extra sexcombs


Targets of Activity

Pc-G genes regulate the segmentation genes of Drosophila. Individuals doubly heterozygous for mutations in polyhomeotic and six other Pc-G genes show gap, pair rule, and segment polarity segmentation defects. Flies carrying duplications of extra sexcombs and heterozygous for mutations of even-skippedrarely survive to adulthood. Embryos and surviving adults of this genotype showed strong segmentation defects in even-numbered segments (McKeon, 1994).

The extra sexcombs gene product is a transcriptional repressor of homeotic genes. Although it is classified in the Polycomb group (PcG) on the basis of phenotypic criteria, it is distinct from most other PcG repressors in the timing of its activity during development. ESC plays an early, transient role in repression of homeotic genes. The predicted ESC protein is composed primarily of multiple copies of a repeat motif, termed the WD40 repeat, which are likely used in protein-protein contact. Individual copies of the ESC WD40 repeats are needed for function in vivo. It is suggested that ESC protein is an adaptor that binds to multiple protein partners and assists in the assembly or targeting of other PcG proteins (Simon, 1995).

Polycomb group proteins bind an engrailed PRE in both the 'ON' and 'OFF' transcriptional states of engrailed

Polycomb group (PcG) and trithorax Group (trxG) proteins maintain the 'OFF' and 'ON' transcriptional states of HOX genes and other targets by modulation of chromatin structure. In Drosophila, PcG proteins are bound to DNA fragments called Polycomb group response elements (PREs). The prevalent model holds that PcG proteins bind PREs only in cells where the target gene is 'OFF'. Another model posits that transcription through PREs disrupts associated PcG complexes, contributing to the establishment of the 'ON' transcriptional state. These two models were tested at the PcG target gene engrailed. engrailed exists in a gene complex with invected, which together have four well-characterized PREs. The data show that these PREs are not transcribed in embryos or larvae. Tests were performed to see Whether PcG proteins are bound to an engrailed PRE in cells where engrailed is transcribed. By FLAG-tagging PcG proteins and expressing them specifically where engrailed is 'ON' or 'OFF', it was determined that components of three major PcG protein complexes are present at an engrailed PRE in both the 'ON' and 'OFF' transcriptional states in larval tissues. These results show that PcG binding per se does not determine the transcriptional state of engrailed (Langlais, 2012).

In this study sought to learn more about PcG protein complex-mediated regulation of en expression, focusing on mechanisms operating through en PREs. First whether the en and inv PREs are transcribed was investigated, and no evidence of transcription of the PREs was found either by in situ hybridization or by analysis of RNAseq data from the region. It is concluded that transcription of inv or en PREs does not play a role in regulation of en/inv by PcG proteins. Second, using FLAG-tagged PcG proteins expressed in either en or ci cells, it was found that PcG proteins are bound to the en PRE2 in both the 'ON' and 'OFF' transcriptional state in imaginal disks. The data suggest that PcG protein binding to PRE2 is constitutive at the en gene in imaginal disks and that PcG repressive activity must be suppressed or bypassed in the cells that express en (Langlais, 2012).

Transcription through a PRE in a transgene has been shown to inactivate it, and, in the case of the Fab7, bxd, and hedgehog PREs turn them into Trithorax-response elements, where they maintain the active chromatin state. However, is this how PREs work in vivo? Available data suggest that this could be the case for the iab7 PRE. Transcription through the PREs of a few non-HOX PcG target genes, including the en, salm, and tll PREs has been shown by in situ hybridization to embryos. However, in contrast to the robust salm and tll staining, the picture of en stripes using the en PRE probe was very weak and corresponded to a stage where transient invaginations occur that could give the appearance of stripes. Further, there was no hybridization of the en PRE probe to regions of the head, where en is also transcribed at this stage. In situ hybridization experiments with probes to detect transcription of the inv or en PREs did not yield specific staining at any embryonic stage, or in imaginal discs. This finding is confirmed by absence of polyA and non-poly RNA signals in this region at any embryonic or larval stage, upon review of RNA-seq data from ModEncode (Langlais, 2012).

The results show that PcG proteins bind to en PRE2 even in cells where en is actively transcribed. In fact, one member of each of the three major PcG protein complexes, Pho from PhoRC, dRing/Sce from PRC1, and Esc from PRC2, as well as Scm, are constitutively bound to en PRE2 in all cells in imaginal discs. It is noted that dRing/Sce is also present in the PcG complex dRAF, which also includes Psc and the demethylase dKDM2. Further experiments would be necessary to see whether Sce-FLAG is bound to en DNA as part of the PRC1 complex, the dRAF complex, or both (Langlais, 2012).

What are the differences between the 'ON' and 'OFF' transcriptional states? The data suggest that there may be some differences in Pho binding to non-PRE fragments. However, this data has to be interpreted with caution. The en-GAL4 driver is an enhancer trap in the inv intron and contains an en fragment extending from -2.4 kb through the en promoter. Thus, it is possible that the en-GAL4 driver alters Pho binding in the en/inv domain. In fact, the increased Pho-binding to non-PRE probes in the 'ON' versus the 'OFF' state in the FLAG-Sce samples suggests that the presence of the en-GAL4 driver alters Pho binding slightly (Langlais, 2012).

One unexpected result from these experiments was that FLAG-Sce binds to PRE2 but not to PRE1. This is an interesting result that needs to be followed up on. Recent ChIP-Seq data in using imaginal disk/brain larval samples and the anti-Pho antibody show five additional Pho binding peaks between en and tou, which could be five additional PREs. Three of these correspond to known Pho binding peaks. ChIP-seq experiments with the FLAG-tagged proteins expressed in the 'ON' and 'OFF' transcriptional states would be necessary to ask whether the distribution of PcG-proteins is altered at any of the PREs or any other region of the en/inv domain (Langlais, 2012).

In conclusion, the data allows two simple models of PcG-regulation of the en/inv genes to be ruled out. First, the en/inv PREs are not transcribed, so this cannot determine their activity state. Second, PcG proteins bind to at least one of the PREs of the en/inv locus in the 'ON' state, therefore a simple model of PcG-binding determining the activity state of en/inv is not correct. Perhaps the proteins that activate en expression modify the PcG-proteins or the 3D structure of the locus and interfere with PcG-silencing. While FLAG-tagged PcG proteins offer a good tool to study PcG-binding particularly in the 'OFF' state, cell-sorting of en positive and negative cells will be necessary to study the 3D structure and chromatin modification of the en/inv locus (Langlais, 2012).

