InteractiveFly: GeneBrief

Polycomblike: Biological Overview | References

PcG protein complex PRC2 is thought to be the histone methyltransferase (HMTase) responsible for H3-K27 trimethylation at Polycomb target genes. This study reports the biochemical purification and characterization of a distinct form of Drosophila PRC2 that contains the Polycomb group protein Polycomblike (Pcl). Like PRC2, Pcl-PRC2 is an H3-K27-specific HMTase that mono-, di- and trimethylates H3-K27 in nucleosomes in vitro. Analysis of Drosophila mutants that lack Pcl unexpectedly reveals that Pcl-PRC2 is required to generate high levels of H3-K27 trimethylation at Polycomb target genes but is dispensable for the genome-wide H3-K27 mono- and dimethylation that is generated by PRC2. In Pcl mutants, Polycomb target genes become derepressed even though H3-K27 trimethylation at these genes is only reduced and not abolished, and even though targeting of the Polycomb protein complexes PhoRC and PRC1 to Polycomb response elements is not affected. Pcl-PRC2 is thus the HMTase that generates the high levels of H3-K27 trimethylation in Polycomb target genes that are needed to maintain a Polycomb-repressed chromatin state (Nekrasov, 2007).

Genetic studies using Drosophila first identified Polycomb group (PcG) genes as regulators that are required for the long-term repression of HOX genes during development. To date, 17 different genes in Drosophila are classified as PcG members because mutations in these genes cause misexpression of HOX genes. All Drosophila PcG genes are also conserved in mammals and at least some of them are also conserved in plants. In all these organisms, PcG gene products function as repressors of HOX and/or other regulatory genes that control specific developmental programs. Moreover, recent studies that analyzed genome-wide binding of PcG proteins in Drosophila and in mammalian cells have identified a large number of target sites, and thus a whole new set of genes that potentially is subject to PcG repression (Nekrasov, 2007).

Biochemical purification and characterization of PcG protein complexes has advanced understanding of the PcG system. To date, three distinct PcG protein complexes have been isolated from Drosophila: PhoRC, PRC1 and PRC2. Biochemically purified Drosophila PRC2 contains the three PcG proteins Enhancer of zeste [E(z)], Suppressor of zeste 12 [Su(z)12] and Extra sex combs (Esc) and, in addition, Nurf55 (FlyBase name: Caf1), a protein that is present in many different chromatin complexes (Czermin, 2002; Müller, 2002). Drosophila PRC2 and the homologue mammalian complex are histone methyltransferases (HMTases) that specifically methylate H3-K27 in nucleosomes (Cao, 2002; Czermin, 2002; Kuzmichev, 2002; Müller, 2002). Chromatin immunoprecipitation (X-ChIP) analyses in Drosophila showed that PRC2 binds in a localized manner at Polycomb response elements (PREs) of target genes, but that H3-K27 trimethylation is present across the whole upstream control, promoter and coding region of these genes (Kahn, 2006; Mohd-Sarip, 2006; Papp, 2006; Schwartz, 2006). Studies that compared the inactive and active state of the HOX gene Ubx in developing Drosophila have found that PRC2 is constitutively bound at PREs and, surprisingly, that the whole upstream control region is constitutively trimethylated at H3-K27 (Papp, 2006). However, presence or absence of H3-K27 trimethylation in the Ubx promoter and coding region correlates tightly with the gene being repressed or active, respectively (Papp, 2006). H3-K27 trimethylation is thus a distinctive mark of PcG-repressed chromatin (Nekrasov, 2007).

Analysis of E(z) mutants suggests that E(z) is also responsible for the genome-wide H3-K27 mono- and dimethylation that has been reported to be present on more than 50% of H3 in Drosophila (Ebert, 2004). However, biochemical analyses showed that E(z) protein alone does not bind to nucleosomes and is virtually inactive as an enzyme; E(z) needs to associate with Su(z)12 and Nurf55 for nucleosome binding and with Esc for enzymatic activity (Czermin, 2002; Müller, 2002; Ketel, 2005; Nekrasov, 2005). This implies that the genome-wide H3-K27 mono- and dimethylation is generated by PRC2 or another E(z)-containing complex that is able to interact in a non-targeted manner with nucleosomes across the whole genome. Conversely, this raises the question whether H3-K27 trimethylation at PcG target genes is simply a consequence of PRC2 being targeted to PREs or whether additional features such as post-translational modifications or associated factors are required (Nekrasov, 2007).

Previous studies reported that the PcG protein Polycomblike (Pcl) interacts with E(z) in GST pull-down, yeast two-hybrid and co-immunoprecipitation assays (O'Connell, 2001; Tie, 2003). Like most other PcG proteins, Pcl has also been found to be bound at PREs in Drosophila (Tie, 2003; Papp, 2006). However, to date, no Pcl-containing complexes have been purified and the role of Pcl in PcG repression has remained enigmatic. This study reports the biochemical purification of Pcl complexes. Pcl is shown to exist in a stable complex with PRC2. This analyses demonstrate that this Pcl complex plays a critical role in generating high levels of repressive H3-K27 trimethylation at PcG target genes (Nekrasov, 2007).

