Alternative ESC and ESC-like subunits of a polycomb group histone methyltransferase complex are differentially deployed during Drosophila development

The Extra sex combs (ESC) protein is a Polycomb group (PcG) repressor that is a key noncatalytic subunit in the ESC-Enhancer of zeste [E(Z)] histone methyltransferase complex. Survival of esc homozygotes to adulthood based solely on maternal product and peak ESC expression during embryonic stages indicate that ESC is most critical during early development. In contrast, two other PcG repressors in the same complex, E(Z) and Suppressor of zeste-12 [SU(Z)12], are required throughout development for viability and Hox gene repression. Here we describe a novel fly PcG repressor, called ESC-Like (ESCL), whose biochemical, molecular, and genetic properties can explain the long-standing paradox of ESC dispensability during postembryonic times. Developmental Western blots show that ESCL, which is 60% identical to ESC, is expressed with peak abundance during postembryonic stages. Recombinant complexes containing ESCL in place of ESC can methylate histone H3 with activity levels, and lysine specificity for K27, similar to that of the ESC-containing complex. Coimmunoprecipitations show that ESCL associates with E(Z) in postembryonic cells and chromatin immunoprecipitations show that ESCL tracks closely with E(Z) on Ubx regulatory DNA in wing discs. Furthermore, reduced escl+ dosage enhances esc loss-of-function phenotypes and double RNA interference knockdown of ESC/ESCL in wing disc-derived cells causes Ubx derepression. These results suggest that ESCL and ESC have similar functions in E(Z) methyltransferase complexes but are differentially deployed as development proceeds.     

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

The ESC-E(Z) complex of Drosophila melanogaster 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. The four-subunit complex is >1,000-fold more active than E(Z) alone. Here we show that ESC and SU(Z)12 play key roles in potentiating E(Z) HMTase activity. We also show that 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. We describe missense mutations 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. We also find that 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.     

A 1-megadalton ESC/E(Z) complex from Drosophila that contains polycomblike and RPD3

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. Here we identify and characterize a 1-MDa ESC/E(Z) complex 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 previously 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.     

The N-terminus of Drosophila ESC mediates its phosphorylation and dimerization

The ESC protein, like other Polycomb Group proteins, is required for heritable silencing of the homeotic genes. ESC is phosphorylated in vivo, but the region of ESC that is phosphorylated and its consequences are not known. Here, we show that the amino-terminal region of ESC (residues 1-60) mediates its phosphorylation and dimerization. Phosphorylation of ESC1-60 in vitro by CK1 and CK2 strongly enhances its dimerization. Both phosphorylation and dimerization are conserved in the mammalian ESC homolog EED, suggesting that they play important roles in vivo. One role is suggested by the effect of phosphatase treatment on native ESC complexes, which does not affect the integrity of the 600 kDa ESC/E(Z) complex, but eliminates the 1 MDa ESC/E(Z) complex, which is distinguished from the former by the presence of the additional subunits PCL and RPD3. Thus, stability and perhaps assembly of larger ESC complexes may depend on ESC phosphorylation.     

Dominant alleles identify SET domain residues required for histone methyltransferase of Polycomb repressive complex 2

Polycomb gene silencing requires histone methyltransferase activity of Polycomb repressive complex 2 (PRC2), which methylates lysine 27 of histone H3. Information on how PRC2 works is limited by lack of structural data on the catalytic subunit, Enhancer of zeste (E(Z)), and the paucity of E(z) mutant alleles that alter its SET domain. Here we analyze missense alleles of Drosophila E(z), selected for molecular study because of their dominant genetic effects. Four missense alleles identify key E(Z) SET domain residues, and a fifth is located in the adjacent CXC domain. Analysis of mutant PRC2 complexes in vitro, and H3-K27 methylation in vivo, shows that each SET domain mutation disrupts PRC2 histone methyltransferase. Based on known SET domain structures, the mutations likely affect either the lysine-substrate binding pocket, the binding site for the adenosylmethionine methyl donor, or a critical tyrosine predicted to interact with the substrate lysine epsilon-amino group. In contrast, the CXC mutant retains catalytic activity, Lys-27 specificity, and trimethylation capacity. Deletion analysis also reveals a functional requirement for a conserved E(Z) domain N-terminal to CXC and SET. These results identify critical SET domain residues needed for PRC2 enzyme function, and they also emphasize functional inputs from outside the SET domain.     

The MES-2/MES-3/MES-6 complex and regulation of histone H3 methylation in C. elegans

The C. elegans proteins MES-2 and MES-6, orthologs of the Polycomb group (PcG) chromatin repressors E(Z) and ESC, exist in a complex with their novel partner MES-3. The MES system participates in silencing the X chromosomes in the hermaphrodite germline. Loss of maternal MES function leads to germline degeneration and sterility. We report here that the MES complex is responsible for di- and trimethylation of histone H3 Lys27 (H3-K27) in the adult germline and in early embryos and that MES-dependent H3-K27 marks are concentrated on the X's. Another H3-K27 HMT functions in adult somatic cells, oocytes, and the PGCs of embryos. In PGCs, the MES complex may specifically convert dimethyl to trimethyl H3-K27. The HMT activity of the MES complex appears to be dependent on the SET domain of MES-2. MES-2 thus joins its orthologs Drosophila E(Z) and human EZH2 among SET domain proteins known to function as HMTs (reviewed in ). Methylation of histones is important for long-term epigenetic regulation of chromatin and plays a key role in diverse processes such as X inactivation and oncogenesis. Our results contribute to understanding the composition and roles of E(Z)/MES-2 complexes across species.     

