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PubMed2003

Assembly of Polycomb complexes and silencing mechanisms.

Polycomb complexes assemble at their target sites and silence neighboring genes when these are not actively transcribed. The action of these complexes and of Trithorax complexes bound to the Polycomb Response Element establish alternative silent or derepressed states that are remembered through cell division and maintained for the rest of development. Recent results that may help explain the properties of these states are reviewed.

Animals↗

PubMed2001

A Drosophila Polycomb group complex includes Zeste and dTAFII proteins.

A goal of modern biology is to identify the physical interactions that define 'functional modules' of proteins that govern biological processes. One essential regulatory process is the maintenance of master regulatory genes, such as homeotic genes, in an appropriate 'on' or 'off' state for the lifetime of an organism. The Polycomb group (PcG) of genes maintain a repressed transcriptional state, and PcG proteins form large multiprotein complexes, but these complexes have not been described owing to inherent difficulties in purification. We previously fractionated a major PcG complex, PRC1, to 20-50% homogeneity from Drosophila embryos. Here, we identify 30 proteins in these preparations, then further fractionate the preparation and use western analyses to validate unanticipated connections. We show that the known PcG proteins Polycomb, Posterior sex combs, Polyhomeotic and dRING1 exist in robust association with the sequence-specific DNA-binding factor Zeste and with numerous TBP (TATA-binding-protein)-associated factors that are components of general transcription factor TFIID (dTAFIIs). Thus, in fly embryos, there is a direct physical connection between proteins that bind to specific regulatory sequences, PcG proteins, and proteins of the general transcription machinery.

Animals↗

PubMed2004

The cell-cycle regulator geminin inhibits Hox function through direct and polycomb-mediated interactions.

Embryonic development is tightly controlled. The clustered genes of the Hox family of homeobox proteins play an important part in regulating this development and also proliferation. They specify embryonic structures along the body axis, and are associated with normal and malignant cell growth. The cell-cycle regulator geminin controls replication by binding to the licensing factor Cdt1, and is involved in neural differentiation. Here, we show that murine geminin associates transiently with members of the Hox-repressing polycomb complex, with the chromatin of Hox regulatory DNA elements and with Hox proteins. Gain- and loss-of-function experiments in the chick neural tube demonstrate that geminin modulates the anterior boundary of Hoxb9 transcription, which suggests a polycomb-like activity for geminin. The interaction between geminin and Hox proteins prevents Hox proteins from binding to DNA, inhibits Hox-dependent transcriptional activation of reporter and endogenous downstream target genes, and displaces Cdt1 from its complex with geminin. By establishing competitive regulation, geminin functions as a coordinator of developmental and proliferative control.

Amino Acid Sequence↗

PubMed2005

Recruitment of Drosophila Polycomb group proteins to chromatin by DSP1.

Polycomb and trithorax group (PcG and trxG) proteins maintain silent and active transcriptional states, respectively, throughout development. In Drosophila, PcG and trxG proteins associate with DNA regions named Polycomb and trithorax response elements (PRE and TRE), but the mechanisms of recruitment are unknown. We previously characterized a minimal element from the regulatory region of the Abdominal-B gene, termed Ab-Fab. Ab-Fab contains a PRE and a TRE and is able to maintain repressed or active chromatin states during development. Here we show that the Dorsal switch protein 1 (DSP1), a Drosophila HMGB2 homologue, binds to a sequence present within Ab-Fab and in other characterized PREs. Addition of this motif to an artificial sequence containing Pleiohomeotic and GAGA factor consensus sites is sufficient for PcG protein recruitment in vivo. Mutations that abolish DSP1 binding to Ab-Fab and to a PRE from the engrailed locus lead to loss of PcG protein binding, loss of silencing, and switching of these PREs into constitutive TREs. The binding of DSP1 to PREs is therefore important for the recruitment of PcG proteins.

Animals↗

PubMed2004

The Arabidopsis thaliana MEDEA Polycomb group protein controls expression of PHERES1 by parental imprinting.