Biochemically purified Pcl complexes contain Pcl together with the four core subunits of PRC2. In contrast, biochemically purified E(z) complexes contain only substoichiometric amounts of Pcl and the previously described purifications of PRC2 failed to reveal Pcl in the purified material (Cao, 2002; Czermin, 2002; Müller, 2002; Kuzmichev, 2002). Moreover, fractionation of crude nuclear extracts by gel filtration indicated that Pcl and PRC2 components Esc, E(z) and Su(z)12 co-fractionate in high-molecular-weight assemblies, but that the bulk of these other PRC2 components is present in lower-molecular-weight fractions that do not contain Pcl (O'Connell, 2001; Tie, 2003). Taken together, these observations suggest that only a fraction of PRC2 is associated with Pcl and that Pcl-PRC2 is a distinct complex (Nekrasov, 2007).

Previous X-ChIP studies showed that Pcl and Su(z)12 colocalize at Ubx and Abd-B PREs (Papp, 2006). This suggests that Pcl-PRC2 is bound at these PREs. In this study, the analysis of Drosophila mutants that lack Pcl protein and therefore lack Pcl-PRC2, provided insight into the function of this complex. The results provide strong evidence that Pcl-PRC2 is needed to generate high levels of H3-K27 trimethylation in the chromatin of PcG target genes. Unlike in E(z) or Su(z)12 mutants, removal of Pcl in embryos or in imaginal discs only reduces but does not eliminate H3-K27 trimethylation. Nevertheless, repression of several PcG target genes is abolished in Pcl mutants. This suggests that not only the mere presence of H3-K27me3, but presence of high levels of H3-K27me3 is crucial for maintaining these PcG target genes in the repressed state. Previous studies on the Ubx gene suggested that presence of H3-K27 trimethylation in the promoter and coding region is critical for PcG repression (Papp, 2006). One possibility would be that it is the overall density of H3-K27me3-marked nucleosomes across the promoter and coding region that determines whether a PcG target gene is repressed. Another possibility would be that even though a whole chromatin domain becomes trimethylated at H3-K27, only a few H3-K27me3-marked nucleosomes at a particular position (e.g., around the transcription start site) are actually required for repression, and failure to maintain this trimethylation results in loss of repression (Nekrasov, 2007).

The observation that Su(z)12 binding and H3-K27 trimethylation are reduced but not lost in the absence of Pcl is consistent with the idea that Pcl might help anchoring PRC2 to PREs, but it also suggests that at least some PRC2 must be targeted to PREs independently of Pcl. It seems likely that the residual H3-K27 trimethylation present in Pcl mutant embryos and in Pcl mutant clones in imaginal discs is generated by PRC2 that is bound at PREs independently of Pcl. In this context it is important to note that not only Pcl-PRC2 but also PRC2 is able to trimethylate H3-K27 in recombinant nucleosomes in vitro. Apart from the suggested role in tethering of PRC2 to PREs, it is possible that Pcl also functions in a post-recruitment step to help PRC2 generate high levels of H3-K27 trimethylation at target genes. For example, the tudor domain and PHD fingers of PRE-bound Pcl might interact with modified nucleosomes in the promoter and coding region of target genes to ensure that they become trimethylated at H3-K27 by the associated PRE-tethered PRC2 (Nekrasov, 2007).

Finally, no evidence was found that Pcl-PRC2 would be required for the genome-wide H3-K27 mono- and di-methylation. X-ChIP analyses suggest that H3-K27 mono- and dimethylation across the genome might even slightly increase in the absence of Pcl. In contrast, there is a loss of all H3-K27 methylation in either E(z) or Suz)12 mutants (Ebert, 2004). This suggests that PRC2 or another E(z)-containing complex generates the genome-wide H3-K27 mono- and dimethylation. The experiments in Pcl mutants thus allowed dissection of the role of different H3-K27 methylation states in Drosophila. The selective reduction of H3-K27me3 levels, and the concomitant loss of repression of PcG target genes in Pcl mutants, provides compelling evidence that only the trimethylated state of H3-K27 is functional in PcG repression in Drosophila. Pcl-PRC2 is evidently critically needed to generate the high levels of H3-K27 trimethylation that are required to maintain a Polycomb-repressed chromatin state (Nekrasov, 2007).

