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PubMed2002

A panel of monoclonal antibodies against human polycomb group proteins.

Polycomb-group (PcG) proteins are chromatin-associated proteins that heritably repress gene activity in many organisms, including man. Two distinct human PcG complexes have been identified. The HPC/HPH PcG complex I contains the HPC, HPH, RING1, and BMI1 proteins, the EED/EZH2 PcG complex II contains the EED, EZH2, and YY1 proteins. Previously we found that the relative expression levels of proteins of the human PcG complexes I and II are severely deregulated in human tumors. These findings signify an important role for antibodies against human PcG proteins as diagnostic tools. To be able to produce standardized anti-human PcG antibodies, we developed a panel of five mouse monoclonal antibodies (MAbs) against the human PcG proteins HPC2, BMI1, RING1A, EED, and EZH2. All MAbs can be used for Western blot analysis and immunofluorescence labeling of tissue culture cells. With the exception of the MAb against HPC2, all MAbs can also be used in immunoprecipitation experiments and immunohistochemistry of human tissues. The novel MAbs are therefore valuable tools for the cell biological, biochemical, and pathological analysis of human PcG proteins.

Animals↗
PubMed2006

Polycomb-group oncogenes EZH2, BMI1, and RING1 are overexpressed in prostate cancer with adverse pathologic and clinical features.

OBJECTIVES: Polycomb group (PcG) proteins are involved in maintenance of cell identity and proliferation. The protein EZH2 is overexpressed in disseminated prostate cancer, implicating a role of PcG complexes in tumor progression. In this study, we evaluated the expression of eight members of both PcG complexes in clinicopathologically defined prostate cancer. METHODS: Components of both PcG protein complexes PRC2 (EZH2, EED, YY1) and PRC1 (BMI1, RING1, HPH1, HPC1, HPC2) were immunohistochemically identified in tissue microarrays of 114 prostate cancer patients. Protein expression was semi-quantitatively scored and correlated with pathologic parameters and recurrence of prostate-specific antigen (PSA). RESULTS: Whereas BMI1, RING1, HPC1 and HPH1 were all abundantly present in normal and malignant prostate epithelium, expression of EZH2 occurred in only <10% of cells. Expression of EZH2, BMI1 and RING1 were all significantly enhanced in tumours with Gleason score (GS) > or = 8, extraprostatic extension, positive surgical margins, and PSA recurrence. When only the subgroup of GS < or = 6 was considered, representing the tumour grade in the majority of needle biopsies, EZH2 and BMI1 were also predictive for PSA recurrence. In a multivariable analysis, BMI1 was the only PcG protein with an independent prognostic value. CONCLUSIONS: PcG proteins EZH2, BMI1, and RING1 are associated with adverse pathologic features and clinical PSA recurrence of prostate cancer. Whereas BMI1 and RING1 are abundantly present in prostate cancer, EZH2 is expressed at relatively low levels, making it a less obvious target for therapy.

Adenocarcinoma↗
PubMed2004

Silencing of human polycomb target genes is associated with methylation of histone H3 Lys 27.

Polycomb group (PcG) complexes 2 and 3 are involved in transcriptional silencing. These complexes contain a histone lysine methyltransferase (HKMT) activity that targets different lysine residues on histones H1 or H3 in vitro. However, it is not known if these histones are methylation targets in vivo because the human PRC2/3 complexes have not been studied in the context of a natural promoter because of the lack of known target genes. Here we report the use of RNA expression arrays and CpG-island DNA arrays to identify and characterize human PRC2/3 target genes. Using oligonucleotide arrays, we first identified a cohort of genes whose expression changes upon siRNA-mediated removal of Suz12, a core component of PRC2/3, from colon cancer cells. To determine which of the putative target genes are directly bound by Suz12 and to precisely map the binding of Suz12 to those promoters, we combined a high-resolution chromatin immunoprecipitation (ChIP) analysis with custom oligonucleotide promoter arrays. We next identified additional putative Suz12 target genes by using ChIP coupled to CpG-island microarrays. We showed that HKMT-Ezh2 and Eed, two other components of the PRC2/3 complexes, colocalize to the target promoters with Suz12. Importantly, recruitment of Suz12, Ezh2 and Eed to target promoters coincides with methylation of histone H3 on Lys 27.

Carrier Proteins↗
PubMed2006

Essential role for activation of the Polycomb group (PcG) protein chromatin silencing pathway in metastatic prostate cancer.

