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PubMed2006

Polycomb group-dependent Cyclin A repression in Drosophila.

Polycomb group (PcG) and trithorax group (trxG) proteins are well known for their role in the maintenance of silent and active expression states of homeotic genes. However, PcG proteins may also be required for the control of cellular proliferation in vertebrates. In Drosophila, PcG factors act by associating with specific DNA regions termed PcG response elements (PREs). Here, we have investigated whether Drosophila cell cycle genes are directly regulated by PcG proteins through PREs. We have isolated a PRE that regulates Cyclin A (CycA) expression. This sequence is bound by the Polycomb (PC) and Polyhomeotic (PH) proteins of the PcG, and also by GAGA factor (GAF), a trxG protein that is usually found associated with PREs. This sequence causes PcG- and trxG-dependent variegation of the mini-white reporter gene in transgenic flies. The combination of FISH with PC immunostaining in embryonic cells shows that the endogenous CycA gene colocalizes with PC at foci of high PC concentration named PcG bodies. Finally, loss of function of the Pc gene and overexpression of Pc and ph trigger up-regulation and down-regulation, respectively, of CycA expression in embryos. These results demonstrate that CycA is directly regulated by PcG proteins, linking them to cell cycle control in vivo.

Animals↗

PubMed1992

The polyhomeotic gene of Drosophila encodes a chromatin protein that shares polytene chromosome-binding sites with Polycomb.

The Polycomb group (PcG) genes in Drosophila melanogaster are required for maintenance of correct spatial expression of homeotic genes, and their products are thought to form either a regulatory network or act as a multimeric complex. Recently, it has been suggested that because of homology between Polycomb (Pc) and Su(var)205, PcG genes encode chromatin proteins required for the maintenance of a determined state in chromatin. The polyhomeotic (ph) gene is a member of the PcG of genes. We present DNA sequence of a ph cDNA, which encodes a 169-kD protein with a single putative zinc finger, a serine/threonine-rich region, and has glutamine repeats, suggesting that ph is a DNA-binding protein. Polyclonal antisera directed against ph protein bind to approximately 80 sites on polytene chromosomes. Most of these sites appear to be the same as those recognized by antibodies to Pc protein. ph protein binds to insertion sites of constructs containing DNA from the bithoraxoid (bxd) region of the Bithorax complex, showing that ph binding to chromatin is DNA dependent. The same bxd constructs are recognized by Pc protein, strongly supporting the hypothesis that ph and Pc interact directly.

Amino Acid Sequence↗

PubMed1994

Transcriptional repression by Drosophila and mammalian Polycomb group proteins in transfected mammalian cells.

The Polycomb group (Pc-G) genes are essential for maintaining the proper spatially restricted expression pattern of the homeotic loci during Drosophila development. The Pc-G proteins appear to function at target loci to maintain a state of transcriptional repression. The murine oncogene bmi-1 has significant homology to the Pc-G gene Posterior sex combs (Psc) and a highly related gene, Suppressor two of zeste [Su(z)2]. We show here that the proteins encoded by bmi-1 and the Pc-G genes Polycomb (Pc) and Psc as well as Su(z)2 mediate repression in mammalian cells when targeted to a promoter by LexA in a cotransfection system. These fusion proteins repress activator function by as much as 30-fold, and the effect on different activation domains is distinct for each Pc-G protein. Repression is observed when the LexA fusion proteins are bound directly adjacent to activator binding sites and also when bound 1,700 bases from the promoter. These data demonstrate that the products of the Pc-G genes can significantly repress activator function on transiently introduced DNA. We suggest that this function contributes to the stable repression of targeted loci during development.

3T3 Cells↗

PubMed1997

RING1 is associated with the polycomb group protein complex and acts as a transcriptional repressor.

