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PubMed2006

Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions.

The Polycomb group (PcG) proteins form chromatin-modifying complexes that are essential for embryonic development and stem cell renewal and are commonly deregulated in cancer. Here, we identify their target genes using genome-wide location analysis in human embryonic fibroblasts. We find that Polycomb-Repressive Complex 1 (PRC1), PRC2, and tri-methylated histone H3K27 co-occupy >1000 silenced genes with a strong functional bias for embryonic development and cell fate decisions. We functionally identify 40 genes derepressed in human embryonic fibroblasts depleted of the PRC2 components (EZH2, EED, SUZ12) and the PRC1 component, BMI-1. Interestingly, several markers of osteogenesis, adipogenesis, and chrondrogenesis are among these genes, consistent with the mesenchymal origin of fibroblasts. Using a neuronal model of differentiation, we delineate two different mechanisms for regulating PcG target genes. For genes activated during differentiation, PcGs are displaced. However, for genes repressed during differentiation, we paradoxically find that they are already bound by the PcGs in nondifferentiated cells despite being actively transcribed. Our results are consistent with the hypothesis that PcGs are part of a preprogrammed memory system established during embryogenesis marking certain key genes for repressive signals during subsequent developmental and differentiation processes.

Carrier Proteins↗

PubMed1998

Maternal control of embryogenesis by MEDEA, a polycomb group gene in Arabidopsis.

The gametophytic maternal effect mutant medea (mea) shows aberrant growth regulation during embryogenesis in Arabidopsis thaliana. Embryos derived from mea eggs grow excessively and die during seed desiccation. Embryo lethality is independent of the paternal contribution and gene dosage. The mea phenotype is consistent with the parental conflict theory for the evolution of parent-of-origin-specific effects. MEA encodes a SET domain protein similar to Enhancer of zeste, a member of the Polycomb group. In animals, Polycomb group proteins ensure the stable inheritance of expression patterns through cell division and regulate the control of cell proliferation.

Alleles↗

PubMed1999

RING1 interacts with multiple Polycomb-group proteins and displays tumorigenic activity.

Polycomb-group (PcG) proteins form large multimeric protein complexes that are involved in maintaining the transcriptionally repressive state of genes. Previously, we reported that RING1 interacts with vertebrate Polycomb (Pc) homologs and is associated with or is part of a human PcG complex. However, very little is known about the role of RING1 as a component of the PcG complex. Here we undertake a detailed characterization of RING1 protein-protein interactions. By using directed two-hybrid and in vitro protein-protein analyses, we demonstrate that RING1, besides interacting with the human Pc homolog HPC2, can also interact with itself and with the vertebrate PcG protein BMI1. Distinct domains in the RING1 protein are involved in the self-association and in the interaction with BMI1. Further, we find that the BMI1 protein can also interact with itself. To better understand the role of RING1 in regulating gene expression, we overexpressed the protein in mammalian cells and analyzed differences in gene expression levels. This analysis shows that overexpression of RING1 strongly represses En-2, a mammalian homolog of the well-characterized Drosophila PcG target gene engrailed. Furthermore, RING1 overexpression results in enhanced expression of the proto-oncogenes c-jun and c-fos. The changes in expression levels of these proto-oncogenes are accompanied by cellular transformation, as judged by anchorage-independent growth and the induction of tumors in athymic mice. Our data demonstrate that RING1 interacts with multiple human PcG proteins, indicating an important role for RING1 in the PcG complex. Further, deregulation of RING1 expression leads to oncogenic transformation by deregulation of the expression levels of certain oncogenes.

3T3 Cells↗

PubMed2000

A Drosophila ESC-E(Z) protein complex is distinct from other polycomb group complexes and contains covalently modified ESC.

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. We report that these two proteins are components of a 600-kDa complex in embryos. Using gel filtration and affinity chromatography, we show that this complex is biochemically distinct from previously described complexes containing the PcG proteins Polyhomeotic, Polycomb, and Sex comb on midleg. In addition, we present evidence 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. We find that mutations in E(z) reduce the ratio of modified to unmodified ESC in vivo. We have also generated germ line transformants 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 it contributes to the function or regulation of these complexes. We discuss how biochemically separable ESC-E(Z) and PC-PH complexes might work together to provide PcG repression.

Animals↗

PubMed2002

The core of the polycomb repressive complex is compositionally and functionally conserved in flies and humans.