Drosophila ESC-like can substitute for ESC and becomes required for Polycomb silencing if ESC is absent

The Drosophila esc-like gene (escl) encodes a protein very similar to ESC. Like ESC, ESCL binds directly to the E(Z) histone methyltransferase via its WD region. In contrast to ESC, which is present at highest levels during embryogenesis and low levels thereafter, ESCL is continuously present throughout development and in adults. ESC/E(Z) complexes are present at high levels mainly during embryogenesis but ESCL/E(Z) complexes are found throughout development. While depletion of either ESCL or ESC by RNAi in S2 and Kc cells has little effect on E(Z)-mediated methylation of histone H3 lysine 27 (H3K27), simultaneous depletion of ESCL and ESC results in loss of di- and trimethyl-H3K27, indicating that either ESC or ESCL is necessary and sufficient for di- and trimethylation of H3K27 in vivo. While E(Z) complexes in S2 cells contain predominantly ESC, in ESC-depleted S2 cells, ESCL levels rise dramatically and ESCL replaces ESC in E(Z) complexes. A mutation in escl that produces very little protein is viable and exhibits no phenotypes but strongly enhances esc mutant phenotypes, suggesting they have similar functions. esc escl double homozygotes die at the end of the larval period, indicating that the well-known “maternal rescue” of esc homozygotes requires ESCL. Furthermore, maternal and zygotic over-expression of escl fully rescues the lethality of esc null mutant embryos that contain no ESC protein, indicating that ESCL can substitute fully for ESC in vivo. These data thus indicate that ESC and ESCL play similar if not identical functions in E(Z) complexes in vivo. Despite this, when esc is expressed normally, escl appears to be entirely dispensable, at least for development into morphologically normal fertile adults. Furthermore, the larval lethality of esc escl double mutants, together with the lack of phenotypes in the escl mutant, further suggests that in wild-type (esc⁺) animals it is the post-embryonic expression of esc, not escl, that is important for development of normal adults. Thus escl appears to function in a backup capacity during development that becomes important only when normal esc expression is compromised.     

The functions of E(Z)/EZH2-mediated methylation of lysine 27 in histone H3

Polycomb group (PcG) proteins are important for maintaining the silenced state of homeotic genes. Biochemical and genetic studies in Drosophila and mammalian cells indicate that PcG proteins function in at least two distinct protein complexes: the ESC-E(Z) or EED-EZH2 complex, and the PRC1 complex. Recent work has shown that at least part of the silencing function of the ESC-E(Z) complex is mediated by its intrinsic activity for methylating histone H3 on lysine 27. In addition to being involved in Hox gene silencing, the complex and its associated histone methyltransferase activity are important in other biological processes including X-inactivation, germline development, stem cell pluripotency and cancer metastasis.     

A phosphorylated form of Mel-18 targets the Ring1B histone H2A ubiquitin ligase to chromatin

Recent studies have shown that PRC1-like Polycomb repressor complexes monoubiquity-late chromatin on histone H2A at lysine residue 119. Here we have analyzed the function of the polycomb protein Mel-18. Using affinity-tagged human MEL-18, we identify a polycomb-like complex, melPRC1, containing the core PRC1 proteins, RING1/2, HPH2, and CBX8. We show that, in ES cells, melPRC1 can functionally substitute for other PRC1-like complexes in Hox gene repression. A reconstituted subcomplex containing only Ring1B and Mel-18 functions as an efficient ubiquitin E3 ligase. This complex ubiquitylates free histone substrates nonspecifically but is highly specific for histone H2A lysine 119 in the context of nucleosomes. Mutational analysis demonstrates that while Ring1B is required for E3 function, Mel-18 directs this activity to H2A lysine 119 in chromatin. Moreover, this substrate-targeting function of Mel-18 is dependent on its prior phosphorylation at multiple residues, providing a direct link between chromatin modification and cell signaling pathways.     

Histone H2A Mono-Ubiquitination Is a Crucial Step to Mediate PRC1-Dependent Repression of Developmental Genes to Maintain ES Cell Identity

Two distinct Polycomb complexes, PRC1 and PRC2, collaborate to maintain epigenetic repression of key developmental loci in embryonic stem cells (ESCs). PRC1 and PRC2 have histone modifying activities, catalyzing mono-ubiquitination of histone H2A (H2AK119u1) and trimethylation of H3 lysine 27 (H3K27me3), respectively. Compared to H3K27me3, localization and the role of H2AK119u1 are not fully understood in ESCs. Here we present genome-wide H2AK119u1 maps in ESCs and identify a group of genes at which H2AK119u1 is deposited in a Ring1-dependent manner. These genes are a distinctive subset of genes with H3K27me3 enrichment and are the central targets of Polycomb silencing that are required to maintain ESC identity. We further show that the H2A ubiquitination activity of PRC1 is dispensable for its target binding and its activity to compact chromatin at Hox loci, but is indispensable for efficient repression of target genes and thereby ESC maintenance. These data demonstrate that multiple effector mechanisms including H2A ubiquitination and chromatin compaction combine to mediate PRC1-dependent repression of genes that are crucial for the maintenance of ESC identity. Utilization of these diverse effector mechanisms might provide a means to maintain a repressive state that is robust yet highly responsive to developmental cues during ES cell self-renewal and differentiation.