The maternally expressed Arabidopsis thaliana Polycomb group protein MEDEA (MEA) controls expression of the MADS-box gene PHERES1 (PHE1). Here, we show that PHE1 is mainly paternally expressed but maternally repressed and that this maternal repression of PHE1 breaks down in seeds lacking maternal MEA activity. Because Polycomb group proteins control parental imprinting in mammals as well, the independent recruitment of similar protein machineries for the imprinting of genes is a notable example of convergent evolution.

Arabidopsis↗

PubMed2006

Genome-wide analysis of Polycomb targets in Drosophila melanogaster.

Polycomb group (PcG) complexes are multiprotein assemblages that bind to chromatin and establish chromatin states leading to epigenetic silencing. PcG proteins regulate homeotic genes in flies and vertebrates, but little is known about other PcG targets and the role of the PcG in development, differentiation and disease. Here, we determined the distribution of the PcG proteins PC, E(Z) and PSC and of trimethylation of histone H3 Lys27 (me3K27) in the D. melanogaster genome. At more than 200 PcG target genes, binding sites for the three PcG proteins colocalize to presumptive Polycomb response elements (PREs). In contrast, H3 me3K27 forms broad domains including the entire transcription unit and regulatory regions. PcG targets are highly enriched in genes encoding transcription factors, but they also include genes coding for receptors, signaling proteins, morphogens and regulators representing all major developmental pathways.

Animals↗

PubMed2006

Structure and E3-ligase activity of the Ring-Ring complex of polycomb proteins Bmi1 and Ring1b.

Polycomb group proteins Ring1b and Bmi1 (B-cell-specific Moloney murine leukaemia virus integration site 1) are critical components of the chromatin modulating PRC1 complex. Histone H2A ubiquitination by the PRC1 complex strongly depends on the Ring1b protein. Here we show that the E3-ligase activity of Ring1b on histone H2A is enhanced by Bmi1 in vitro. The N-terminal Ring-domains are sufficient for this activity and Ring1a can replace Ring1b. E2 enzymes UbcH5a, b, c or UbcH6 support this activity with varying processivity and selectivity. All four E2s promote autoubiquitination of Ring1b without affecting E3-ligase activity. We solved the crystal structure of the Ring-Ring heterodimeric complex of Ring1b and Bmi1. In the structure the arrangement of the Ring-domains is similar to another H2A E3 ligase, the BRCA1/BARD1 complex, but complex formation depends on an N-terminal arm of Ring1b that embraces the Bmi1 Ring-domain. Mutation of a critical residue in the E2/E3 interface shows that catalytic activity resides in Ring1b and not in Bmi1. These data provide a foundation for understanding the critical enzymatic activity at the core of the PRC1 polycomb complex, which is implicated in stem cell maintenance and cancer.

Amino Acid Sequence↗

PubMed2001

The ENL moiety of the childhood leukemia-associated MLL-ENL oncoprotein recruits human Polycomb 3.

The translocation t(11;19) is frequently found in acute leukemia in infants. This event truncates the proto-oncogene MLL and fuses the 5' end of MLL in frame with the ENL gene. ENL contributes a crucial protein-protein interaction domain to the resulting oncoprotein MLL-ENL. Here we show by yeast two-hybrid assays, GST-pull-down experiments and in a far western blot analysis that this domain is necessary and sufficient to recruit a novel member of the human Polycomb protein family (hPc3). hPc3 RNA was detected throughout the human hematopoietic system. Similar to other Polycomb proteins hPc3 acts as a transcriptional repressor. The ENL-hPc3 interaction was verified by mutual co-precipitation of the proteins from cell extracts. ENL and hPc3 tagged with fluorescent proteins co-localized in living cells in a nuclear dot pattern. An internal region of hPc3 was responsible for binding to ENL. Finally, hPc3 binds to the C-terminus of AF9, another common MLL fusion partner. The recruitment of a repressive function by ENL opens up a new insight into a possible mechanism of leukemogenesis by the fusion protein MLL-ENL.