The Polycomb group (PcG) genes are required to maintain homeotic genes in a silenced state during development in drosophila and mammals and are thought to form several distinct silencing complexes that maintain homeotic gene repression during development. Mutations in the PcG genes result in developmental defects and have been implicated in human cancer. Although some PcG protein domains are conserved between flies and humans, substantial regions of several PcG proteins are divergent and humans contain multiple versions of each PcG gene. To determine the effects of these changes on the composition and function of a PcG complex, we have purified a human Polycomb repressive complex from HeLa cells (hPRC-H) that contains homologues of PcG proteins found in drosophila embryonic PRC1 (dPRC1). hPRC-H was found to have fewer components than dPRC1, retaining the PcG core proteins of dPRC1 but lacking most non-PcG proteins. Preparations of hPRC-H contained either two or three different homologues of most of the core PcG proteins, including a new Ph homologue we have named HPH3. Despite differences in composition, dPRC1 and hPRC-H have similar functions: hPRC-H is able to efficiently block remodeling of nucleosomal arrays through a mechanism that does not block the ability of nucleases to access and cleave the arrays.

Amino Acid Sequence↗

PubMed2002

Pleiohomeotic can link polycomb to DNA and mediate transcriptional repression.

Polycomb group (PcG) proteins function through cis-acting DNA elements called PcG response elements (PREs) to stably silence developmental regulators, including the homeotic genes. However, the mechanism by which they are targeted to PREs remains largely unclear. Pleiohomeotic (PHO) is a sequence-specific DNA-binding PcG protein and therefore may function to tether other PcG proteins to the DNA. Here, we show that PHO can directly bind to a Polycomb (PC)-containing complex as well as the Brahma (BRM) chromatin-remodeling complex. PHO contacts the BRM complex through its zinc finger DNA-binding domain and a short N-terminal region. A distinct domain of PHO containing a conserved motif contacts the PcG proteins PC and Polyhomeotic (PH). With mobility shift assays and DNA pulldown experiments, we demonstrated that PHO is able to link PC, which lacks sequence-specific DNA-binding activity, to the DNA. Importantly, we found that the PC-binding domain of PHO can mediate transcriptional repression in transfected Drosophila Schneider cells. Concomitant overexpression of PC resulted in stronger PHO-directed repression that was dependent on its PC-binding domain. Together, these results suggest that PHO can contribute to PRE-mediated silencing by direct recruitment of a PC complex to repress transcription.

Animals↗

PubMed2006

Histone deacetylase inhibitors deplete enhancer of zeste 2 and associated polycomb repressive complex 2 proteins in human acute leukemia cells.

Human enhancer of zeste 2 (EZH2) protein belongs to the multiprotein polycomb repressive complex 2, which also includes suppressor of zeste 12 (SUZ12) and embryonic ectoderm development (EED). The polycomb repressive complex 2 complex possesses histone methyltransferase activity mediated by the Su(var)3-9, enhancer of zeste, and trithorax domain of EZH2, which methylates histone H3 on lysine (K)-27 (H3K27). In the present studies, we determined that treatment with the hydroxamate histone deacetylase inhibitor LBH589 or LAQ824 depleted the protein levels of EZH2, SUZ12, and EED in the cultured (K562, U937, and HL-60) and primary human acute leukemia cells. This was associated with decreased levels of trimethylated and dimethylated H3K27, with concomitant depletion of the homeobox domain containing HOXA9 and of MEIS1 transcription factors. Knockdown of EZH2 by EZH2 small interfering RNA also depleted SUZ12 and EED, inhibited histone methyltransferase activity, and reduced trimethylated and dimethylated H3K27 levels, with a concomitant loss of clonogenic survival of the cultured acute myelogenous leukemia (AML) cells. EZH2 small interfering RNA sensitized the AML cells to LBH589-mediated depletion of EZH2, SUZ12, and EED; loss of clonogenic survival; and LBH589-induced differentiation of the AML cells. These findings support the rationale to test anti-EZH2 treatment combined with hydroxamate histone deacetylase inhibitors as an antileukemia epigenetic therapy, especially against AML with coexpression of EZH2, HOXA9, and MEIS1 genes.

Acetylation↗

PubMed2006

Corto and DSP1 interact and bind to a maintenance element of the Scr Hox gene: understanding the role of Enhancers of trithorax and Polycomb.