The Polycomb group (PcG) gene BMI1 is required for the proliferation and self-renewal of normal and leukemic stem cells. Overexpression of Bmi1 oncogene causes neoplastic transformation of lymphocytes and plays essential role in pathogenesis of myeloid leukemia. Another PcG protein, Ezh2, was implicated in metastatic prostate and breast cancers, suggesting that PcG pathway activation is relevant for epithelial malignancies. Whether an oncogenic role of the BMI1 and PcG pathway activation may be extended beyond the leukemia and may affect progression of solid tumors as well remains unknown. Here we demonstrate that activation of the BMI1 oncogene-associated PcG pathway plays an essential role in metastatic prostate cancer, thus mechanistically linking the pathogenesis of leukemia, self-renewal of stem cells, and prostate cancer metastasis. To characterize the functional status of the PcG pathway in metastatic prostate cancer, we utilized advanced cell- and whole animal-imaging technologies, gene and protein expression profiling, stable siRNA-gene targeting, and tissue microarray (TMA) analysis in relevant experimental and clinical settings. We demonstrate that in multiple experimental models of metastatic prostate cancer both BMI1 and Ezh2 genes are amplified and gene amplification is associated with increased expression of corresponding mRNAs and proteins. For the first time, we provide images of human prostate carcinoma metastasis precursor cells isolated from blood and shown to overexpress both BMI1 and Ezh2 oncoproteins. Consistent with the PcG pathway activation hypothesis, increased BMI1 and Ezh2 expression in metastatic cancer cells is associated with elevated levels of H2AubiK119 and H3metK27 histones. Quantitative immunofluorescence colocalization analysis and expression profiling experiments documented increased BMI1 and Ezh2 expression in clinical prostate carcinoma samples and demonstrated that high levels of BMI1 and Ezh2 expression are associated with markedly increased likelihood of therapy failure and disease relapse after radical prostatectomy. Gene-silencing analysis reveals that activation of the PcG pathway is mechanistically linked with highly malignant behavior of human prostate carcinoma cells and is essential for in vivo growth and metastasis of human prostate cancer. We conclude that the results of experimental and clinical analyses indicate the important biological role of the PcG pathway activation in metastatic prostate cancer. Our work suggests that the PcG pathway activation is a common oncogenic event in pathogenesis of metastatic solid tumors and provides justification for development of small molecule inhibitors of the PcG chromatin silencing pathway as a novel therapeutic modality for treatment of metastatic prostate cancer.

Adenocarcinoma↗
PubMed2006

The Polycomb group protein Eed protects the inactive X-chromosome from differentiation-induced reactivation.

The Polycomb group (PcG) encodes an evolutionarily conserved set of chromatin-modifying proteins that are thought to maintain cellular transcriptional memory by stably silencing gene expression. In mouse embryos that are mutated for the PcG protein Eed, X-chromosome inactivation (XCI) is not stably maintained in extra-embryonic tissues. Eed is a component of a histone-methyltransferase complex that is thought to contribute to stable silencing in undifferentiated cells due to its enrichment on the inactive X-chromosome in cells of the early mouse embryo and in stem cells of the extra-embryonic trophectoderm lineage. Here, we demonstrate that the inactive X-chromosome in Eed(-/-) trophoblast stem cells and in cells of the trophectoderm-derived extra-embryonic ectoderm in Eed(-/-) embryos remain transcriptionally silent, despite lacking the PcG-mediated histone modifications that normally characterize the facultative heterochromatin of the inactive X-chromosome. Whereas undifferentiated Eed(-/-) trophoblast stem cells maintained XCI, reactivation of the inactive X-chromosome occurred when these cells were differentiated. These results indicate that PcG complexes are not necessary to maintain transcriptional silencing of the inactive X-chromosome in undifferentiated stem cells. Instead, PcG proteins seem to propagate cellular memory by preventing transcriptional activation of facultative heterochromatin during differentiation.

Animals↗
PubMed2001

Xenopus Enhancer of Zeste (XEZ); an anteriorly restricted polycomb gene with a role in neural patterning.