Amino Acid Sequence↗

PubMed2002

The Polycomb-group gene eed regulates thymocyte differentiation and suppresses the development of carcinogen-induced T-cell lymphomas.

The mouse Polycomb-group gene, embryonic ectoderm development (eed), appears to regulate cellular growth and differentiation in a developmental and tissue specific manner. During embryogenesis, eed regulates axial patterning, whereas in the adult eed represses proliferation of myeloid and B cell precursors. The present report demonstrates two novel functional activities of eed: alteration of thymocyte maturation and suppression of thymic lymphoma development. Mice that inherit the viable hypomorphic 17Rn5(1989SB) eed allele sustain a partial developmental block at or before the CD4(-)CD8(-)CD44(-)CD25(+) stage of thymocyte differentiation. Furthermore, mice that are homozygous or heterozygous for the hypomorphic eed allele have an increased incidence and decreased latency of N-methyl-N-nitrosourea-induced thymic lymphoma compared to wild-type littermates. These findings support the notion that Polycomb-group genes exert pleiotropic effects dictated by developmental stage and cellular context.

Animals↗

PubMed2006

Polycomb recruitment to DNA in vivo by the YY1 REPO domain.

Polycomb group (PcG) proteins are responsible for maintaining transcriptional repression of developmentally important genes. However, the mechanism of PcG recruitment to specific DNA sequences is poorly understood. Transcription factor YY1 is one of the few PcG proteins with sequence-specific DNA binding activity. We previously showed that YY1 can recruit other PcG proteins to DNA, leading to histone posttranslational modifications and stable transcriptional repression. Using Drosophila transgenic approaches, we identified YY1 sequences 201-226 as necessary and sufficient for PcG transcriptional repression in vivo. When fused to a heterologous DNA-binding domain, this short 26-aa motif was sufficient for transcriptional repression, recruitment of PcG proteins to DNA, and methylation of histone H3 lysine 27. Deletion of this short YY1 motif did not affect transient transcriptional repression but ablated PcG repression, PcG protein recruitment to DNA, and methylation of H3 lysine 27. We propose that this motif be named the REPO domain for its function in recruitment of Polycomb. The REPO domain is well conserved in YY1 orthologs and in related proteins.

Animals↗

PubMed2006

The Arabidopsis thaliana vernalization response requires a polycomb-like protein complex that also includes VERNALIZATION INSENSITIVE 3.

In Arabidopsis thaliana, the promotion of flowering by cold temperatures, vernalization, is regulated via a floral-repressive MADS box transcription factor, FLOWERING LOCUS C (FLC). Vernalization leads to the epigenetic repression of FLC expression, a process that requires the polycomb group (PcG) protein VERNALIZATION 2 (VRN2) and the plant homeodomain protein VERNALIZATION INSENSITIVE 3 (VIN3). We demonstrate that the repression of FLC by vernalization requires homologues of other Polycomb Repressive Complex 2 proteins and VRN2. We show in planta that VRN2 and VIN3 are part of a large protein complex that can include the PcG proteins FERTILIZATION INDEPENDENT ENDOSPERM, CURLY LEAF, and SWINGER. These findings suggest a single protein complex is responsible for histone deacetylation at FLC and histone methylation at FLC in vernalized plants. The abundance of the complex increases during vernalization and declines after plants are returned to higher temperatures, consistent with the complex having a role in establishing FLC repression.

Amino Acid Sequence↗

PubMed1998

The distribution of polycomb-group proteins during cell division and development in Drosophila embryos: impact on models for silencing.