The Polycomb (Pc) protein is a component of a multimeric, chromatin-associated Polycomb group (PcG) protein complex, which is involved in stable repression of gene activity. The identities of components of the PcG protein complex are largely unknown. In a two-hybrid screen with a vertebrate Pc homolog as a target, we identify the human RING1 protein as interacting with Pc. RING1 is a protein that contains the RING finger motif, a specific zinc-binding domain, which is found in many regulatory proteins. So far, the function of the RING1 protein has remained enigmatic. Here, we show that RING1 coimmunoprecipitates with a human Pc homolog, the vertebrate PcG protein BMI1, and HPH1, a human homolog of the PcG protein Polyhomeotic (Ph). Also, RING1 colocalizes with these vertebrate PcG proteins in nuclear domains of SW480 human colorectal adenocarcinoma and Saos-2 human osteosarcoma cells. Finally, we show that RING1, like Pc, is able to repress gene activity when targeted to a reporter gene. Our findings indicate that RING1 is associated with the human PcG protein complex and that RING1, like PcG proteins, can act as a transcriptional repressor.

Amino Acid Sequence↗

PubMed1998

The Drosophila polycomb group protein Psc contacts ph and Pc through specific conserved domains.

The Polycomb group proteins are transcriptional repressors that are thought to act through multimeric nuclear complexes. We show that ph and Psc coprecipitate with Pc from nuclear extracts. We have analyzed the domains required for the association of Psc with ph and Pc by using the yeast two-hybrid system and an in vitro protein-binding assay. Psc and ph interact through regions of sequence conservation with mammalian homologs, i.e., the H1 domain of ph (amino acids 1297 to 1418) and the helix-turn-helix-containing region of Psc (amino acids 336 to 473). Psc contacts Pc primarily at the helix-turn-helix-containing region of Psc (amino acids 336 to 473), but also at the ring finger (amino acids 250 to 335). The Pc chromobox is not required for this interaction. We discuss the implication of these results for the nature of the complexes formed by Polycomb group proteins.

Amino Acid Sequence↗

PubMed1999

The Drosophila polycomb protein interacts with nucleosomal core particles In vitro via its repression domain.

The proteins of the Polycomb group (PcG) are required for maintaining regulator genes, such as the homeotic selectors, stably and heritably repressed in appropriate developmental domains. It has been suggested that PcG proteins silence genes by creating higher-order chromatin structures at their chromosomal targets, thus preventing the interaction of components of the transcriptional machinery with their cis-regulatory elements. An unresolved issue is how higher order-structures are anchored at the chromatin base, the nucleosomal fiber. Here we show a direct biochemical interaction of a PcG protein-the Polycomb (PC) protein-with nucleosomal core particles in vitro. The main nucleosome-binding domain coincides with a region in the C-terminal part of PC previously identified as the repression domain. Our results suggest that PC, by binding to the core particle, recruits other PcG proteins to chromatin. This interaction could provide a key step in the establishment or regulation of higher-order chromatin structures.

Animals↗

PubMed2005

Analysis of a polycomb group protein defines regions that link repressive activity on nucleosomal templates to in vivo function.

Polycomb group (PcG) genes propagate patterns of transcriptional repression throughout development. The products of several such genes are part of Polycomb repressive complex 1 (PRC1), which inhibits chromatin remodeling and transcription in vitro. Genetic and biochemical studies suggest the product of the Posterior sex combs (Psc) gene plays a central role in both PcG-mediated gene repression in vivo and PRC1 activity in vitro. To dissect the relationship between the in vivo and in vitro activities of Psc, we identified the lesions associated with 11 genetically characterized Psc mutations and asked how the corresponding mutant proteins affect Psc activity on nucleosomal templates in vitro. Analysis of both single-mutant Psc proteins and recombinant complexes containing mutant protein revealed that Psc encodes at least two functions, complex formation and the inhibition of remodeling and transcription, which require different regions of the protein. There is an excellent correlation between the in vivo phenotypes of mutant Psc alleles and the structure and in vitro activities of the corresponding proteins, suggesting that the in vitro activities of PRC1 reflect essential functions of Psc in vivo.

Animals↗

PubMed1997

In situ dissection of the Fab-7 region of the bithorax complex into a chromatin domain boundary and a Polycomb-response element.