Protein Interactions

Evidence is presented for direct physical interaction between the Esc and Enhancer of zeste [E(z)] proteins using yeast two-hybrid and in vitro binding assays. Coimmunoprecipitation from embryo extracts demonstrates association of Esc and E(z) in vivo. The Esc-binding domain of E(z) has been delimited to an N-terminal 33-amino-acid region. Only 6 of 33 amino acids in this region are identical among fly E(z), the murine homologs Ezh1 and Ezh2, and the human homologs EZH1 and EZH2. Site-directed mutations in the Esc protein previously shown to impair Esc function in vivo disrupt Esc-E(z) interactions in vitro. An in vitro interaction also occurs between the human eed (heed) and EZH1 proteins, which are human homologs of Esc and E(z), respectively. The amino acid sequences among these E(z) homologs are much more highly conserved in domains outside the 33 amino acid region. This result suggests that amino acids other than those that are evolutionarily conserved between E(z) and EZH1 participate in the interaction with Esc. It also suggests that Esc residues outside the absolutely conserved portions of the loops connecting blades 3, 4, and 5 of the WD repeat region are involved in binding to E(z) and EZH1. Additional studies will be required to further map the interacting domains of heed and EZH1 and to demonstrate their in vivo association. These results suggest that the Esc-E(z) molecular partnership has been conserved in evolution. Previous studies suggested that Esc is primarily involved in the early stages of Pc-G-mediated silencing during embryogenesis. However, E(z) is continuously required in order to maintain chromosome binding by other Pc-G proteins. In light of these earlier observations and the molecular data presented here, the paper discusses how Esc-E(z) protein complexes may contribute to transcriptional silencing by the Pc-G (Jones, 1998).

At least two lines of evidence indicate that Drosophila E(z) and Esc are not obligate partners at all loci and during all developmental stages. (1) Although both proteins display uniform spatial distributions in nuclei of blastoderm and early gastrulation-stage embryos, the Esc protein is much more limited than E(z) in mid-to-late-stage embryos. (2) There are target genes other than homeotic genes that require E(z), but not Esc, for repression during development. These examples show that the E(z) protein can be recruited to and function at target loci without assistance from the Esc protein. Presumably, E(z) action at these loci involves alternative binding partners. The ability of E(z) to sometimes act independently of Esc is one possible explanation for the differential efficiencies of reciprocal coimmunoprecipitations observed with the two proteins. It has been suggested that in addition to its role in Pc-G repression, E(z) may also be involved in the maintenance of transcriptional activity by trithorax-Group proteins. In contrast, the known role of the Esc protein is limited to repression. A dual role for E(z) could reflect participation in more than one type of protein complex. For example, when E(z) is bound to Esc, it is a component of silencing complexes. However, when E(z) is associated with other, as-yet-unidentified proteins, it may contribute to trx-G-mediated transcriptional activation. In support of this idea, heat inactivation of temperature-sensitive E(z) proteins reduces chromosomal binding by the Trx protein. To assess these possibilities, it will be necessary to define the number and constituents of E(z)-containing complexes isolated from fly embryos (Jones, 1998).

The extra sexcombs (esc) gene is expressed maternally and its product is most abundant during the early embryonic stages. It encodes a protein of the WD-40 repeat family, which localizes predominantly to the nucleus. During germ band extension, it is expressed in a stereotypic pattern of neuroblasts. A model has been proposed in which Esc is recruited by gap proteins both to act as a corepressor that competes with the TAFII80 coactivator to block transcription and also to mediate the transition to permanent repression by Polycomb-group proteins. This model is based on a possible analogy to yeast protein Tup1, which includes tandemly repeated WD-40 domains at its C-terminal portion. Tup1 protein has been shown to act as a corepressor when recruited by DNA binding proteins. Although it is unclear how Tup1 represses transcription, it has been shown that Tup1 interferes with the general transcription machinery. The interference of Tup1 with the assembly of an active transcription complex depends on the WD-40 domains. TAFII80, one of the factors associated with the TATA binding protein in the TFIID complex includes seven C-terminal WD-40 repeats. The corepressor activity of ESC appears to be mediated through its WD-40 domains that compete with those of TAFII80 for binding to other TAFs, TBP or other parts of the basal transcription apparatus, thus displacing TAFII80 from the TFIID complex (Gutjahr, 1995).

The Extra sex combs (Esc) and Enhancer of zeste [E(z)] proteins, members of the Polycomb group (PcG) of transcriptional repressors, interact directly and are coassociated in fly embryos. These two proteins are components of a 600-kDa complex in embryos. Using gel filtration and affinity chromatography, it has been shown that this complex is biochemically distinct from previously described complexes containing the PcG proteins Polyhomeotic, Polycomb, and Sex comb on midleg. In addition, evidence is presented that Esc is phosphorylated in vivo and that this modified Esc is preferentially associated in the complex with E(z). Modified Esc accumulates between 2 and 6 h of embryogenesis, which is the developmental time when esc function is first required. Mutations in E(z) reduce the ratio of modified to unmodified Esc in vivo. Germ line transformants were generated that express Esc proteins bearing site-directed mutations that disrupt Esc-E(z) binding in vitro. These mutant Esc proteins fail to provide esc function, show reduced levels of modification in vivo, and are still assembled into complexes. Taken together, these results suggest that Esc phosphorylation normally occurs after assembly into Esc-E(z) complexes and that Esc contributes to the function or regulation of these complexes (Ng, 2000).

ESC mRNA is expressed primarily during early development, with the highest levels being found before 4 h of embryogenesis. This early expression has prompted the hypothesis that esc functions in the transition between initiation of homeotic gene repression by gap proteins, such as hunchback, and maintenance of this repression by PcG proteins. This transition occurs at about 4 h, when gap gene products decay. Esc protein is expressed at peak levels at 6 to 12 h, after ESC mRNA has decayed to low levels. In addition, Esc is detected until the end of embryogenesis. The presence of substantial levels of Esc in mid- to late-stage embryos suggests that Esc may play a greater role than simply in the transition between gap protein and PcG protein repression. In addition, a second peak of Esc protein is detected during larval and pupal stages, consistent with its nonessential function in imaginal discs (Ng, 2000).

The predicted Esc structure identifies two surface-accessible regions likely to contain phosphorylation sites: the highly charged N terminus and the surface loops of the ß-propeller. The Esc phosphorylation sites have not been mapped. However, it is predicted that Esc is serine/threonine phosphorylated, because many of the Ser and Thr residues are surface accessible. In particular, the N-terminal tail is very rich in Ser and Thr residues (35%), a feature which has been conserved in Esc during evolution. A scan of the accessible Esc regions for consensus kinase recognition motifs identifies numerous possible modification sites and is therefore not particularly instructive (Ng, 2000).

Gel filtration experiments also show that modified Esc is found preferentially in the Esc-E(Z) complex while unmodified Esc behaves predominantly as unassociated monomer. Interestingly, mutant Esc proteins with reduced levels of modification also associate in complexes with the same apparent molecular mass as the wild-type complex. This suggests that Esc modification is not required for its stable association in complexes. Consistent with this idea, low levels of unmodified wild-type Esc are reproducibly detected in the 600-kDa complex. Based on these data, a model is favored in which Esc modification contributes to function rather than to assembly of the complex. The finding that E(z) function is required for wild-type levels of Esc modification further suggests that this modification occurs after Esc has complexed with its partners (Ng, 2000).