BACKGROUND: Polycomb-group genes (PcG) encode proteins that maintain homeotic (Hox) gene repression throughout development. Conversely, trithorax-group (trxG) genes encode positive factors required for maintenance of long term Hox gene activation. Both kinds of factors bind chromatin regions called maintenance elements (ME). Our previous work has shown that corto, which codes for a chromodomain protein, and dsp1, which codes for an HMGB protein, belong to a class of genes called the Enhancers of trithorax and Polycomb (ETP) that interact with both PcG and trxG. Moreover, dsp1 interacts with the Hox gene Scr, the DSP1 protein is present on a Scr ME in S2 cells but not in embryos. To understand better the role of ETP, we addressed genetic and molecular interactions between corto and dsp1. RESULTS: We show that Corto and DSP1 proteins co-localize at 91 sites on polytene chromosomes and co-immunoprecipitate in embryos. They interact directly through the DSP1 HMG-boxes and the amino-part of Corto, which contains a chromodomain. In order to search for a common target, we performed a genetic interaction analysis. We observed that corto mutants suppressed dsp11 sex comb phenotypes and enhanced AntpScx phenotypes, suggesting that corto and dsp1 are simultaneously involved in the regulation of Scr. Using chromatin immunoprecipitation of the Scr ME, we found that Corto was present on this ME both in Drosophila S2 cells and in embryos, whereas DSP1 was present only in S2 cells. CONCLUSION: Our results reveal that the proteins Corto and DSP1 are differently recruited to a Scr ME depending on whether the ME is active, as seen in S2 cells, or inactive, as in most embryonic cells. The presence of a given combination of ETPs on an ME would control the recruitment of either PcG or TrxG complexes, propagating the silenced or active state.

Animals↗

PubMed2024

Refractory testicular germ cell tumors are highly sensitive to the targeting of polycomb pathway demethylases KDM6A and KDM6B.

Testicular germ cell tumors (TGCTs) can be treated with cisplatin-based therapy. However, a clinically significant number of cisplatin-resistant patients die from progressive disease as no effective alternatives exist. Curative cisplatin therapy results in acute and life-long toxicities in the young TGCT patient population providing a rationale to decrease cisplatin exposure. In contrast to genetic alterations, recent evidence suggests that epigenetics is a major driving factor for TGCT formation, progression, and response to chemotherapy. Hence, targeting epigenetic pathways with "epidrugs" is one potential relatively unexplored strategy to advance TGCT treatment beyond cisplatin. In this report, we demonstrate for the first time that targeting polycomb demethylases KDM6A and KDM6B with epidrug GSK-J4 can treat both cisplatin-sensitive and -resistant TGCTs. While GSK-J4 had minimal effects alone on TGCT tumor growth in vivo, it dramatically sensitized cisplatin-sensitive and -resistant TGCTs to cisplatin. We validated KDM6A/KDM6B as the target of GSK-J4 since KDM6A/KDM6B genetic depletion had a similar effect to GSK-J4 on cisplatin-mediated anti-tumor activity and transcriptome alterations. Pharmacologic and genetic targeting of KDM6A/KDM6B potentiated or primed the p53-dominant transcriptional response to cisplatin, with also evidence for basal activation of p53. Further, several chromatin modifier genes, including BRD4, lysine demethylases, chromodomain helicase DNA binding proteins, and lysine methyltransferases, were repressed with cisplatin only in KDM6A/KDM6B-targeted cells, implying that KDM6A/KDM6B inhibition sets the stage for extensive chromatin remodeling of TGCT cells upon cisplatin treatment. Our findings demonstrate that targeting polycomb demethylases is a new potent pharmacologic strategy for treating cisplatin resistant TGCTs that warrants clinical development.

Testicular Neoplasms↗

PubMed2001

The Drosophila Polycomb Group proteins ESC and E(Z) are present in a complex containing the histone-binding protein p55 and the histone deacetylase RPD3.

The Drosophila Polycomb Group (PcG) proteins are required for stable long term transcriptional silencing of the homeotic genes. Among PcG genes, esc is unique in being critically required for establishment of PcG-mediated silencing during early embryogenesis, but not for its subsequent maintenance throughout development. We previously showed that ESC is physically associated in vivo with the PcG protein E(Z). We report here that ESC, together with E(Z), is present in a 600 kDa complex that is distinct from complexes containing other PcG proteins. We have purified this ESC complex and show that it also contains the histone deacetylase RPD3 and the histone-binding protein p55, which is also a component of the chromatin remodeling complex NURF and the chromatin assembly complex CAF-1. The association of ESC and E(Z) with p55 and RPD3 is conserved in mammals. We show that RPD3 is required for silencing mediated by a Polycomb response element (PRE) in vivo and that E(Z) and RPD3 are bound to the Ubx PRE in vivo, suggesting that they act directly at the PRE. We propose that histone deacetylation by this complex is a prerequisite for establishment of stable long-term silencing by other continuously required PcG complexes.