Recruitment of Drosophila Polycomb-group proteins by Polycomblike, a component of a novel protein complex in larvae

Polycomb-group (PcG) proteins are highly conserved epigenetic transcriptional repressors that play central roles in numerous examples of developmental gene regulation. Four PcG repressor complexes have been purified from Drosophila embryos: PRC1, PRC2, Pcl-PRC2 and PhoRC. Previous studies described a hierarchical recruitment pathway of PcG proteins at the bxd Polycomb Response Element (PRE) of the Ultrabithorax (Ubx) gene in larval wing imaginal discs. The DNA-binding proteins Pho and/or Phol are required for target site binding by PRC2, which in turn is required for chromosome binding by PRC1. This study identified a novel larval complex that contains the PcG protein Polycomblike (Pcl) that is distinct from PRC1 and PRC2 and which is also dependent on Pho and/or Phol for binding to the bxd PRE in wing imaginal discs. RNAi-mediated depletion of Pcl in larvae disrupts chromosome binding by E(z), a core component of PRC2, but Pcl does not require E(z) for chromosome binding. These results place the Pcl complex (PCLC) downstream of Pho and/or Phol and upstream of PRC2 and PRC1 in the recruitment hierarchy (Savla, 2008).

Drosophila Polycomb-group (PcG) genes were originally identified as negative regulators of Hox genes. PcG-mediated silencing in Drosophila occurs in essentially two broadly defined stages: assumption of transcriptional repression responsibilities from gene-specific transcription factors in early embryos, followed by maintenance of the silenced state through many cycles of cell division beginning in mid-late-stage embryos and continuing throughout the remainder of development (Savla, 2008).

Although much of the genetic analysis of PcG functions and studies of the mechanisms by which PcG proteins are targeted to specific genomic sites have focused on their activities in larval tissues, in vitro biochemical analyses have focused on PcG complexes isolated from embryos: PRC1, PRC2 and PhoRC. PRC1 possesses multiple chromatin modifying activities in vitro suggesting that it, among PcG complexes, might be most directly responsible for preventing transcription. The primary functions of PhoRC and PRC2 appear to be to recruit and/or stabilize target site binding by PRC1, and potentially other PcG proteins. PhoRC includes the DNA-binding PcG protein Pleiohomeotic (Pho), which binds to sites within Polycomb Response Elements (PREs) that serve as docking platforms for PcG proteins. Pho directly interacts with components of both PRC1 and PRC2, and is required for recruitment of both complexes (Mohd-Sarip, 2002; Mohd-Sarip, 2005; Wang, 2004). The E(z) subunit of PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), facilitating recruitment of PRC1 (Savla, 2008).

A variant of PRC2 has recently been described that includes the PcG protein Polycomblike (Pcl) (Nekrasov, 2007). On the basis of gel filtration analysis of native complexes in embryo nuclear extracts and the stoichiometry of the purified Pcl-PRC2 complex, it appears that the majority of embryonic Pcl is present in Pcl-PRC2, but that the other PRC2 core subunits, E(z), Su(z)12, Esc and NURF55 (also known as Caf1 - FlyBase), predominantly are in a complex(es) lacking Pcl (Nekrasov, 2007; O'Connell, 2001; Tie, 2003). It has been proposed that inclusion of Pcl in PRC2 is required for high levels of H3K27me3 in vivo, although the in vitro histone methyltransferase activity of Pcl-PRC2 is indistinguishable from that of PRC2 lacking Pcl (Nekrasov, 2007). In this study, a larval Pcl-containing complex is identified that is distinct from PRC2 and PRC1 and shown to be required for chromosome binding by these PcG complexes (Savla, 2008).

In order to examine potential differences between embryonic and larval stage PcG complexes, larval nuclear extracts were fractionated over a Superose 6 gel filtration column, and western blots of the fractions were probed with anti-E(z) and anti-Pcl antibodies. Larval E(z)-containing complexes have a relative mass of ~500 to 600 kDa, similar to that of embryonic PRC2 complexes that lack Pcl (Ng, 2000). However, Pcl was undetectable in E(z)-containing fractions and appeared to be in a complex with a relative mass of ~1500 kDa. This is different from the fractionation profile of Pcl from embryo extracts, in which it co-fractionates with E(z) in native complexes with relative mass estimates in the range of ~650 kDa (O'Connell, 2001) to 1000 kDa (Tie, 2003), suggesting that, unlike its association with a subset of PRC2 complexes in embryos, Pcl functions as a component of a distinct complex in larvae, which will be referred to as the Pcl-Complex (PCLC) (Savla, 2008).

In order to further investigate the relationship of Pcl with other PcG proteins and its role in PcG-mediated silencing in larvae, chromatin immunoprecipitation (ChIP) assays were performed on wing imaginal discs. The PcG maintains the transcriptional silence of the Hox gene Ultrabithorax (Ubx) in the epithelial cells of wing discs. Other PcG proteins, including the DNA-binding proteins Pho and Phol and components of the PRC1 and PRC2 complexes, have previously been shown to be present at the major PRE in the Ubx cis-regulatory bxd region in this tissue. Consistent with a previous report (Papp, 2006), Pcl also was detected at the bxd PRE, and appears to largely colocalize with E(z) and Phol (Savla, 2008).