The mutant Esc proteins described in this study show reduced Esc-E(z) binding in vitro. Therefore, it was surprising to find that these mutants assemble into complexes of apparently wild-type size. It is suggested that Esc may bind to multiple protein partners in the Esc-E(z) complex, such that specific disruption of Esc-E(z) interaction still allows complex assembly. In support of this idea, ß-propeller proteins have been shown to make simultaneous contacts with multiple partners (Ng, 2000).

The PcG proteins Pc and Ph are associated in a complex estimated to be 2 MDa. In addition, Ph and Ph coimmunoprecipitate and interact with another PcG protein, Psc. The Esc-E(Z) complex is biochemically distinct from complexes containing Ph. In agreement with this, a Ph-Pc-Psc complex does not contain E(z). Taken together, these results support a model in which there are at least two distinct PcG complexes in vivo, one containing Esc and E(z) and the other containing Ph, Pc, and Psc. Consistent with this idea, the mammalian Esc and E(z) homologs, EED and EZH2, fail to coimmunoprecipitate with the mammalian Ph, Psc, and Pc homologs. In addition, EED and EZH2 do not colocalize with mammalian PH, PSC, and PC within nuclei of osteosarcoma cells. Furthermore, the patterns of pairwise interactions among Drosophila PcG proteins are reiterated among their mammalian counterparts, which suggests that this division of labor in the PcG has been conserved in evolution (Ng, 2000 and references therein).

Although the existence of at least two different PcG complexes has been established, the complete spectrum of PcG protein interactions has not yet been elucidated. There appears to be further division among Ph-Pc-Psc complexes, which have different compositions at different target genes. In addition, multiple complexes containing the mammalian Ph, Pc, and Psc proteins have been detected. Moreover, there are additional PcG proteins, such as Asx, Pcl, and Pho, whose in vivo associations have yet to be described. Some of these proteins may correspond to as yet unidentified components of Esc-E(z) or Ph-Pc-Psc complexes, or they may sort into additional distinct complexes. In particular, complexes containing Pho, the only known DNA-binding member of the PcG, may be important for targeting other PcG complexes to sites of action. It is noted that Pho is not detected as a stable member of either the Esc-E(z) or Ph-Pc-Psc complexes (Ng, 2000).

Despite the presence of biochemically separable PcG complexes, the similar mutant phenotypes and genetic interactions of PcG genes indicate that they work together at some level. Any model for PcG repression must therefore accommodate both the biochemical separability and functional synergy of PcG complexes. One possibility is that repression requires multiple chromatin-modifying events by the different PcG complexes. This would be similar to the in vivo synergy between the chromatin-modifying SWI-SNF and SAGA complexes, which are both required for maintenance of HO expression in yeast. An alternative possibility is that one PcG complex directly modifies chromatin while the other complex counteracts trithorax group activation by inhibiting the chromatin-remodeling activity of the brahma complex. Indeed, the first evidence that a PcG complex may covalently modify chromatin is provided by the recent report of histone deacetylase activity associated with mammalian homologs of Esc and E(z) (Ng, 2000 and references therein).

These mechanisms are inconsistent with an esc role limited to the transition from gap repressors to PcG repressors. Instead, it is suggested that Esc is more globally involved in chromatin regulation and that this involvement is most critical early in fly development. Consistent with a global role, EED mRNA is expressed in many tissues during mouse development. Furthermore, the C. elegans homolog of Esc, MES-6, is a transcriptional repressor that functions in germ line development. MES-6 in worms therefore plays a distinct developmental role from Esc in flies. This suggests that Esc participates in a general repression mechanism that has been adapted for use in different cell lineages, rather than in the specific transition between gap protein and PcG protein repression (Ng, 2000 and references therein).

If Esc-E(z) complexes function as general chromatin regulators, the early requirement for Esc in Drosophila must be reconciled with the need for long-term PcG repression during development. One possibility is that another protein replaces Esc in the Esc-E(z) complex at late developmental stages, when Esc is no longer critically required. Alternatively, E(z) may associate with a completely different set of PcG proteins to supply the biochemical function provided by Esc-E(z) complexes during embryogenesis. To address these possibilities, the nature of E(z) complexes at postembryonic stages will have to be investigated (Ng, 2000).

A tethering assay was developed to study the effects of Polycomb group (PcG) proteins on gene expression in vivo. This system employed the Su(Hw) DNA-binding domain (ZnF) to direct PcG proteins to transposons that carried the white and yellow reporter genes. These reporters constituted naive sensors of PcG effects, since bona fide PcG response elements (PREs) were absent from the constructs. To assess the effects of different genomic environments, reporter transposons integrated at nearly 40 chromosomal sites were analyzed. Three PcG fusion proteins, ZnF-PC, ZnF-SCM, and ZnF-ESC, were studied, since biochemical analyses place these PcG proteins in distinct complexes. Tethered ZnF-PcG proteins repress white and yellow expression at the majority of sites tested, with each fusion protein displaying a characteristic degree of silencing. Repression by ZnF-PC is stronger than ZnF-SCM, which is stronger than ZnF-ESC, as judged by the percentage of insertion lines affected and the magnitude of the conferred repression. ZnF-PcG repression is more effective at centric and telomeric reporter insertion sites, as compared to euchromatic sites. ZnF-PcG proteins tethered as far as 3.0 kb away from the target promoter produce silencing, indicating that these effects are long range. Repression by ZnF-SCM requires a protein interaction domain, the SPM domain, which suggests that this domain is not primarily used to direct SCM to chromosomal loci. This targeting system is useful for studying protein domains and mechanisms involved in PcG repression in vivo (Roseman, 2001).

Biochemical studies indicate that PcG repression involves multiple, distinct PcG complexes. Thus, an underlying assumption of the assay system that was used is that gene silencing by the tethered ZnF-PcG protein involves assembly with endogenous PcG proteins at the reporter site. This hypothesis leads to the prediction that repression by a tethered ZnF-PcG protein should be compromised by loss of function for an endogenous PcG partner. It was difficult to test this prediction for the comprehensive set of endogenous PcG proteins because the basic assay system involved generating a very complex genotype. Nevertheless, the requirement was tested for endogenous PH protein, which is encoded by an X-linked gene and for which a hemizygous viable allele is available (Roseman, 2001).

A reporter integration site was identified that normally lacks PH binding, as scored on polytene chromosomes: it was repressed by all three ZnF-PcG proteins. Genetic tests show that reduction in PH dosage relieves tether-based repression by PC and SCM at this site. These results can be reconciled with the known PC-PH and SCM-PH molecular interactions. Surprisingly, ZnF-ESC repression is sensitive to PH dosage. This result was not expected since ESC-PH interactions have not been reported and there is evidence that ESC and PH are in separate complexes in embryos (Roseman, 2001).

Polycomb group (PcG) proteins maintain transcriptional repression during development, likely by creating repressive chromatin states. The Extra Sex Combs (Esc) and Enhancer of Zeste [E(z)] proteins are partners in an essential PcG complex, but its full composition and biochemical activities are not known. A SET domain in E(z) suggests this complex might methylate histones. An Esc-E(z) complex has been purified from Drosophila embryos and four major subunits were found: Esc, E(z), NURF-55, and the PcG repressor, Su(z)12. A recombinant complex reconstituted from these four subunits methylates lysine-27 of histone H3. Mutations in the E(z) SET domain disrupt methyltransferase activity in vitro and HOX gene repression in vivo. These results identify E(Z) as a PcG protein with enzymatic activity and implicate histone methylation in PcG-mediated silencing (Müller, 2002).