We have identified the Xenopus homologue of Drosophila Enhancer of Zeste using a differential display strategy designed to identify genes involved in early anterior neural differentiation. XEZ codes for a protein of 748 amino acids that is very highly conserved in evolution and is 96% identical to both human and mouse EZ(H)2. In common with most other Xenopus Pc-G genes and unlike mammalian Pc-G genes, XEZ is anteriorly restricted. Zygotic expression of XEZ commences during gastrulation, much earlier than other anteriorly localized Pc-G genes; expression is restricted to the anterior neural plate and is confined later to the forebrain, eyes and branchial arches. XEZ is induced in animal caps overexpressing noggin; up-regulation of XEZ therefore represents a response to inhibition of BMP signalling in ectodermal cells. We show that the midbrain/hindbrain junction marker En-2,and hindbrain marker Krox-20, are target genes of XEZ and that XEZ functions to repress these anteroposterior marker genes. Conversely, XEZ does not repress the forebrain marker Otx-2. XEZ overexpression results in a greatly thickened floor of the forebrain. These results implicate an important role for XEZ in the patterning of the nervous system.

Amino Acid Sequence↗
PubMed2005

EZH2 and histone 3 trimethyl lysine 27 associated with Il4 and Il13 gene silencing in Th1 cells.

Differentiation of naïve CD4 T cells toward the T helper 1 (T(H)1) and T helper 2 (T(H)2) fates involves the transcriptional repression and enhancement, respectively, of Il4 and Il13, adjacent chromosome 11 genes encoding the canonical T(H)2 cytokines interleukin-4 and interleukin-13. Proper execution of this developmental fate choice during immune responses is critical to host defense and, when misregulated, leads to susceptibility to infectious microbes and to allergic and autoimmune diseases. Here, using chromatin immunoprecipitation and real time reverse transcription PCR we identify the Polycomb family histone methyltransferase EZH2 as the enzyme responsible for methylating lysine 27 of histone H3 at the Il4-Il13 locus of T(H)1 but not T(H)2 cells, implicating EZH2 in the mechanism of Il4 and Il13 transcriptional silencing.

Animals↗
PubMed2006

Distinct roles of Polycomb group gene products in transcriptionally repressed and active domains of Hoxb8.

To address the molecular mechanisms underlying Polycomb group (PcG)-mediated repression of Hox gene expression, we have focused on the binding patterns of PcG gene products to the flanking regions of the Hoxb8 gene in expressing and non-expressing tissues. In parallel, we followed the distribution of histone marks of transcriptionally active H3 acetylated on lysine 9 (H3-K9) and methylated on lysine 4 (H3-K4), and of transcriptionally inactive chromatin trimethylated on lysine 27 (H3-K27). Chromatin immunoprecipitation revealed that the association of PcG proteins, and H3-K9 acetylation and H3-K27 trimethylation around Hoxb8 were distinct in tissues expressing and not expressing the gene. We show that developmental changes of these epigenetic marks temporally coincide with the misexpression of Hox genes in PcG mutants. Functional analyses, using mutant alleles impairing the PcG class 2 component Rnf2 or the Suz12 mutation decreasing H3-K27 trimethylation, revealed that interactions between class 1 and class 2 PcG complexes, mediated by trimethylated H3-K27, play decisive roles in the maintenance of Hox gene repression outside their expression domain. Within the expression domains, class 2 PcG complexes appeared to maintain the transcriptionally active status via profound regulation of H3-K9 acetylation. The present study indicates distinct roles for class 2 PcG complexes in transcriptionally repressed and active domains of Hoxb8 gene.

Animals↗
PubMed2004

Participation of Polycomb group gene extra sex combs in hedgehog signaling pathway.

Polycomb group (PcG) genes are required for stable inheritance of epigenetic states across cell divisions, a phenomenon termed cellular memory. PcG proteins form multimeric nuclear complex which modifies the chromatin structure of target site. Drosophila PcG gene extra sex combs (esc) and its vertebrate orthologs constitute a member of ESC-E(Z) complex, which possesses histone methyltransferase activity. Here we report isolation and characterization of medaka esc homolog, termed oleed. Hypomorphic knock-down of oleed using morpholino antisense oligonucleotides resulted in the fusion of eyes, termed cyclopia. Prechordal plate formation was not substantially impaired, but expression of hedgehog target genes was dependent on oleed, suggesting some link with hedgehog signaling. In support of this implication, histone methylation, which requires the activity of esc gene product, is increased in hedgehog stimulated mouse NIH-3T3 cells. Our data argue for the novel role of esc in hedgehog signaling and provide fundamental insight into the epigenetic mechanisms in general.