The subcellular three-dimensional distribution of three polycomb-group (PcG) proteins-polycomb, polyhomeotic and posterior sex combs-in fixed whole-mount Drosophila embryos was analyzed by multicolor confocal fluorescence microscopy. All three proteins are localized in complex patterns of 100 or more loci throughout most of the interphase nuclear volume. The rather narrow distribution of the protein intensities in the vast majority of loci argues against a PcG-mediated sequestration of repressed target genes by aggregation into subnuclear domains. In contrast to the case for PEV repression (Csink, A.K., and S. Henikoff. 1996. Nature. 381:529-531), there is a lack of correlation between the occurrence of PcG proteins and high concentrations of DNA, demonstrating that the silenced genes are not targeted to heterochromatic regions within the nucleus. There is a clear distinction between sites of transcription in the nucleus and sites of PcG binding, supporting the assumption that most PcG binding loci are sites of repressive complexes. Although the PcG proteins maintain tissue-specific repression for up to 14 cell generations, the proteins studied here visibly dissociate from the chromatin during mitosis, and disperse into the cytoplasm in a differential manner. Quantitation of the fluorescence intensities in the whole mount embryos demonstrate that the dissociated proteins are present in the cytoplasm. We determined that <2% of PH remains attached to late metaphase and anaphase chromosomes. Each of the three proteins that were studied has a different rate and extent of dissociation at prophase and reassociation at telophase. These observations have important implications for models of the mechanism and maintenance of PcG- mediated gene repression.

Animals↗

PubMed2006

Mel-18, a polycomb group protein, regulates cell proliferation and senescence via transcriptional repression of Bmi-1 and c-Myc oncoproteins.

Polycomb group (PcG) protein Bmi-1 is an important regulator of cell proliferation. It regulates cellular senescence and proliferation of cells via the transcriptional repression of INK4a/ARF locus and other target genes. Here, we report that Mel-18, a PcG ring finger protein (PCGF) transcriptionally down-regulates Bmi-1. Furthermore, the expression of Bmi-1 and Mel-18 inversely correlates in proliferating and senescent human fibroblasts. Bmi-1 down-regulation by Mel-18 results in accelerated senescence and shortening of the replicative life span in normal human cells. Importantly, using promoter-reporter, chromatin immunoprecipitation, and quantitative real-time primary transcript RT-PCR assays, and an RNA interference approach, we demonstrate that Bmi-1 is a bona fide target of c-Myc oncoprotein. Finally, our data suggest that Mel-18 regulates Bmi-1 expression during senescence via down-regulation of c-Myc. These studies link c-Myc and polycomb function in cell proliferation and senescence.

Binding Sites↗

PubMed1995

Comparison of germline mosaics of genes in the Polycomb group of Drosophila melanogaster.

The genes of the Polycomb group (PcG) repress the genes of the bithorax and Antennapedia complexes, among others. To observe a null phenotype for a PcG gene, one must remove its maternal as well as zygotic contribution to the embryo. Five members of the PcG group are compared here: Enhancer of Polycomb [E(Pc)], Additional sex combs (Asx), Posterior sex combs (Psc), Suppressor of zeste 2 [Su (z) 2] and Polycomblike (Pcl). The yeast recombinase (FLP) system was used to induce mitotic recombination in the maternal germline. Mutant embryos were analyzed by staining with antibodies against six target genes of the PcG. The loss of the maternal component leads to enhanced homeotic phenotypes and to unique patterns of misexpression. E(Pc) and Su(z) 2 mutations had only subtle effects on the target genes, even when the maternal contributions were removed. Asx and Pcl mutants show derepression of the targets only in specific cell types. Psc shows unusual effects on two of the targets, Ultrabithorax and abdominal-A. These results show that the PcG genes do not act only in a common complex or pathway; they must have some independent functions.

Animals↗

PubMed2003

GAGA facilitates binding of Pleiohomeotic to a chromatinized Polycomb response element.

Polycomb response elements (PREs) are chromosomal elements, typically comprising thousands of base pairs of poorly defined sequences that confer the maintenance of gene expression patterns by Polycomb group (PcG) repressors and trithorax group (trxG) activators. Genetic studies have indicated a synergistic requirement for the trxG protein GAGA and the PcG protein Pleiohomeotic (PHO) in silencing at several PREs. However, the molecular basis of this cooperation remains unknown. Here, using DNaseI footprinting analysis, we provide a high-resolution map of sites for the sequence- specific DNA-binding PcG protein PHO, trxG proteins GAGA and Zeste and the gap protein Hunchback (HB) on the 1.6 kb Ultrabithorax (Ubx) PRE. Although these binding elements are present throughout the PRE, they display clear patterns of clustering, suggestive of functional collaboration at the level of PRE binding. We found that while GAGA could efficiently bind to a chromatinized PRE, PHO alone was incapable of binding to chromatin. However, PHO binding to chromatin, but not naked DNA, was strongly facilitated by GAGA, indicating interdependence between GAGA and PHO already at the level of PRE binding. These results provide a biochemical explanation for the in vivo cooperation between GAGA and PHO and suggest that PRE function involves the integrated activities of genetically antagonistic trxG and PcG proteins.