Histone H3 is an attractive candidate to be the primary functional target for the Esc-E(z) complex in vivo. Previous work on heterochromatin proteins has established a molecular model that links H3 methylation and gene repression. In this case, SUV39H HMTase methylates H3-K9 and this modification creates a binding site for the chromodomain of HP1. Similarly, H3 methylation by Esc-E(z) might provide a binding site for another chromodomain protein, such as Polycomb (PC), a component of the PRC1 complex, whose chromodomain is required for chromatin localization in vivo. Targeting of PRC1 via histone methylation performed by Esc-E(z) is consistent with several in vivo results that imply synergy between these complexes. Rather than supplying binding sites for specific proteins, Esc-E(z) mediated methylation could influence chromatin more generally by altering adjacent nucleosome interactions needed to package the chromatin fiber (Müller, 2002).

Remarkably, Esc, E(z), and SU(z)12 are conserved in many organisms, where they appear to function together as repressors in a wide array of developmental processes. Mammalian complexes that resemble the Esc-E(z) complex are implicated in multiple processes including early embryonic patterning, HOX gene regulation, and hematopoiesis. Intriguingly, mouse homologs of Esc and E(z) associate with the inactive X chromosome in trophoblast stem cells, suggesting a direct role in X-inactivation. In C. elegans, the Esc- and E(z)-related proteins MES-6 and MES-2 form a stable complex and are required for germline development and gene silencing. The conserved partnership extends to plants, where proteins related to Esc, E(z), and SU(z)12 are cohort regulators in several Arabidopsis developmental pathways. A striking example is VRN2, a SU(z)12 relative, which is required for long-term gene silencing in response to vernalization. Further work in these systems should address if histone methylation by Esc-E(z) complexes represent an evolutionary ancient mechanism to mark chromatin for heritable repression during development (Müller, 2002).

The Polycomb Group proteins are required for stable long-term maintenance of transcriptionally repressed states. Two distinct Polycomb Group complexes have been identified, a 2-MDa PRC1 complex and a 600-kDa complex containing the ESC and E(Z) proteins together with the histone deacetylase RPD3 and the histone-binding protein p55. There are at least two embryonic ESC/E(Z) complexes that undergo dynamic changes during development and a third larval E(Z) complex that forms after disappearance of ESC. A larger embryonic ESC complex has been identified containing RPD3 and p55, along with E(Z), that is present only until mid-embryogenesis, while the previously identified 600-kDa ESC/E(Z) complex persists until the end of embryogenesis. Constitutive overexpression of ESC does not promote abnormal persistence of the larger or smaller embryonic complexes and does not delay a dissociation of E(Z) from the smaller ESC complex or delay appearance of the larval E(Z) complex, indicating that these changes are developmentally programmed and not regulated by the temporal profile of ESC itself. Genetic removal of ESC prevents appearance of E(Z) in the smaller embryonic complex, but does not appear to affect formation of the large embryonic ESC complex or the PRC1 complex. The ESC complex is already bound to chromosomes in preblastoderm embryos and genetic evidence is presented that ESC is required during this very early period (Furuyama, 2003).

Polycomb group (PcG) proteins are required to maintain stable repression of the homeotic genes and others throughout development. The PcG proteins ESC and E(Z) are present in a prominent 600-kDa complex as well as in a number of higher-molecular-mass complexes. A 1-MDa ESC/E(Z) complex has been identified and characterized that is distinguished from the 600-kDa complex by the presence of the PcG protein Polycomblike (PCL) and the histone deacetylase RPD3. In addition, the 1-MDa complex shares with the 600-kDa complex the histone binding protein p55 and the PcG protein SU(Z)12. Coimmunoprecipitation assays performed on embryo extracts and gel filtration column fractions indicate that, during embryogenesis E(Z), SU(Z)12, and p55 are present in all ESC complexes, while PCL and RPD3 are associated with ESC, E(Z), SU(Z)12, and p55 only in the 1-MDa complex. Glutathione transferase pulldown assays demonstrate that RPD3 binds directly to PCL via the conserved PHD fingers of PCL and the N terminus of RPD3. PCL and E(Z) colocalize virtually completely on polytene chromosomes and are associated with a subset of RPD3 sites. As shown for E(Z) and RPD3, PCL and SU(Z)12 are also recruited to the insertion site of a minimal Ubx Polycomb response element transgene in vivo. Consistent with these biochemical and cytological results, Rpd3 mutations enhance the phenotypes of Pcl mutants, further indicating that RPD3 is required for PcG silencing and possibly for PCL function. These results suggest that there may be multiple ESC/E(Z) complexes with distinct functions in vivo (Tie, 2003).

Subunit contributions to histone methyltransferase activities of fly and worm polycomb group complexes

The ESC-E(Z) complex of Drosophila Polycomb group (PcG) repressors is a histone H3 methyltransferase (HMTase). This complex silences fly Hox genes, and related HMTases control germ line development in worms, flowering in plants, and X inactivation in mammals. The fly complex contains a catalytic SET domain subunit, E(Z), plus three noncatalytic subunits, SU(Z)12, ESC, and NURF-55/CAF-1. The four-subunit complex is >1,000-fold more active than E(Z) alone. ESC and SU(Z)12 play key roles in potentiating E(Z) HMTase activity. Loss of ESC disrupts global methylation of histone H3-lysine 27 in fly embryos. Subunit mutations identify domains required for catalytic activity and/or binding to specific partners. Missense mutations are described in surface loops of ESC, in the CXC domain of E(Z), and in the conserved VEFS domain of SU(Z)12, which each disrupt HMTase activity but preserve complex assembly. Thus, the E(Z) SET domain requires multiple partner inputs to produce active HMTase. A recombinant worm complex containing the E(Z) homolog, MES-2, has robust HMTase activity, which depends upon both MES-6, an ESC homolog, and MES-3, a pioneer protein. Thus, although the fly and mammalian PcG complexes absolutely require SU(Z)12, the worm complex generates HMTase activity from a distinct partner set (Ketel, 2005).

In vitro and in vivo data indicate that the noncatalytic ESC subunit makes a critical contribution to HMTase function of the ESC-E(Z) complex. In particular, since global levels of H3-K27 methylation are similarly reduced by genetic loss of ESC or E(Z), ESC appears to be an obligate functional partner for E(Z) HMTase activity (Ketel, 2005).