Amino Acid Sequence↗
PubMed2006

Mammalian Polycomb complexes are required for Peyer's patch development by regulating lymphoid cell proliferation.

The vertebrate Polycomb Group (PcG) genes encode proteins that form large multimeric and chromatin-associated complexes implicated in the stable repression of developmentally essential genes. Rnf110 and Phc2 are shown to be components of mammalian PcG multimeric complexes in HeLa cells. Here we report defects in Peyer's patch (PP) development in Rnf110 mutant mice, which is synergically exaggerated by Phc2 mutation. PP development involves a series of inductive interactions and subsequent differentiation and proliferation between lymphoid and mesenchymal cells in late gestational stage. Rnf110 and Phc2 mutations impair development of PP anlagen by affecting proliferation of lymphoid lineage cells populated in PP anlagen in gene-dosage dependent manner. We suggest that PcG complexes may act to mediate certain inductive signals maybe through IL-7Ralpha to allow sufficient proliferation of lymphoid inducer cells during PP organogenesis.

Animals↗
PubMed2004

Hierarchical recruitment of polycomb group silencing complexes.

Polycomb group (PcG) proteins maintain the transcriptional silence of target genes through many cycles of cell division. Here, we provide evidence for the sequential binding of PcG proteins at a Polycomb response element (PRE) in proliferating cells in which the sequence-specific DNA binding Pho and Phol proteins directly recruit E(z)-containing complexes, which in turn methylate histone H3 at lysine 27 (H3mK27). This provides a tag that facilitates binding by a Pc-containing complex. In wing imaginal discs, these PcG proteins also are present at discrete locations at or downstream of the promoter of a silenced target gene, Ubx. E(z)-dependent H3mK27 is also present near the Ubx promoter and is needed for Pc binding. The location of E(z)- and Pc-containing complexes downstream of the Ubx transcription start site suggests that they may inhibit transcription by interfering with assembly of the preinitiation complex or by blocking transcription initiation or elongation.

Animals↗
PubMed2002

Mitotically stable association of polycomb group proteins eed and enx1 with the inactive x chromosome in trophoblast stem cells.

X inactivation in female mammals is one of the best studied examples of heritable gene silencing and provides an important model for studying maintenance of patterns of gene expression during differentiation and development. The process is initiated by a cis-acting RNA, the X inactive specific transcript (Xist). Xist RNA is thought to recruit silencing complexes to the inactive X, which then serve to establish and maintain the inactive state in all subsequent cell divisions. Most lineages undergo random X inactivation, there being an equal probability of either the maternally (Xm) or paternally (Xp) inherited X chromosome being inactivated in a given cell. In the extraembryonic trophectoderm and primitive endoderm lineages of mouse embryos, however, there is imprinted X inactivation of Xp. This process is also Xist dependent. A recent study has shown that imprinted X inactivation in trophectoderm is not maintained in embryonic ectoderm development (eed) mutant mice. Here we show that Eed and a second Polycomb group protein, Enx1, are directly localized to the inactive X chromosome in XX trophoblast stem (TS) cells. The association of Eed/Enx1 complexes is mitotically stable, suggesting a mechanism for the maintenance of imprinted X inactivation in these cells.

Animals↗
PubMed2003

Establishment of histone h3 methylation on the inactive X chromosome requires transient recruitment of Eed-Enx1 polycomb group complexes.

Previous studies have implicated the Eed-Enx1 Polycomb group complex in the maintenance of imprinted X inactivation in the trophectoderm lineage in mouse. Here we show that recruitment of Eed-Enx1 to the inactive X chromosome (Xi) also occurs in random X inactivation in the embryo proper. Localization of Eed-Enx1 complexes to Xi occurs very early, at the onset of Xist expression, but then disappears as differentiation and development progress. This transient localization correlates with the presence of high levels of the complex in totipotent cells and during early differentiation stages. Functional analysis demonstrates that Eed-Enx1 is required to establish methylation of histone H3 at lysine 9 and/or lysine 27 on Xi and that this, in turn, is required to stabilize the Xi chromatin structure.

Amino Acid Sequence↗
PubMed2003

SETting the stage. Eed-Enx1 leaves an epigenetic signature on the inactive X chromosome.

Despite evidence implicating the Polycomb group protein, Eed (embryonic ectoderm development protein) in imprinted X inactivation, a similar role in random X inactivation in the embryo has remained an open question. Brockdorff and colleagues now report that Eed, along with its binding partner Enx1, transiently associates with the inactive X chromosome (Xi) and likely contributes to the epigenetic signature and long-term stability of the Xi heterochromatin.