A hierarchical relationship among PcG proteins at the bxd PRE in which Pho and/or Phol are required, but are not necessarily sufficient, for recruitment of PRC2, which in turn facilitates recruitment of PRC1 (Wang, 2004). In order to determine how Pcl might fit into this recruitment pathway, ChIP assays were performed on E(z) mutant wing imaginal discs. E(z)⁶¹ is a temperature-sensitive allele that displays nearly wild-type activity at 18°C, but strongly reduced activity at 29°C. Following shift from 18°C to 29°C, bxd PRE binding by E(z)⁶¹ protein is rapidly lost and along with it the detection of H3K27me3 and Pc in this region (Cao, 2002; Wang, 2004). ChIP assays of wing discs dissected from E(z)⁶¹ larvae 24 hours following shift from 18° to 29°C confirmed loss of E(z) from the PRE, but revealed no effect on Pcl and Phol binding to PRE fragments 3 and 4, but a slight decrease of both proteins at the PRE 2 fragment. It is speculated that Pcl and Phol signals at this proximal edge of the PRE are partly due to protein-protein cross-links, which might be reduced in the absence of PRC2. Retention of Pcl at the PRE in the absence of E(z) and by extension absence of PRC1, which requires PRC2 for binding to this region, confirms that Pcl is not a stable subunit of larval versions of either PRC1 or PRC2 and is consistent with its inclusion in a distinct complex (Savla, 2008).

Flies that are homozygous for null Pcl alleles die as embryos and no conditional Pcl alleles exist, precluding reciprocal experiments on Pcl mutant larvae. Therefore, transgenic fly lines were generated that contain inserts of a pWIZ-Pcl construct, which expresses Pcl shRNA under the control of Gal4, permitting inducible RNAi-mediated knockdown of Pcl in combination with Gal4 drivers. Individuals that contain both pWIZ-Pcl and P{GAL4-da.G32}, which constitutively expresses Gal4, died as early pupae and exhibited dramatically reduced levels of Pcl in wing imaginal discs. E(z) levels were not affected. ChIP assays of these Pcl-depleted wing discs confirmed reduced Pcl levels at the bxd PRE and revealed commensurate loss of E(z). Thus, although Pcl does not require PRC2 for PRE binding, Pcl, presumably functioning as a subunit of PCLC, is needed for stable binding of PRC2 to the bxd PRE. Phol remains at the PRE in the absence of Pcl (Savla, 2008).

In order to determine whether Pcl (like components of PRC1 and PRC2) requires Pho and/or Phol for PRE binding, ChIP assays were performed using wing imaginal discs from phol^81A; pho¹ larvae. Consistent with their role in recruiting other PcG proteins (Mohd-Sarip, 2002; Mohd-Sarip, 2005; Wang, 2004), Pcl was lost from the bxd PRE in the absence of Pho and Phol. These observations at the bxd PRE also appear to generally apply to PcG-binding sites throughout the genome (Savla, 2008).

These results demonstrate the existence of a distinct Pcl protein complex in larvae that is required for recruitment of PRC2 to chromosomal target sites and/or to stabilize its binding. As previously described, E(z), as a core subunit of PRC2, is required for target site binding by PRC1 (Cao, 2002; Wang, 2004). Therefore, Pcl is indirectly required for chromosome binding by PRC1 as well, although direct interaction with PRC1 cannot be ruled out, similar to the way in which Pho may contribute to target site binding by PRC1 by interacting both with PRC2 subunits (Wang, 2004) and with Pc, a core subunit of PRC1 (Mohd-Sarip, 2002; Mohd-Sarip, 2005; Savla, 2008 and references therein).

In vitro histone methyltransferase assays of Pcl-PRC2 show that its activity and specificity for methylation of H3K27 are essentially indistinguishable from that of PRC2 complexes lacking Pcl. ChIP analysis of Pcl mutant embryos has shown that Pcl does not seem to be required for target site binding by other PRC2 subunits, but that it may be needed for high levels of trimethylation of H3K27 (Nekrasov, 2007). One explanation for these observations is that the contribution of Pcl to Pcl-PRC2 in embryos might be to mediate interaction with other proteins that are yet to be identified. In larvae, Pcl exists as a subunit of a distinct complex. Given the ability of Pcl to directly interact with several PRC2 subunits (Nekrasov, 2007; O'Connell, 2001), colocalization of Pcl and E(z) at the PRE (Papp, 2006), and dependence of E(z) on Pcl for binding to the bxd PRE and other genomic sites, it is likely that PCLC is closely associated with PRC2 at target sites in larvae. In both embryos and larvae, some of the activities attributed to Pcl might, upon further inspection, be due to the activities of other Pcl-associated proteins, the close apposition of which with PRC2 and other PcG complexes may be mediated by Pcl. The differential deployment of Pcl as a subunit of PRC2 and as a subunit of PCLC at distinct developmental stages is intriguing and might reflect the different molecular activities needed for establishment of silencing in embryos and maintenance of the silenced state in larval tissues. A more detailed understanding of the mechanisms by which Pcl contributes to PcG silencing will require identification of the other proteins contained within the larval PCLC complex and the potential biochemical activities of the complex (Savla, 2008).