Two main molecular explanations are envisioned for the ESC requirement. (1) ESC could potentiate HMTase activity through direct interaction with E(Z). ESC binding could trigger a conformational change in E(Z) that improves catalytic efficiency, and/or ESC residues could directly interact with and influence the E(Z) active site. (2) Alternatively, the main role of ESC could be to bind nucleosomes. In this scenario, ESC would boost HMTase activity by facilitating interaction of the enzyme complex with its substrate. Based on several lines of evidence, a mechanism is favored that works through direct ESC-E(Z) contact. (1) The ESC M236K and V289M mutations, which significantly reduce HMTase activity, are located in surface loops previously shown to mediate direct ESC contact with E(Z). Furthermore, M236K displays dominant-negative properties in vivo. This genetic behavior is consistent with an enzyme complex that assembles normally but is compromised in catalytic function. (2) A recent report documents that ESC lacks nucleosome-binding activity on its own and that addition of ESC to a trimeric NURF-55/SU(Z)12/E(Z) complex has little additive effect on ability to bind nucleosomes. (3) ESC potentiation through direct E(Z) binding is supported by evolutionary considerations. Every organism examined that has an E(Z) homolog, ranging from plants to worms, flies, and humans, has at least one ESC homolog. In addition, 28 residues within the ESC surface loops implicated in E(Z) binding are identical from flies to humans. This conservation may reflect a tight functional requirement wherein direct ESC-E(Z) partnership, combining to produce HMTase activity, is maintained by evolutionary pressure. Future studies will be needed to define the precise biochemical mechanism by which ESC potentiates HMTase activity, including tests for binding-induced conformational changes in E(Z) (Ketel, 2005).

Studies on the mammalian homolog of ESC, called EED, have also highlighted its important role as a regulatory subunit. In human cells, multiple EED isoforms are expressed, which differ in the extents of their N-terminal tails. These isoforms are generated by alternative start codon usage of the same EED mRNA. Intriguingly, incorporation of particular EED isoforms into EZH2 complexes can shift the enzyme specificity so that K26 of histone H1 is methylated in addition to H3-K27. Thus, it appears that the ESC/EED subunit can influence both catalytic efficiency and lysine substrate preference. In the fly system, ESC isoforms produced from the same mRNA have not been detected. Instead, alternative ESC isoforms could be supplied by an esc-related gene (CG5202) located about 150 kb proximal to esc. Since mutations in this second esc gene, called esc-like (escl), have not yet been reported, its in vivo contributions remain to be assessed. However, since genetic loss of ESC alone dramatically reduces global methylation of H3-K27 in fly embryos, it is concluded that ESC is the predominant functional E(Z) partner during embryonic stages (Ketel, 2005).

Studies on recombinant complexes show that fly SU(Z)12 is absolutely required for HMTase activity of the ESC-E(Z) complex. A key requirement for SU(Z)12 in mammalian EZH2 complexes has also been established based upon in vitro tests and loss-of-function studies in vivo. How does SU(Z)12 contribute molecularly to HMTase activity? Again, two main possibilities are envisioned: influence through direct contact with E(Z) or by mediating nucleosome binding. To address this, it is instructive to consider the SU(Z)12 mutants affecting the conserved VEFS domain. Deletion of the entire VEFS domain eliminates assembly of the fly complex by disrupting SU(Z)12-E(Z) binding. Pairwise binding assays with mammalian SU(Z)12 have similarly shown that the VEFS domain is needed for binding to EZH2 in vitro. Thus, a conserved function of this domain is to contact E(Z). However, missense mutations within the VEFS domain, D546A and E550A, preserve full complex assembly yet have reduced levels of HMTase. Taken together, these results implicate the VEFS domain in both binding to E(Z) and potentiating its enzyme activity, which suggests a connection between these two functions. In contrast, a recent report provides evidence that SU(Z)12 contributes to affinity for nucleosomes. Although SU(Z)12 cannot bind to nucleosomes by itself, the SU(Z)12/NURF-55 dimer has nucleosome-binding properties that are similar to those of the four-subunit complex. Thus, as also suggested for the human PRC2 complex, at least one role of SU(Z)12 is to mediate nucleosome binding. Further work will be needed to define the SU(Z)12 functional domains required for interactions with NURF-55 and with nucleosomes. Based on the available data, SU(Z)12 potentiation of E(Z) HMTase activity may involve both direct E(Z) contact and facilitated binding to nucleosome substrate (Ketel, 2005).

Although the SET domain is the most well-characterized functional domain of E(Z), the adjacent cysteine-rich CXC domain is also remarkably conserved from flies to humans. To address CXC function, in vitro properties of two missense mutants, C545Y and C603Y were analyzed. Both mutations correspond to E(z) loss-of-function alleles; in particular, in vivo effects of the E(z)61 mutation (C603Y) have been well documented. This mutation disrupts global H3-K27 methylation in embryos and causes loss of methyl-H3-K27 from a Hox target gene in imaginal discs. Mutant complexes bearing E(Z)-C603Y can assemble normally but show an approximately 10-fold reduction in HMTase levels. C545Y causes a more modest HMTase reduction, which parallels results obtained with the analogous substitution (C588Y) in human EZH2. These results suggest that the CXC domain interfaces with the SET domain to produce robust HMTase activity. In this regard, the CXC domain could be considered similar to cysteine-rich "preSET" domains required for robust HMTase activity in other SET domain proteins. Another effect of these CXC mutations in vivo is that they dislodge E(Z) from target sites in chromatin. Although the molecular basis for this dissociation is not known, in vitro assembly results suggest that it is not due to wholesale destabilization of the ESC-E(Z) complex. The dissociation may reflect another proposed role for the CXC domain, which is to interact with the PcG targeting factor PHO (Ketel, 2005).

In vitro tests were performed to investigate the role of E(Z) domain II. Both the complete domain deletion and the C363Y missense mutation show that domain II is required for stable association of E(Z) with SU(Z)12. Thus, the composite domain organization of E(Z) reflects division of labor among catalytic functions and requirements for complex assembly. In addition, it appears that none of the E(Z) domains are specifically built for nucleosome interactions; E(Z) plays little or no role itself in stable binding of the complex to nucleosomes (Ketel, 2005).

The NURF-55 subunit is distinct from the other three subunits in several ways. (1) It makes only minimal contributions to in vitro HMTase activity in both the fly and mammalian complexes. (2) Whereas the other three subunits appear dedicated to PcG function, NURF-55 is present in diverse chromatin-modifying complexes, including NURF, chromatin assembly factor 1 (CAF-1), and histone deacetylase complexes. The ability of the mammalian NURF-55 homologs RbAp46 and RbAp48 to bind to free histone H4 has led to the suggestion that NURF-55 may help chromatin complexes interact with substrate. Indeed, the absence of a NURF-55-related protein from the trimeric worm MES complex could help explain its inability to methylate free histones. The free histone-binding property of NURF-55 has also prompted the intriguing suggestion that silencing by the ESC-E(Z) complex in vivo could involve methylation of histones prior to nucleosome assembly. Since NURF-55 loss-of-function alleles have not been described in flies, many questions about roles of NURF-55 remain to be addressed. Even with alleles available, the multiplicity of NURF-55-containing complexes will likely complicate in vivo dissection of its PcG functions (Ketel, 2005).