Amino Acid Sequence↗

PubMed2001

Mice doubly deficient for the Polycomb Group genes Mel18 and Bmi1 reveal synergy and requirement for maintenance but not initiation of Hox gene expression.

Polycomb group genes were identified as a conserved group of genes whose products are required in multimeric complexes to maintain spatially restricted expression of Hox cluster genes. Unlike in Drosophila, in mammals Polycomb group (PcG) genes are represented as highly related gene pairs, indicative of duplication during metazoan evolution. Mel18 and Bmi1 are mammalian homologs of Drosophila Posterior sex combs. Mice deficient for Mel18 or Bmi1 exhibit similar posterior transformations of the axial skeleton and display severe immune deficiency, suggesting that their gene products act on overlapping pathways/target genes. However unique phenotypes upon loss of either Mel18 or Bmi1 are also observed. We show using embryos doubly deficient for Mel18 and Bmi1 that Mel18 and Bmi1 act in synergy and in a dose-dependent and cell type-specific manner to repress Hox cluster genes and mediate cell survival of embryos during development. In addition, we demonstrate that Mel18 and Bmi1, although essential for maintenance of the appropriate expression domains of Hox cluster genes, are not required for the initial establishment of Hox gene expression. Furthermore, we show an unexpected requirement for Mel18 and Bmi1 gene products to maintain stable expression of Hox cluster genes in regions caudal to the prospective anterior expression boundaries during subsequent development.

Animals↗

PubMed2004

Requirement for sex comb on midleg protein interactions in Drosophila polycomb group repression.

The Drosophila Sex Comb on Midleg (SCM) protein is a transcriptional repressor of the Polycomb group (PcG). Although genetic studies establish SCM as a crucial PcG member, its molecular role is not known. To investigate how SCM might link to PcG complexes, we analyzed the in vivo role of a conserved protein interaction module, the SPM domain. This domain is found in SCM and in another PcG protein, Polyhomeotic (PH), which is a core component of Polycomb repressive complex 1 (PRC1). SCM-PH interactions in vitro are mediated by their respective SPM domains. Yeast two-hybrid and in vitro binding assays were used to isolate and characterize >30 missense mutations in the SPM domain of SCM. Genetic rescue assays showed that SCM repressor function in vivo is disrupted by mutations that impair SPM domain interactions in vitro. Furthermore, overexpression of an isolated, wild-type SPM domain produced PcG loss-of-function phenotypes in flies. Coassembly of SCM with a reconstituted PRC1 core complex shows that SCM can partner with PRC1. However, gel filtration chromatography showed that the bulk of SCM is biochemically separable from PH in embryo nuclear extracts. These results suggest that SCM, although not a core component of PRC1, interacts and functions with PRC1 in gene silencing.

Amino Acid Sequence↗

PubMed2005

To SIR with Polycomb: linking silencing mechanisms.

Yeast SIR2, the most evolutionarily conserved deacetylase, plays an essential role in epigenetic silencing at the silent mating type loci and telomeres. SIR2 has been implicated in chromatin silencing and lifespan determination in several organisms. Discovery that Drosophila SIR2 is also involved in epigenetic silencing mediated by the Polycomb group proteins and is physically associated with a complex containing the E(Z) histone methyltransferase has wide implications. These findings suggest possible link of Polycomb system to diverse cellular processes including senescence.

Animals↗

PubMed1995

Drosophila Polycomb-group regulated chromatin inhibits the accessibility of a trans-activator to its target DNA.

The genes of the Polycomb-group (Pc-G) are responsible for maintaining the inactive expression state of homeotic genes. They act through specific cis-regulatory DNA elements termed PREs (Pc-G Response Elements). Multimeric complexes containing the Pc-G proteins are thought to induce heterochromatin-like structures, which stably and heritably inactivate transcription. We have tested the functional role of the FAB fragment, a PRE of the bithorax complex. We find that this element behaves as an orientation dependent silencer, capable of inducing mosaic gene expression on neighboring genes. Transgenic fly lines were constructed containing a PRE adjacent to a reporter gene inducible by the yeast GAL4 trans-activator. The competition between the activator and Pc-G-containing chromatin was visualized on polytene chromosomes using immunocytochemistry. The Pc-G protein Polycomb and GAL4 have mutually exclusive binding patterns, supporting the notion that Pc-G-induced chromatin structures can prevent activators from binding to their target sequences. However, this antagonistic function can be overcome by high doses of GAL4, even in the absence of DNA replication.

Animals↗

PubMed1995

Transcriptional silencing by the Polycomb protein in Drosophila embryos.

