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.     

MicroRNA regulation of Cbx7 mediates a switch of Polycomb orthologs during ESC differentiation

The Polycomb Group (PcG) of chromatin modifiers regulates pluripotency and differentiation. Mammalian genomes encode multiple homologs of the Polycomb repressive complex 1 (PRC1) components, including five orthologs of the Drosophila Polycomb protein (Cbx2, Cbx4, Cbx6, Cbx7, and Cbx8). We have identified Cbx7 as the primary Polycomb ortholog of PRC1 complexes in embryonic stem cells (ESCs). The expression of Cbx7 is downregulated during ESC differentiation, preceding the upregulation of Cbx2, Cbx4, and Cbx8, which are directly repressed by Cbx7. Ectopic expression of Cbx7 inhibits differentiation and X chromosome inactivation and enhances ESC self-renewal. Conversely, Cbx7 knockdown induces differentiation and derepresses lineage-specific markers. In a functional screen, we identified the miR-125 and miR-181 families as regulators of Cbx7 that are induced during ESC differentiation. Ectopic expression of these miRNAs accelerates ESC differentiation via regulation of Cbx7. These observations establish a critical role for Cbx7 and its regulatory miRNAs in determining pluripotency.     

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.     

Drosophila enhancer of Zeste/ESC complexes have a histone H3 methyltransferase activity that marks chromosomal Polycomb sites

Enhancer of Zeste is a Polycomb Group protein essential for the establishment and maintenance of repression of homeotic and other genes. In the early embryo it is found in a complex that includes ESC and is recruited to Polycomb Response Elements. We show that this complex contains a methyltransferase activity that methylates lysine 9 and lysine 27 of histone H3, but the activity is lost when the E(Z) SET domain is mutated. The lysine 9 position is trimethylated and this mark is closely associated with Polycomb binding sites on polytene chromosomes but is also found in centric heterochromatin, chromosome 4, and telomeric sites. Histone H3 methylated in vitro by the E(Z)/ESC complex binds specifically to Polycomb protein.     

Polycomb-like 3 promotes polycomb repressive complex 2 binding to CpG islands and embryonic stem cell self-renewal

Polycomb repressive complex 2 (PRC2) trimethylates lysine 27 of histone H3 (H3K27me3) to regulate gene expression during diverse biological transitions in development, embryonic stem cell (ESC) differentiation, and cancer. Here, we show that Polycomb-like 3 (Pcl3) is a component of PRC2 that promotes ESC self-renewal. Using mass spectrometry, we identified Pcl3 as a Suz12 binding partner and confirmed Pcl3 interactions with core PRC2 components by co-immunoprecipitation. Knockdown of Pcl3 in ESCs increases spontaneous differentiation, yet does not affect early differentiation decisions as assessed in teratomas and embryoid bodies, indicating that Pcl3 has a specific role in regulating ESC self-renewal. Consistent with Pcl3 promoting PRC2 function, decreasing Pcl3 levels reduces H3K27me3 levels while overexpressing Pcl3 increases H3K27me3 levels. Furthermore, chromatin immunoprecipitation and sequencing (ChIP-seq) reveal that Pcl3 co-localizes with PRC2 core component, Suz12, and depletion of Pcl3 decreases Suz12 binding at over 60% of PRC2 targets. Mutation of conserved residues within the Pcl3 Tudor domain, a domain implicated in recognizing methylated histones, compromises H3K27me3 formation, suggesting that the Tudor domain of Pcl3 is essential for function. We also show that Pcl3 and its paralog, Pcl2, exist in different PRC2 complexes but bind many of the same PRC2 targets, particularly CpG islands regulated by Pcl3. Thus, Pcl3 is a component of PRC2 critical for ESC self-renewal, histone methylation, and recruitment of PRC2 to a subset of its genomic sites.     

Elements of the Polycomb Repressor SU(Z)12 Needed for Histone H3-K27 Methylation,the Interface with E(Z), and In Vivo Function

Polycomb repressive complex 2 (PRC2) is an essential chromatin-modifying enzyme that implements gene silencing. PRC2 methylates histone H3 on lysine-27 and is conserved from plants to flies to humans. In Drosophila melanogaster, PRC2 contains four core subunits: E(Z), SU(Z)12, ESC, and NURF55. E(Z) bears a SET domain that houses the enzyme active site. However, PRC2 activity depends upon critical inputs from SU(Z)12 and ESC. The stimulatory mechanisms are not understood. We present here functional dissection of the SU(Z)12 subunit. SU(Z)12 contains two highly conserved domains: an ∼140-amino-acid VEFS domain and a Cys₂-His₂ zinc finger (ZnF). Analysis of recombinant PRC2 bearing VEFS domain alterations, including some modeled after leukemia mutations, identifies distinct elements needed for SU(Z)12 assembly with E(Z) and stimulation of histone methyltransferase. The results define an extensive VEFS subdomain that organizes the SU(Z)12-E(Z) interface. Although the SU(Z)12 ZnF is not needed for methyltransferase in vitro, genetic rescue assays show that the ZnF is required in vivo. Chromatin immunoprecipitations reveal that this ZnF facilitates PRC2 binding to a genomic target. This study defines functionally critical SU(Z)12 elements, including key determinants of SU(Z)12-E(Z) communication. Together with recent findings, this illuminates PRC2 modulation by conserved inputs from its noncatalytic subunits.     

Characterization of Aquilegia Polycomb Repressive Complex 2 homologs reveals absence of imprinting

Epigenetic regulation is important for maintaining gene expression patterns in multicellular organisms. The Polycomb Group (PcG) proteins form several complexes with important and deeply conserved epigenetic functions in both the plant and animal kingdoms. The plant Polycomb Repressive Complex 2 (PRC2) contains four core proteins, Enhancer of Zeste (E(z)), Suppressor of Zeste 12 (Su(z)12), Extra Sex Combs (ESC), and Multicopy Suppressor of IRA 1 (MSI1), and functions in many developmental transitions. In some plant species, including rice and Arabidopsis, duplications in the core PRC2 proteins allow the formation of PRC2s with distinct developmental functions. In addition, members of the plant specific VEL PHD family have been shown to associate with the PRC2 complex in Arabidopsis and may play a role in targeting the PRC2 to specific loci. Here we examine the evolution and expression of the PRC2 and VEL PHD families in Aquilegia, a member of the lower eudicot order Ranunculales and an emerging model for the investigation of plant ecology, evolution and developmental genetics. We find that Aquilegia has a relatively simple PRC2 with only one homolog of Su(z)12, ESC and MSI1 and two ancient copies of E(z), AqSWN and AqCLF. Aquilegia has four members of the VEL PHD family, three of which appear to be closely related to Arabidopsis proteins known to associate with the PRC2. The PRC2 and VEL PHD family proteins are expressed at a relatively constant level throughout Aquilegia vulgaris development, with the VEL PHD family and MSI1 expressed at higher levels during and after vernalization and in the inflorescence. Both AqSWN and AqCLF are expressed in Aquilegia endosperm but neither copy is imprinted.     

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.     

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.