The basic enzymatic function of the ESC-E(Z) complex, to methylate H3-K27, is shared between the worm, fly, and mammalian versions. Another similarity revealed from this study of recombinant worm complexes is that robust HMTase activity depends critically upon the two noncatalytic subunits. Although MES-6 and MES-3 can each individually bind to the catalytic subunit, MES-2, all three subunits are required together to produce enzyme activity. Since worm MES-6 is a WD repeat protein related to fly ESC, it seems likely that MES-6 and ESC potentiate HMTase activity through similar mechanisms. It is suggested that this mechanism entails direct subunit interactions rather than an influence upon affinity for nucleosomes. However, a major puzzle is presented by the dissimilarity between worm MES-3 and fly SU(Z)12. Though each is required for HMTase activity in their respective complexes, no relatedness was recognized between these two proteins in primary sequence or predicted secondary structure arrangement. From an evolutionary standpoint, it appears that SU(Z)12 represents the more ancient partner, since it is functionally conserved across plant and animal kingdoms. MES-3, a novel protein, may have evolved more recently to replace SU(Z)12 in the worm complex. Since a molecular role attributed to SU(Z)12 in the fly complex is nucleosome binding, it is speculated that MES-3 may supply this function for the worm complex. There are many strategies for building nucleosome contact domains, as represented among divergent chromatin proteins, so MES-3 could have acquired functional similarity without overt sequence similarity to SU(Z)12. In this view, MES-3 function in the worm complex would require, at minimum, affinity for nucleosomes and ability to bind MES-2. In this regard, it is interesting that MES-2 appears to lack domain II, which is needed in E(Z) for stable binding to SU(Z)12. Presumably, MES-2 has instead acquired a site for stable MES-3 interaction. In summary, it is suggested that the E(Z)/ESC and MES-2/MES-6 dimers have been conserved as core subunits of the HMTase complex, whereas the additional required partners in each complex, SU(Z)12 and MES-3, have been allowed to diverge. Future studies will be needed, including functional tests of chimeric worm and fly proteins, to address such a model (Ketel, 2005).

Nucleosome binding and histone methyltransferase activity of Drosophila PRC2

The Drosophila Polycomb group protein E(z) is a histone methyltransferase (HMTase) that is essential for maintaining HOX gene silencing during development. E(z) exists in a multiprotein complex called Polycomb repressive complex 2 (PRC2) that also contains Su(z)12, Esc and Nurf55. Reconstituted recombinant PRC2 methylates nucleosomes in vitro, but recombinant E(z) on its own shows only poor HMTase activity on nucleosomes. This study investigated the function of the PRC2 subunits. It was shown that PRC2 binds to nucleosomes in vitro but that individual PRC2 subunits alone do not bind to nucleosomes. By analysing PRC2 subcomplexes, it was shown that Su(z)12-Nurf55 is the minimal nucleosome-binding module of PRC2 and that Esc contributes to high-affinity binding of PRC2 nucleosomes. Nucleosome binding of PRC2 is not sufficient for histone methylation and only complexes that contain Esc protein show robust HMTase activity. These observations suggest that different subunits provide mechanistically distinct functions within the PRC2 HMTase: the nucleosome-binding subunits Su(z)12 and Nurf55 anchor the E(z) enzyme on chromatin substrates, whereas Esc is needed to boost enzymatic activity (Nekrasov, 2005).

Baculovirus expression vectors have been used to reconstitute and purify recombinant tetrameric PRC2 from Sf9 cells. To identify intermolecular interactions within this complex, tests were performed for reconstitution of PRC2 subcomplexes on coexpression of two or more subunits in Sf9 cells. In each case, the Flag-epitope tag present on one of the subunits was used for affinity purification from Sf9 cell extracts. The following stable dimeric complexes could be purfied: Esc-E(z), E(z)-Su(z)12, E(z)-Nurf55 and Su(z)12-Nurf55. In contrast, purification either from cells expressing Flag-Esc and Nurf55 or from cells expressing Flag-Esc and Su(z)12 resulted in the isolation of Flag-Esc protein only, suggesting that these proteins do not bind directly to each other. Purification from Sf9 cells expressing three subunits allowed the isolation of trimeric Esc-E(z)-Su(z)12 and E(z)-Su(z)12-Nurf55 complexes. Finally, tetrameric PRC2 was isolated, using either Flag-E(z) or Flag-Su(z)12 for affinity purification. Importantly, each of these complexes was stable in buffers containing up to 1 M KCl. Taken together, these data suggest that E(z) binds tightly to Su(z)12, Esc and Nurf55 and that Su(z)12 also binds to Nurf55. The failure to isolate dimeric complexes that contain Esc and Su(z)12 or those that contain Esc and Nurf55 indicates that E(z), Su(z)12 and Nurf55 form a trimeric core complex to which Esc binds through interaction with E(z). The observation that E(z) forms stable dimeric complexes with either Esc or Nurf55 in this reconstitution assay is consistent with earlier studies that reported physical interactions between these proteins in glutathione-S-transferase (GST) pull-down assays (Nekrasov, 2005).

It is noted that the molecular architecture of mammalian PRC2 is unclear at present; conflicting data on intermolecular interactions between subunits have been reported. Specifically, it has been reported that human EZH2, SU(Z)12 and RbAp48 all bind to EED, the Esc homologue, and that EZH2 does not interact with SU(Z)12 or RbAp48 in GST pull-down assays; it has been proposed that EED is the core component of the complex and EZH2 associates with other components through EED. It has also been reported that EZH2 binds to SUZ12 in GST pull-down assays, consistent with the finding that Drosophila E(z) and Su(z)12 form a stable complex (Nekrasov, 2005).