Polycomb group (Pc-G) proteins act to keep homeotic genes stably and heritably silenced during Drosophila development. Here, it is shown that Polycomb (Pc), one of the Pc-G proteins, acts as a transcriptional silencer in Drosophila embryos if tethered to reporter genes by the DNA binding domain of GAL4 (i.e. as a GAL-Pc fusion protein). The results suggest that silencing by GAL-Pc requires the C-terminal portion of Pc, but not the chromodomain. If a pulse of Gal-Pc is provided, synthetic reporter genes are repressed, though only transiently. In contrast, reporter genes containing homeotic gene sequences remain stably and heritably silenced in a Pc-G gene-dependent fashion, even when GAL-Pc is no longer present. This implies that GAL-Pc recruits Pc-G proteins to DNA and suggests that maintenance of silencing requires the anchoring of Pc-G proteins to specific cis-regulatory sequences present in homeotic genes. The extent of DNA over which the Pc-G machinery acts is quite selective, as silencing established on one enhancer does not necessarily 'spread' to a juxtaposed synthetic enhancer.

ATP-Binding Cassette Transporters↗

PubMed2007

PARP-3 associates with polycomb group bodies and with components of the DNA damage repair machinery.

Poly(ADP-ribose) polymerase 3 (PARP-3) is a novel member of the PARP family of enzymes that synthesize poly(ADP-ribose) on themselves and other acceptor proteins. Very little is known about this PARP, which is closely related to PARP-1 and PARP-2. By sequence analysis, we find that PARP-3 may be expressed in two isoforms which we studied in more detail to gain insight into their possible functions. We find that both PARP-3 isoforms, transiently expressed as GFP or FLAG fusions, are nuclear. Detection of endogenous PARP-3 with a specific antibody also shows a widespread nuclear distribution, appearing in numerous small foci and a small number of larger foci. Through co-localization experiments and immunoprecipitations, the larger nuclear foci were identified as Polycomb group bodies (PcG bodies) and we found that PARP-3 is part of Polycomb group protein complexes. Furthermore, using a proteomics approach, we determined that both PARP-3 isoforms are part of complexes comprising DNA-PKcs, PARP-1, DNA ligase III, DNA ligase IV, Ku70, and Ku80. Our findings suggest that PARP-3 is a nuclear protein involved in transcriptional silencing and in the cellular response to DNA damage.

Amino Acid Sequence↗

PubMed1994

Gene inactivation in Drosophila mediated by the Polycomb gene product or by position-effect variegation does not involve major changes in the accessibility of the chromatin fibre.

In Drosophila position effect variegation and Polycomb-dependent regulation of homeotic gene expression are phenomena in which genes are inactivated in a clonally inherited manner. In both processes inactivation involves proteins that interact with the chromosome at or close to the position of inactivated genes. Two models have been proposed to explain this form of genetic silencing. In one, cooperative concatamerisation of a large multisubunit protein complex packages the chromatin fibre into a higher order structure, which is inaccessible for the transcription apparatus. In the second, the chromatin fibre is left unaltered but the region to be silenced is assigned to a compartment within the nucleus to which not all transcription factors have access. To distinguish between these types of model we have used the ligation-mediated PCR procedure to quantitate the accessibility of restriction sites in the chromatin fibre in both the active and inactivated forms. By making use of appropriate mutations and tissues we show that the inactivation of genes by Polycomb or by position effect variegation is not accompanied by a substantial change in the accessibility of the fibre. These results favour models in which the inactivation is achieved by sequestration of the silenced region in a particular nuclear compartment rather than by a chromatin packaging model.

Animals↗

PubMed1995

Mapping functional domains of the polycomb protein of Drosophila melanogaster.

In Drosophila the Polycomb group (Pc-G) proteins are responsible for the stable and heritable silencing of genes. The Pc-G apparently uses heterochromatin-like mechanisms to transcriptionally inactivate developmental regulators such as the homeotic genes. The Polycomb (Pc) protein is part of a large multimeric complex composed of other members of the Pc-G. We have identified functionally relevant domains of the Pc protein by sequencing different Pc alleles. Additionally, using a Pc-beta gal fusion protein with deleted internal histidine repeats, we found that this mutant protein cannot bind to four particular target loci, but otherwise does not change the remaining overall binding pattern. We show that, in contrast to the dotted subnuclear localization of the wild-type protein, the nuclear distribution of mutant proteins becomes homogeneous. Surprisingly, in Pc mutants the polyhomeotic protein, another member of the Pc-G, is also redistributed in the nucleus. Our results indicate that the appropriate subnuclear localization of the two proteins is critical for the silencing function of the Pc-G complex.

Alleles↗