes (Leeb, 2010).

The data show that the deletion of Ring1B and Eed in ES cells disrupts the catalytic function of PRC1 or PRC2, respectively. Although no H3K27me3 was detected, it is noted that Ezh2 protein is still present at a reduced amount and could be functional to an extent below detection. Since the same level of Ezh2 is also present in dKO cells, the differentiation defect does not arise due to further loss of Ezh2. Ring1B-/- ES cells are deficient for PRC1 function, since Ring1A, a functional homolog of Ring1B, is not expressed in ES cells and does not restore genomic ubH2A levels. In addition, Ring1B deletion leads to a loss of several PRC1 proteins, including Rybp, Cbx4, Mel18, and Bmi1. At present, it is not resolved to what extent the catalytic activity toward ubH2A and the structural role of PRC1 proteins contribute to the phenotype. From these observations, it is suggested that the function of PRC1 and PRC2 is largely eliminated by disruption of Ring1B and Eed, respectively (Leeb, 2010).

Ring1B-deficient or Eed-deficient ES cells can differentiate into cell types of all three germ layers, suggesting that the dynamic modulation of gene expression required for differentiation can be performed by a single PcG complex. However, the complete loss of PcG function abolishes the tumor formation potential of dKO ES cells. The ability of an EedGFP transgene to restore tumor formation indicates that dKO ES cells can regain pluripotency. Upon differentiation of dKO ES cells, pluripotency markers are down-regulated and differentiation markers are up-regulated. Thus, the differentiation defect of PcG-deficient ES cells does not result from a block to enter differentiation, but is due to a failure to maintain differentiated cells. This suggests that the Polycomb system is critical for fine-tuning gene expression, and that epigenetic patterns required to progress through differentiation cannot be set up in the absence of PcG regulation (Leeb, 2010).

ES cells lacking Ring1B and Eed can self-renew and maintain pluripotency marker expression. They can also contribute to the inner cell mass when injected into blastocysts. However, these ES cells are unstable and tend to spontaneously differentiate in culture. Furthermore, they fail to execute differentiation programs appropriately. Thus, Polycomb complexes stabilize ES cell identity. Only a slight decrease in the proliferation rate of dKO ES cells was measured, as compared with controls, indicating that ES cell self-renewal is largely independent of epigenetic gene regulation. This is also consistent with the finding that ES cells lacking DNA cytosine methyltransferases are viable. Recently, it has been suggested that ES cells represent the ground state of pluripotency, which would not require epigenetic regulation. These data support this notion by showing that the network of transcription factors in ES cells is stable enough to maintain self renewal in the absence of Polycomb regulation. The ability to maintain ES cells in the absence of PRC1 and PRC2 catalytic activity now provides an opportunity for studying the function of the PcG system in gene repression, chromatin organization, and genome stability. In the future, this will facilitate characterizing the role of the Polycomb system for the nuclear architecture of mammals (Leeb, 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 that Jarid2 (see Drosophila 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).

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).


extra sexcombs: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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