The HMTase activity of recombinant E(z) protein is significantly lower than the activity observed with recombinant tetrameric PRC2. A simple mechanistic explanation would be that one or several PRC2 subunits are needed for nucleosome binding to facilitate interaction of the E(z) HMTase with its substrate, the histone H3 tail. Since it is not known whether any of the PRC2 subunits binds to nucleosomes, tests were performed to see whether complex components alone or in combination could form stable complexes with mononucleosomes, in a bandshift assay. To this end, mononucleosomes were reconstituted with recombinant core histones that were expressed in E. coli and a 201 base pairs (bp) long radioactively labelled DNA template that contained a strong nucleosome-binding sequence called '601'. When recombinant tetrameric PRC2 was incubated with such mononucleosomes and the reaction mixture was resolved on a polyacrylamide gel, distinct, slowly migrating complexes were observed that appeared in a concentration-dependent manner. In contrast, when PRC2 was incubated with naked 601 DNA template, it was not possible to resolve specific protein-DNA complexes. Together, these observations suggest that PRC2 binds to mononucleosomes and that these protein-nucleosome complexes remain stably associated under electrophoretic conditions. Individual PRC2 subunits were tested for nucleosome binding, but no formation of protein-nucleosome complexes was detected with any of the four proteins. This suggests that more than one subunit is needed for nucleosome binding and therefore the different di- and trimeric PRC2 subcomplexes were tested. Among the different subcomplexes, only incubation with the Su(z)12-Nurf55 or E(z)-Su(z)12-Nurf55 complexes results in the appearance of distinct, slowly migrating protein-nucleosome complexes. These protein-nucleosome complexes migrate similarly to the complexes observed with tetrameric PRC2, but two- to threefold higher concentrations of the Su(z)12-Nurf55 or E(z)-Su(z)12-Nurf55 complex are needed to shift all of the nucleosome probe. Thus, the presence of Esc in PRC2 increases the affinity of the complex for nucleosomes or allows the complex to bind more stably under the experimental conditions compared with PRC2 subcomplexes that lack Esc. In contrast to the distinct protein-nucleosome complexes observed with the Su(z)12-Nurf55 or E(z)-Su(z)12-Nurf55 complex, no specific protein-nucleosome complexes are formed if nucleosomes are incubated with the E(z)-Su(z)12, E(z)-Nurf55, Esc-E(z) or Esc-E(z)-Su(z)12 complexes. However, incubation with high concentrations of the trimeric Esc-E(z)-Su(z)12 complex also shifts almost all of the nucleosome probe, and much of the probe is retained in the well of the gel. Thus, it seems that the Esc-E(z)-Su(z)12 complex also binds to nucleosomes but that it binds in a manner distinct from the other PRC2 (sub)complexes. Taken together, these binding assays suggest that several subunits need to cooperate for nucleosome binding of PRC2, as follows: (1) Su(z)12 is essential for nucleosome binding because only complexes containing Su(z)12 bind; (2) the minimal nucleosome-binding complex contains Su(z)12 and Nurf55, and only complexes that contain these two proteins give rise to distinct, slowly migrating protein-nucleosome complexes; Su(z)12 and Nurf55 together thus form the minimal nucleosome-binding module of PRC2; (3) as discussed above, Esc also contributes to nucleosome binding because (a.) tetrameric PRC2 binds more strongly than the E(z)-Su(z)12-Nurf55 complex and (b.) in the absence of Nurf55, that is, in the Esc-E(z)-Su(z)12 complex, Esc seems to cooperate with Su(z)12 to cause retention of the nucleosome probe. E(z) is thus the only subunit for which no contribution to nucleosome binding was detected. Note that comparable concentrations of the Su(z)12-Nurf55 or E(z)-Su(z)12-Nurf55 complex are required to shift 50% of the nucleosome probe (Nekrasov, 2005).

Nurf55 and Esc are both WD40 repeat proteins. RbAp46 and RbAp48, the mammalian homologues of Nurf55, have been reported to bind directly to helix 1 of histone H4, a portion of H4 that is thought to be inaccessible within the nucleosome, and, consistent with this, RbAp46 and RbAp48 are unable to bind to H4 in nucleosomal templates. As shown in this study, Drosophila Nurf55 or Esc alone are not able to bind to mononucleosomes but they bind in combination with Su(z)12, which, by itself, also does not bind to nucleosomes. It is possible that the combination of Su(z)12 and Nurf55 or Esc is needed to create the necessary surface for stable nucleosome binding. Alternatively, it could be that these proteins act in a cooperative manner to disrupt histone-DNA contacts locally to expose the histone core (i.e. H4) for binding by Nurf55 or Esc (Nekrasov, 2005).

These results suggest that Su(z)12 together with Nurf55 or Esc tethers the complex to nucleosomes, whereas E(z), the catalytic subunit of the complex, contributes little to nucleosome binding. Next the HMTase activity of the different PRC2 (sub)complexes were analyzed. As substrates for these reactions, non-radiolabelled mononucleosomes were used identical to those used in the bandshift assay. Recombinant tetrameric PRC2 methylates H3 in mononucleosomes. In contrast, E(z) protein alone, the dimeric Esc-E(z), and the Su(z)12-E(z) or E(z)-Nurf55 complexes do not detectably methylate mononucleosomes. Strikingly, the Su(z)12-E(z)-Nurf55 complex also shows no detectable HMTase activity, whereas the Esc-E(z)-Su(z)12 complex methylates H3 in mononucleosomes with efficacy similar to tetrameric PRC2. Thus, no straightforward correlation is observed between nucleosome binding in bandshift assays and HMTase activity. In particular, the Su(z)12-E(z)-Nurf55 complex seems to bind to nucleosomes with only two- to threefold lower affinity than tetrameric PRC2, but shows markedly reduced HMTase activity. In contrast, the Esc-E(z)-Su(z)12 complex, which shows almost no nucleosome binding at the concentration used in the HMTase assay, shows HMTase activity comparable with PRC2. Together, these data suggest that nucleosome binding is not sufficient for HMTase activity and that Esc has a crucial role in boosting the enzymatic activity of E(z). It is possible that Esc is required to dock the complex in a specific orientation on the nucleosome that presents the H3 tail in a particularly favourable position to the E(z) enzyme. Alternatively, Esc could directly increase the catalytic activity of E(z) by inducing a conformational change in the enzyme. However, it is important to note that the Esc-E(z) complex shows no detectable HMTase activity on mononucleosomes and the presence of Su(z)12 is thus essential for HMTase activity of the Esc-E(z)-Su(z)12 complex. The bandshift data show that Su(z)12 is strictly needed for nucleosome binding of PRC2. At the low complex concentrations used in the HMTase assay, there is probably very little nucleosome binding of the Esc-E(z)-Su(z)12 complex; nevertheless, it seems probable that the nucleosome interactions that Su(z)12 shows in cooperation with Esc contribute to the HMTase activity of the Esc-E(z)-Su(z)12 complex. Finally, it is puzzling that the absence of Nurf55 from the complex (i.e. in the Esc-E(z)-Su(z)12 complex) does not seem to diminish HMTase activity because Nurf55 is important for the formation of stable PRC2-nucleosome complexes in bandshift assays. It is possible that Nurf55 is not needed for HMTase activity under the assay conditions that were used but that it is important for methylation of chromatin in vivo (Nekrasov, 2005).

Recent studies have shown that SU(Z)12 is crucial for HMTase activity of mammalian PRC2 in vitro, but the molecular basis for this requirement has remained unclear. This study reports two main findings: (1) Su(z)12 has a crucial role in nucleosome binding of Drosophila PRC2; (2) PRC2 subcomplexes that bind to nucleosomes but lack Esc are poorly active; Esc thus has an important role in boosting HMTase activity of the complex (Nekrasov, 2005).

The requirement of Su(z)12 for HMTase activity can for the most part be explained by its ability to tether PRC2 to nucleosomes, and the data suggest that nucleosome binding requires Su(z)12 in combination with either Nurf55 or Esc. Although both Nurf55 and Esc contribute to nucleosome binding of the complex, only inclusion of Esc leads to an increase in HMTase activity. The contribution of Esc to HMTase activity thus goes beyond the activity that one would expect if Esc were required only for nucleosome binding. In summary, these data suggest that the Su(z)12, Nurf55 and Esc subunits all contribute to nucleosome binding of PRC2 but that these three subunits make distinct contributions to the activation of the E(z) HMTase (Nekrasov, 2005).