Animals↗
PubMed2004

Role of histone H2A ubiquitination in Polycomb silencing.

Covalent modification of histones is important in regulating chromatin dynamics and transcription. One example of such modification is ubiquitination, which mainly occurs on histones H2A and H2B. Although recent studies have uncovered the enzymes involved in histone H2B ubiquitination and a 'cross-talk' between H2B ubiquitination and histone methylation, the responsible enzymes and the functions of H2A ubiquitination are unknown. Here we report the purification and functional characterization of an E3 ubiquitin ligase complex that is specific for histone H2A. The complex, termed hPRC1L (human Polycomb repressive complex 1-like), is composed of several Polycomb-group proteins including Ring1, Ring2, Bmi1 and HPH2. hPRC1L monoubiquitinates nucleosomal histone H2A at lysine 119. Reducing the expression of Ring2 results in a dramatic decrease in the level of ubiquitinated H2A in HeLa cells. Chromatin immunoprecipitation analysis demonstrated colocalization of dRing with ubiquitinated H2A at the PRE and promoter regions of the Drosophila Ubx gene in wing imaginal discs. Removal of dRing in SL2 tissue culture cells by RNA interference resulted in loss of H2A ubiquitination concomitant with derepression of Ubx. Thus, our studies identify the H2A ubiquitin ligase, and link H2A ubiquitination to Polycomb silencing.

Amino Acid Sequence↗
PubMed2001

Imprinted X inactivation maintained by a mouse Polycomb group gene.

In mammals, dosage compensation of X-linked genes is achieved by the transcriptional silencing of one X chromosome in the female (reviewed in ref. 1). This process, called X inactivation, is usually random in the embryo proper. In marsupials and the extra-embryonic region of the mouse, however, X inactivation is imprinted: the paternal X chromosome is preferentially inactivated whereas the maternal X is always active. Having more than one active X chromosome is deleterious to extra-embryonic development in the mouse. Here we show that the gene eed (embryonic ectoderm development), a member of the mouse Polycomb group (Pc-G) of genes, is required for primary and secondary trophoblast giant cell development in female embryos. Results from mice carrying a paternally inherited X-linked green fluorescent protein (GFP) transgene implicate eed in the stable maintenance of imprinted X inactivation in extra-embryonic tissues. Based on the recent finding that the Eed protein interacts with histone deacetylases, we suggest that this maintenance activity involves hypoacetylation of the inactivated paternal X chromosome in the extra-embryonic tissues.

Acetylation↗
PubMed2004

MAPKAP kinase 3pK phosphorylates and regulates chromatin association of the polycomb group protein Bmi1.

Polycomb group (PcG) proteins form chromatin-associated, transcriptionally repressive complexes, which are critically involved in the control of cell proliferation and differentiation. Although the mechanisms involved in PcG-mediated repression are beginning to unravel, little is known about the regulation of PcG function. We showed previously that PcG complexes are phosphorylated in vivo, which regulates their association with chromatin. The nature of the responsible PcG kinases remained unknown. Here we present the novel finding that the PcG protein Bmi1 is phosphorylated by 3pK (MAPKAP kinase 3), a convergence point downstream of activated ERK and p38 signaling pathways and implicated in differentiation and developmental processes. We identified 3pK as an interaction partner of PcG proteins, in vitro and in vivo, by yeast two-hybrid interaction and co-immunoprecipitation, respectively. Activation or overexpression of 3pK resulted in phosphorylation of Bmi1 and other PcG members and their dissociation from chromatin. Phosphorylation and subsequent chromatin dissociation of PcG complexes were expected to result in de-repression of targets. One such reported Bmi1 target is the Cdkn2a/INK4A locus. Cells overexpressing 3pK showed PcG complex/chromatin dissociation and concomitant de-repression of p14(ARF), which was encoded by the Cdkn2a/INK4A locus. Thus, 3pK is a candidate regulator of phosphorylation-dependent PcG/chromatin interaction. We speculate that phosphorylation may not only affect chromatin association but, in addition, the function of individual complex members. Our findings linked for the first time MAPK signaling pathways to the Polycomb transcriptional memory system. This suggests a novel mechanism by which a silenced gene status can be modulated and implicates PcG-mediated repression as a dynamically controlled process.

Cell Line↗