Histone trimethylation and the maintenance of transcriptional ON and OFF states by trxG and PcG proteins

Polycomb group (PcG) and trithorax group (trxG) proteins act as antagonistic regulators to maintain transcriptional OFF and ON states of HOX and other target genes. To study the molecular basis of PcG/trxG control, the chromatin of the HOX gene Ultrabithorax (Ubx) was analyzed in Ubx^OFF and Ubx^ONcells purified from developing Drosophila. PcG protein complexes PhoRC, PRC1, and PRC2 and the Trx protein are all constitutively bound to Polycomb response elements (PREs) in the OFF and ON state. In contrast, the trxG protein Ash1 is only bound in the ON state; not at PREs but downstream of the transcription start site. In the OFF state, extensive trimethylation was found at H3-K27, H3-K9, and H4-K20 across the entire Ubx gene; i.e., throughout the upstream control, promoter, and coding region. In the ON state, the upstream control region is also trimethylated at H3-K27, H3-K9, and H4-K20, but all three modifications are absent in the promoter and 5' coding region. These analyses of mutants that lack the PcG histone methyltransferase (HMTase) E(z) or the trxG HMTase Ash1 provide strong evidence that differential histone lysine trimethylation at the promoter and in the coding region confers transcriptional ON and OFF states of Ubx. In particular, these results suggest that PRE-tethered PcG protein complexes act over long distances to generate Pc-repressed chromatin that is trimethylated at H3-K27, H3-K9, and H4-K20, but that the trxG HMTase Ash1 selectively prevents this trimethylation in the promoter and coding region in the ON state (Papp, 2006; Full text of article).

Previous studies have shown that PhoRC contains the DNA-binding PcG protein Pho that targets the complex to PREs, and dSfmbt, a novel PcG protein that selectively binds to histone H3 and H4 tail peptides that are mono- or dimethylated at H3-K9 or H4-K20 (H3-K9me1/2 and H4-K20me1/2, respectively) (Klymenko, 2006). PRC1 contains the PcG proteins Ph, Psc, Sce/Ring, and Pc. PRC1 inhibits nucleosome remodeling and transcription in in-vitro assays and its subunit Pc specifically binds to trimethylated K27 in histone H3 (H3-K27me3). PRC2 contains the PcG proteins E(z), Su(z)12, and Esc as well as Nurf55, and this complex functions as a histone methyltransferase (HMTase) that specifically methylates K27 in histone H3 (H3-K27) in nucleosomes (Papp, 2006).

This study used quantitative X-ChIP analysis to examine the chromatin of the HOX gene Ubx in its ON and OFF state in developing Drosophila larvae. Previous genetic studies had established that all of the PcG and trxG proteins analyzed in this study are critically needed to maintain Ubx OFF and ON states in the very same imaginal disc cells in which their binding to Ubx was analyzed in this study. The following conclusions can be drawn from the analyses reported in this study. (1) The PcG protein complexes PhoRC, PRC1, and PRC2 (recognized using antibodies against Su(z)12 and Pcl) and the Trx protein are all highly localized at PREs, but they are all constitutively bound at comparable levels in the OFF and ON state. (2) The trxG protein Ash1 is bound only in the ON state, where it is specifically localized ~1 kb downstream of the transcription start site. (3) In the OFF state, PRC2 and other unknown HMTases trimethylate H3-K27, H3-K9, and H4-K20 over an extended 100-kb domain that spans the whole Ubx gene. (4) In the ON state, comparable H3-K27, H3-K9, and H4-K20 trimethylation is restricted to the upstream control regions and Ash1 selectively prevents this trimethylation in the promoter and coding region. (5) Repressed Ubx chromatin is extensively tri- but not di- or monomethylated at H3-K27, H3-K9, and H4-K20. (6) Trimethylation of H3-K27, H3-K9, and H4-K20 at imaginal disc enhancers in the upstream control region does not impair the function of these enhancers in the ON state. (7) TBP and Spt5 are bound at the Ubx transcription start site in the ON and OFF state, but Kis is only bound in the ON state. This suggests that in the OFF state, transcription is blocked at a late step of transcriptional initiation, prior to the transition to elongation. A schematic representation of PcG and trxG protein complex binding and histone methylation at the Ubx gene in the OFF and ON state is presented (Papp, 2006).