Base Sequence↗

PubMed2005

An Sp1/KLF binding site is important for the activity of a Polycomb group response element from the Drosophila engrailed gene.

Polycomb-group response elements (PREs) are DNA elements through which the Polycomb-group (PcG) of transcriptional repressors act. Many of the PcG proteins are associated with two protein complexes that repress gene expression by modifying chromatin. Both of these protein complexes specifically associate with PREs in vivo, however, it is not known how they are recruited or held at the PRE. PREs are complex elements, made up of binding sites for many proteins. Our laboratory has been working to define all the sequences and DNA binding proteins required for the activity of a 181 bp PRE from the Drosophila engrailed gene. Here we show that one of the sites necessary for PRE activity, Site 2, can be bound by members of the Sp1/KLF family of zinc finger proteins. There are 10 Sp1/KLF family members in Drosophila, and nine of them bind to Site 2. We derive a consensus binding site for the Sp1/KLF Drosophila family members and show that this consensus sequence is present in most of the molecularly characterized PREs. These data suggest that one or more Sp1/KLF family members play a role in PRE function in Drosophila.

Amino Acid Sequence↗

PubMed1997

Identification of Bmi1-interacting proteins as constituents of a multimeric mammalian polycomb complex.

The Bmi1 gene has been identified as a mouse Polycomb group (Pc-G) gene implicated in the regulation of Hox gene expression. Here we describe the characterization of a Bmi binding protein Mph1, which shares similarity to Drosophila polyhomeotic. Coimmunoprecipitation experiments indicate that Bmi1 and Mph1, as well as the Mel18 and M33 proteins described previously, are constituents of a multimeric protein complex in mouse embryos and human cells. A central domain of Bmi1 interacts with the carboxyl terminus of Mph1, whereas a conserved alpha-helical domain in the Mph1 protein is required for its homodimerization. Transgenic mice overexpressing various mutant Bmi1 proteins demonstrate that the central domain of Bmil is required for the induction of anterior transformations of the axial skeleton. Bmi1, M33, and Mph1 show an overlapping speckled distribution in interphase nuclei. These data provide molecular evidence for the existence of a mammalian Polycomb complex.

Amino Acid Sequence↗

PubMed2005

Intergenic transcription through a polycomb group response element counteracts silencing.

Polycomb group response elements (PREs) mediate the mitotic inheritance of gene expression programs and thus maintain determined cell fates. By default, PREs silence associated genes via the targeting of Polycomb group (PcG) complexes. Upon an activating signal, however, PREs recruit counteracting trithorax group (trxG) proteins, which in turn maintain target genes in a transcriptionally active state. Using a transgenic reporter system, we show that the switch from the silenced to the activated state of a PRE requires noncoding transcription. Continuous transcription through the PRE induced by an actin promoter prevents the establishment of PcG-mediated silencing. The maintenance of epigenetic activation requires transcription through the PRE to proceed at least until embryogenesis is completed. At the homeotic bithorax complex of Drosophila, intergenic PRE transcripts can be detected not only during embryogenesis, but also at late larval stages, suggesting that transcription through endogenous PREs is required continuously as an anti-silencing mechanism to prevent the access of repressive PcG complexes to the chromatin. Furthermore, all other PREs outside the homeotic complex we tested were found to be transcribed in the same tissue as the mRNA of the corresponding target gene, suggesting that anti-silencing by transcription is a fundamental aspect of the cellular memory system.

Animals↗