The findings reported here imply that E(z) HMTase activity in vivo could be regulated at the level of chromatin binding and/or enzyme activity by modulating the abundance or activity of different PRC2 subunits. It is important to discuss the results reported here in the context of the in vivo requirement for different PRC2 subunits. Genetic studies have shown that Su(z)12 and E(z) are required throughout development to maintain silencing of HOX genes in Drosophila and that this process requires the enzymatic activity of E(z). In contrast, Esc protein is required early in development and then becomes to a large extent, although not completely, dispensable for maintenance of HOX gene silencing during postembryonic development. There are two possible explanations for the paradoxical observation that Esc is required for strong HMTase activity in vitro but that the protein seems to be largely dispensable for the HMTase activity of E(z) that is needed to maintain HOX gene silencing during larval development. (1) It is possible that strong HMTase activity of PRC2 is required primarily early in embryogenesis and that, once it is established, H3-K27 methylation can be maintained with a catalytically less active form of the complex (i.e., lacking Esc). (2) It is possible that another protein substitutes for Esc at later developmental stages. It is noted that the Drosophila genome encodes a second Esc-like protein (CG5202) but, at present, it is not known whether this protein is required for HOX gene silencing (Nekrasov, 2005).

Histone H3 Serine 28 is essential for efficient Polycomb-mediated gene repression in Drosophila

Trimethylation at histone H3K27 is central to the polycomb repression system. Juxtaposed to H3K27 is a widely conserved phosphorylatable serine residue (H3S28) whose function is unclear. To assess the importance of H3S28, a Drosophila H3 histone mutant was generated with a serine-to-alanine mutation at position 28. H3S28A mutant cells lack H3S28ph on mitotic chromosomes but support normal mitosis. Strikingly, all methylation states of H3K27 drop in H3S28A cells, leading to Hox gene derepression and to homeotic transformations in adult tissues. These defects are not caused by active H3K27 demethylation nor by the loss of H3S28ph. Biochemical assays show that H3S28A nucleosomes are a suboptimal substrate for PRC2 (containing Esc, Su(z)12, E(z) and Nurf55), suggesting that the unphosphorylated state of serine 28 is important for assisting in the function of polycomb complexes. Collectively, these data indicate that the conserved H3S28 residue in metazoans has a role in supporting PRC2 catalysis (Yung, 2015).

This report has established a H3S28A histone mutant in Drosophila. In theory, this mutation could have two different effects on the polycomb system. (1) It could be that PcG proteins are not evicted from H3K27me3-binding sites in the absence of H3S28ph, and thus, PcG target genes might become ectopically repressed or (2) the mutation at H3S28 or the absence of H3S28ph could compromise PcG functions, resulting in derepression of PcG target genes. No evidence was found for the first possibility, although it is formally possible that H3S28 is phosphorylated under certain developmental conditions or in response to particular stimuli to counteract polycomb silencing. Instead, the data point to an inhibition of PRC2 activity by the H3S28A mutation. This inhibition is independent of active H3K27 demethylation by dUtx. Besides, RNAi against Aurora B kinase and hence depletion of H3S28ph did not hamper polycomb silencing. On the other hand, H3S28A nucleosomes proved to be a suboptimal substrate for in vitro PRC2 HMT activity. Although a 3D structure of the human Ezh2 SET domain is available, the exact contribution of the hydroxyl group of H3S28 for H3K27 methylation is difficult to deduce from the available data. vSET, the only other protein capable of H3K27 methylation in the absence of PRC2 subunits, does not require H3S28 for catalysis, whereas it does use H3A29 to define substrate specificity. Clearly, more work will be required to determine the exact structural and biochemical role of H3S28 in PRC2 catalysis. Consistent with the in vitro HMT assays, in vivo the H3S28A mutant exhibits defects in H3K27 methylation and shows similar, though milder, Hox derepression profiles and transformation phenotypes to those observed in H3K27R mutant flies (Yung, 2015).

Interestingly, the 'KS' module is frequently found in Ezh2 substrates other than K27S28 of histone H3. These include K26S27 of human histone H1 variant H1b (H1.4), K38S39 of the nuclear orphan receptor RORα, and K180S181 of STAT3. Whether these serine residues act similarly to H3S28 to support methylation of the adjacent lysine residue remains unknown. Of note, some other Ezh2 substrates can be methylated despite the lack of a 'KS' module. These include K26 of mouse histone H1 variant H1e, K49 of STAT3, and K116 of Jarid2, where the lysine residue is followed by an alanine, glutamate, and phenylalanine, respectively. Moreover, the link between peptide sequence and enzymology of Ezh2 was shown to differ in non-histone substrates. Hence, the role of serine following the Ezh2 methylation target amino acid might not be extrapolated to all other Ezh2 substrates and should be tested individually (Yung, 2015).

Previous reports revealed discrepancies in Drosophila PcG protein localization on mitotic chromosomes depending on staining protocols and tissue types. Nonetheless, live imaging of Pc-GFP, Ph-GFP, and E(z)-GFP in early Drosophila embryos has suggested that the majority of these PcG components are dissociated from mitotic chromosomes. Because stress-induced H3S28ph evicts PcG complexes during interphase, one might expect rebinding of PcG proteins on mitotic chromosomes depleted of H3S28ph. Whereas loss of Ph from mitotic chromosomes was observed in WT background, significant Ph association was not observed in H3S28A mutant condition. The reduced levels of H3K27me3 in the H3S28A mutant could contribute to this observation. Alternatively, other mechanisms might operate to dissociate the majority of PcG proteins during mitosis (Yung, 2015).

The establishment of the histone replacement system in Drosophila has proven to be an important tool to complement functional characterization of chromatin modifiers. Whereas depletion of H3K27 methylation, either by mutation of the histone mark writer E(z) or by mutation of the histone itself in the H3K27R mutant, leads to similar loss of polycomb-dependent silencing, other histone mutations revealed different phenotypes than the loss of their corresponding histone mark writers. For example, H3K4R mutations in both H3.2 and H3.3, hence a complete loss of H3K4 methylation, did not hamper active transcription. Also, the loss of H4K20 methylation upon H4K20R mutation unexpectedly supports development and does not phenocopy cell cycle and gene silencing defects reported upon the loss of the H4K20 methylase PR-Set7. In this study, by comparing the phenotype of Aurora B knockdown and H3S28A mutation in vivo, together with in vitro HMT assay, the requirement of the unmodified H3S28 residue is specifically attributed to supporting PRC2 deposition of H3K27 methylation (Yung, 2015).

Whereas the published data suggest that H3S28 phosphorylation might be important for eviction of PcG components for derepression of PcG target genes upon stimulatory cues, the data reveal a so far unacknowledged function of the unphosphorylated state of H3S28. This study shows that serine 28 is required to enable proper methylation of H3K27 by PRC2 and thus to establish polycomb-dependent gene silencing. Serine 28 of histone H3 is universally conserved in species that display canonical PRC2-dependent silencing mechanisms. Given the fact that no major mitotic defects are found upon its mutation, it is proposed that the major role of this residue is to ensure optimal PRC2 function while facilitating the removal of polycomb proteins in response to signals that induce phosphorylation (Yung, 2015).

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

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