Unexpectedly, ChIP analysis by qPCR used in this study and in a similar study by the laboratory of Vincent Pirrotta (V. Pirrotta, pers. comm. to Papp, 2006) reveals that the relationship between PcG and trxG proteins and histone methylation is quite different from the currently held views. Specifically, X-ChIP studies have reported that H3-K27 trimethylation is localized at PREs and this led to the model that recruitment of PRC1 to PREs occurs through H3-K27me3 (i.e., via the Pc chromodomain). In contrast, the current study and that by Vincent Pirrotta found H3-K27 trimethylation to be present across the whole inactive Ubx gene, both in wing discs and in S2 cells (V. Pirrotta, pers. comm. to Papp, 2006). No specific enrichment of H3-K27 trimethylation at PREs has been detected; rather, a reduction of H3-K27me3 signals is observed at PREs, consistent with the reduced signals of H3 that are detected at these sites. Consistent with these results, genome-wide analyses of PcG protein binding and H3-K27me3 profiles in S2 cells revealed that, at most PcG-binding sites in the genome, PcG proteins are tightly localized, whereas H3-K27 trimethylation is typically present across an extended domain that often spans the whole coding region. How could the differences between this study and the earlier studies be explained? It should be noted that in contrast to the qPCR analysis used in the current study, previous studies all relied on nonquantitative end-point PCR after 36 or more cycles to assess the X-ChIP results. It is possible that these experimental differences account for the discrepancies (Papp, 2006).

PhoRC, PRC1, and PRC2 are all tightly localized at PREs but they are all constitutively bound at the inactive and active Ubx gene. This suggests that recruitment of PcG complexes to PREs occurs by default. Although all three complexes are bound at comparable levels to the bxd PRE in the inactive and active state and PhoRC is also bound at comparable levels at the bx PRE, it should be pointed out that the levels of PRC1 and PRC2 binding at the bx PRE are about twofold reduced in the active Ubx gene compared with the inactive Ubx gene. Even though there is still high-level binding of PRC1 and PRC2 at the bx PRE, it cannot be excluded that the observed reduction in binding helps to prevent default PcG repression of the active Ubx gene. It is possible that transcription through the bx PRE reduces PRC1 and PRC2 binding at this PRE. Transcription through PREs has been proposed to serve as an 'anti-silencing' mechanism that prevents default silencing of active genes by PREs (Papp, 2006),

The highly localized binding of all three PcG protein complexes at PREs, together with earlier studies on PRE targeting of PcG protein complexes supports the idea that not only PhoRC but also PRC1 and PRC2 are targeted to PRE DNA through interactions with Pho and/or other sequence-specific DNA-binding proteins. In the case of trxG proteins, the binding modes are more diverse. In particular, recruitment of Trx protein to PREs and to the promoter is also constitutive in both states but recruitment of Ash1 to the coding region is clearly observed only at the active Ubx gene. At present, it is not known how Trx or Ash1 are targeted to these sites. It is possible that a transcription-coupled process recruits Ash1 to the position 1 kb downstream of the transcription start site (Papp, 2006).

In contrast to the localized and constitutive binding of PcG protein complexes and the Trx protein, it was found that the patterns of histone trimethylation are very distinct in the active and inactive Ubx gene. The results also suggest that the locally bound PcG and trxG HMTases act across different distances to methylate chromatin. For example, H3-K4 trimethylation is confined to the first kilobase of the Ubx coding region where Ash1 and Trx are bound, whereas H3-K27 trimethylation is present across an extended 100-kb domain of chromatin that spans the whole Ubx gene. This suggests that PRE-tethered PRC2 is able to trimethylate H3-K27 in nucleosomes that are as far as 30 kb away from the bxd or bx PREs. Unexpectedly, it was found that the H3-K9me3 and H4-K20me3 profiles closely match the H3-K27me3 profile. At present it is not known which HMTases are responsible for H3-K9 and H4-K20 trimethylation, but analysis of E(z) mutants indicate that these modifications may be generated in a sequential manner, following H3-K27 trimethylation by PRC2. The molecular mechanisms that permit locally tethered HMTases such as PRE-bound PRC2 to maintain such extended chromatin stretches in a trimethylated state are only poorly understood. However, a recent study showed that the PhoRC subunit dSfmbt selectively binds to mono- and dimethylated H3-K9 and H4-K20 in peptide-binding assays (Klymenko. 2006). One possibility would be that dSfmbt participates in the process that ensures that repressed Ubx chromatin is trimethylated at H3-K27, H3-K9, and H4-K20. For example, dSfmbt, tethered to PREs by Pho, may interact with nucleosomes of lower methylated states (i.e., H3-K9me1/2 or H4-K20me1/2) in the flanking chromatin and thereby bring them into the vicinity of PRE-anchored HMTases that will hypermethylate them to the trimethylated state (Papp, 2006).

These analyses suggest that H3-K27, H3-K9, and H4-K20 trimethylation in the promoter and coding region is critical for Polycomb repression. (1) Although H3-K27, H3-K9, and H4-K20 trimethylation is present at the inactive and active Ubx gene, it is specifically depleted in the promoter and coding region in the active Ubx gene. (2) Misexpression of Ubx in wing discs with impaired E(z) activity correlates well with loss of H3-K27 and H3-K9 trimethylation at the promoter and 5' coding region. It is possible that the persisting H3-K27 and H3-K9 trimethylation in the 3' coding region is responsible for maintenance of repression in those E(z) mutant wing discs cells that do not show misexpression of Ubx. (3) In haltere and third-leg discs of ash1 mutants, the promoter and coding region become extensively trimethylated at H3-K27 and H3-K9, and this correlates with loss of Ubx expression. Previous studies showed that Ubx expression is restored in ash1 mutants cells that also lack E(z) function. Together, these findings therefore provide strong evidence that Ash1 is required to prevent PRC2 and other HMTases from trimethylating the promoter and coding region at H3-K27 and H3-K9. The loss of H3-K4 trimethylation in ash1 mutants is formally consistent with the idea that Ash1 exerts its antirepressor function by trimethylating H3-K4 in nucleosomes in the promoter and 5' coding region, but other explanations are possible (Papp, 2006).

But how might H3-K27, H3-K9, and H4-K20 trimethylation in the promoter and coding region repress transcription? The observation that TBP and Spt5 are also bound to the promoter in the OFF state suggests that these methylation marks do not prevent assembly of the basic transcription apparatus at the promoter. However, the nucleosome remodeling factor Kis is not recruited in the OFF state, and transcription thus appears to be blocked at a late step of transcriptional intiation prior to elongation. It was found that the low-level binding of Pc in the coding region correlates with the presence of H3-K27 trimethylation; i.e., Pc and H3-K27me3 are both present in the OFF state, but are absent in the ON state. One possible scenario would thus be that H3-K27 trimethylation in the promoter and coding region permits direct recruitment of PRC1. According to this view, locally recruited PRC1 would then repress transcription; e.g., by inhibiting nucleosome remodeling in the promoter region. However, several observations are not easily reconciled with such a simple 'recruitment-by-methylation' model. First, peak levels of all PRC1 components are present at PREs and, apart from Pc, very little binding is observed outside of PREs. Second, excision of PRE sequences from a PRE reporter gene during development leads to a rapid loss of silencing, suggesting that transcriptional repression requires the continuous presence of PREs and the proteins that are bound to them. A second, more plausible scenario would therefore be that DNA-binding factors first target PcG protein complexes to PREs, and that these PRE-tethered complexes then interact with trimethylated nucleosomes in the flanking chromatin in order to repress transcription. For example, it is possible that bridging interactions between the Pc chromodomain in PRE-tethered PRC1 and H3-K27me3-marked chromatin in the promoter or coding region permit other PRE-tethered PcG proteins to recognize the chromatin interval across which they should act, e.g., to inhibit nucleosome remodeling in the case of PRC1 or to trimethylate H3-K27 at hypomethylated nucleosomes in the case of PRC2 (Papp, 2006).

The analysis of a HOX gene in developing Drosophila suggests that histone trimethylation at H3-K27, H3-K9, and H4-K20 in the promoter and coding region plays a central role in generating and maintaining of a PcG-repressed state. Contrary to previous reports, the current findings provide no evidence that H3-K27 trimethylation is specifically localized at PREs and could thus recruit PRC1 to PREs; widespread H3-K27 trimethylation is found across the whole transcription unit. The data presented in this study provide evidence that PREs serve as assembly platforms for PcG protein complexes such as PRC2 that act over considerable distances to trimethylate H3-K27 across long stretches of chromatin. The presence of this trimethylation mark in the chromatin that flanks PREs may in turn serve as a signal to define the chromatin interval that is targeted by other PRE-tethered PcG protein complexes such as PRC1. The results reported here also provide a molecular explanation for the previously reported antirepressor function of trxG HMTases; selective binding of Ash1 to the active HOX gene blocks PcG repression by preventing PRC2 from trimethylating the promoter and coding region. It is possible that the extended domain of combined H3-K27, H3-K9, and H4-K20 trimethylation creates not only the necessary stability for transcriptional repression, but that it also provides the molecular marks that permits PcG repression to be heritably maintained through cell division (Papp, 2006).

Polycomblike PHD fingers mediate conserved interaction with enhancer of zeste protein

The products of Polycomb group (PcG) genes are required for the epigenetic repression of a number of important developmental regulatory genes, including homeotic genes. Enhancer of zeste [E(Z)] is a Drosophila PcG protein that binds directly to another PcG protein, Extra Sex Combs (ESC), and is present along with ESC in a 600-kDa complex in Drosophila embryos. Using yeast two-hybrid and in vitro binding assays, it was shown that E(Z) binds directly to another PcG protein, Polycomblike (PCL). PCL.E(Z) interaction is shown to be mediated by the plant homeodomain (PHD) fingers domain of PCL, providing evidence that this motif can act as an independent protein interaction domain. An association was also observed between PHF1 and EZH2, human homologs of PCL and E(Z), respectively, demonstrating the evolutionary conservation of this interaction. E(Z) was found to not interact with the PHD domains of three Drosophila trithorax group (trxG) proteins, which function to maintain the transcriptional activity of homeotic genes, providing evidence for the specificity of the interaction of E(Z) with the PCL PHD domain. Coimmunoprecipitation and gel filtration experiments demonstrate in vivo association of PCL with E(Z) and ESC in Drosophila embryos. The implications of PCL association with ESC.E(Z) complexes is discussed and the possibility that PCL may either be a subunit of a subset of ESC.E(Z) complexes or a subunit of a separate complex that interacts with ESC.E(Z) complexes (O'Connell, 2001; full text of article).

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. 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; full text of article).

Search PubMed for articles about Drosophila Polycomblike

Cao, R., et al. (2002). Role of Histone H3 lysine 27 methylation in Polycomb-Group silencing. Science 298: 1039-1043. 12351676

Czermin, B., et al. (2002). Drosophila Enhancer of Zeste/Esc complexes have a histone H3 methyltransferase activity that marks chromosomal polycomb sites. Cell 111: 185-196. 12408863

Ebert, A., et al. (2004). Su(var) genes regulate the balance between euchromatin and heterochromatin in Drosophila. Genes Dev. 18: 2973-2983. PubMed citation; Online text

Kahn, T. G., Schwartz, Y. B., Dellino, G. I. and Pirrotta, V. (2006). Polycomb complexes and the propagation of the methylation mark at the Drosophila Ubx gene. J. Biol. Chem. 281(39): 29064-75. Medline abstract: 16887811

Ketel, C. S., et al. (2005). Subunit contributions to histone methyltransferase activities of fly and worm polycomb group complexes. Mol. Cell. Biol. 25: 6857-6868. 16055700

Kuzmichev, A., et al. (2002). Histone methyltransferase activity associated with a human multiprotein complex containing the Enhancer of Zeste protein. Genes Dev. 16: 2893-2905. 12435631

Mohd-Sarip, A., Venturini, F., Chalkley, G.E. and Verrijzer, C.P. (2002). Pleiohomeotic can link polycomb to DNA and mediate transcriptional repression. Mol. Cell Biol. 22: 7473-7483. 12370294

Mohd-Sarip, A., et al. (2005). Synergistic recognition of an epigenetic DNA element by Pleiohomeotic and a Polycomb core complex. Genes Dev. 19: 1755-1760. 16077005

Mohd-Sarip, A., van der Knaap, J. A., Wyman, C., Kanaar, R., Schedl, P. and Verrijzer, C. P. (2006). Architecture of a polycomb nucleoprotein complex. Mol. Cell 24(1): 91-100. Medline abstract: 17018295

Müller, J., et al. (2000). Histone methyltransferase activity of a Drosophila polycomb group repressor complex. Cell 111: 197-208. 12408864

Nekrasov, M., Wild, B., and Müller, J. (2005). Nucleosome binding and histone methyltransferase activity of Drosophila PRC2. EMBO Rep. 6: 348-353. PubMed citation; Online text

Nekrasov, M., et al. (2007). Pcl-PRC2 is needed to generate high levels of H3-K27 trimethylation at Polycomb target genes. EMBO J. 26(18): 4078-88. PubMed citation; Online text

Ng, J., et al. (2000). A Drosophila ESC-E(Z) protein complex is distinct from other polycomb group complexes and contains covalently modified ESC. Mol. Cell. Biol. 20: 20(9): 3069-78. PubMed citation; Online text

O'Connell S., et al. (2001). Polycomblike PHD fingers mediate conserved interaction with enhancer of zeste protein. J. Biol. Chem. 276(46): 43065-73. 11571280

Papp, B. and Muller, J. (2006). Histone trimethylation and the maintenance of transcriptional ON and OFF states by trxG and PcG proteins. Genes Dev. 20(15): 2041-54. PubMed citation; Online text

Savla, U., Benes, J., Zhang, J. and Jones, R. S. (2008). Recruitment of Drosophila Polycomb-group proteins by Polycomblike, a component of a novel protein complex in larvae. Development 135(5): 813-7. PubMed citation; Online text

Schwartz, Y., et al. (2006). Genome-wide analysis of Polycomb targets in Drosophila melanogaster. Nat Genet 38: 700-705. PubMed citation; Online text

Tie, F., Prasad-Sinha, J., Birve, A., Rasmuson-Lestander, A. and Harte, P. J. (2003). A 1-megadalton ESC/E(Z) complex from Drosophila that contains polycomblike and RPD3. Mol. Cell. Biol. 23(9): 3352-62. PubMed citation; Online text

Wang, L., Brown, J. L., Cao, R., Zhang, Y., Kassis, J. A. and Jones, R. S. (2004). Hierarchical recruitment of polycomb group silencing complexes. Mol. Cell 14: 637-646. Medline abstract: 15175158

date revised: 20 February 2008

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