Parasegmental (PS)-specific expression of the homeotic genes of the bithorax-complex (BX-C) appears to depend upon the subdivision of the complex into a series of functionally independent cis-regulatory domains. Fab-7 is a regulatory element that lies between iab-6 and iab-7 (the PS11- and PS12-specific cis-regulatory domains, respectively). Deletion of Fab-7 causes ectopic expression of iab-7 in PS11 (where normally only iab-6 is active). Two models have been proposed to account for the dominant Fab-7 phenotype. The first considers that Fab-7 functions as a boundary element that insulates iab-6 and iab-7. The second model envisages that Fab-7 contains a silencer element that keeps iab-7 repressed in parasegments anterior to PS12. Using a P-element inserted in the middle of the Fab-7 region (the bit transposon), we have generated an extensive collection of new Fab-7 mutations that allow us to subdivide Fab-7 into a boundary element and a Polycomb-respond element (PRE). The boundary lies within 1 kb of DNA on the proximal side of the bit transposon (towards iab-6). Deletions removing this element alone cause a complex gain- and loss-of-function phenotype in PS11; in some groups of cells, both iab-6 and iab-7 are active, while in others both iab-6 and iab-7 are inactive. Thus, deletion of the boundary allows activating as well as repressing activities to travel between iab-6 and iab-7. We also provide evidences that the boundary region contains an enhancer blocker element. The Polycomb-response element lies within 0.5 kb of DNA immediately distal to the boundary (towards iab-7). Deletions removing the PRE alone do not typically cause any visible phenotype as homozygotes. Interestingly, weak ectopic activation of iab-7 is observed in hemizygous PRE deletions, suggesting that the mechanisms that keep iab-7 repressed in the absence of this element may depend upon chromosome pairing. These results help to reconcile the previously contradictory models on Fab-7 function and to shed light on how a chromatin domain boundary and a nearby PRE concur in the setting up of the appropriate PS-specific expression of the Abd-B gene of the BX-C.

Abdomen↗

PubMed2001

Polycomb group proteins and heritable silencing of Drosophila Hox genes.

Early in Drosophila embryogenesis, transcriptional repressors encoded by Gap genes prevent the expression of particular combinations of Hox genes in each segment. During subsequent development, those Hox genes that were initially repressed in each segment remain off in all the descendent cells, even though the Gap repressors are no longer present. This phenomenon of heritable silencing depends on proteins of the Polycomb Group (PcG) and on cis-acting Polycomb response elements (PREs) in the Hox gene loci. We have removed individual PcG proteins from proliferating cells and then resupplied these proteins after a few or several cell generations. We show that most PcG proteins are required throughout development: when these proteins are removed, Hox genes become derepressed. However, we find that resupply of at least some PcG proteins can cause re-repression of Hox genes, provided that it occurs within a few cell generations of the loss of repression. These results suggest a functional distinction between transcriptional repression and heritable silencing: in at least some contexts, Hox genes can retain the capacity to be heritably silenced, despite being transcribed and replicated. We propose that silenced Hox genes bear a heritable, molecular mark that targets them for transcriptional repression. Some PcG proteins may be required to define and propagate this mark; others may function to repress the transcription of Hox genes that bear the mark.

Animals↗

PubMed2002

The Drosophila trithorax protein is a coactivator required to prevent re-establishment of polycomb silencing.

Polycomb group (PcG) and Trithorax (TRX) complexes assemble at Polycomb response elements (PREs) and maintain respectively the repressed and active state of homeotic genes. Although PcG and TRX complexes are distinct, their binding to some PRE fragments in vitro depends on GAGA motifs. GAGA factor immunoprecipitates with both complexes. In presence of a PRE, TRX stimulates expression and prevents the return of repression at later stages. When TRX levels are reduced, repression is re-established in inappropriate regions of imaginal discs, suggesting that TRX insufficiency impairs the epigenetic memory of the active state. Targeting a GAL-TRX fusion shows that TRX is a coactivator that stimulates expression of an active gene but cannot initiate expression by itself. Targeting a histone acetylase to a PRE does not affect embryonic silencing but causes a loss of memory in imaginal discs, suggesting that deacetylation is required to establish the memory of the repressed state.

Acetylation↗

PubMed2002

Transcription through the iab-7 cis-regulatory domain of the bithorax complex interferes with maintenance of Polycomb-mediated silencing.

The Fab-7 chromatin domain boundary insures functional autonomy of the iab-6 and iab-7 cis-regulatory domains in the bithorax complex (BX-C). We have previously shown that chromatin insulators such as gypsy or scs(min) are potent insulators that cannot substitute for Fab-7 function within the BX-C. During the early stages of these swapping experiments, we initially used a fragment of scs that was slightly larger than a minimal scs element (scs(min)). We report that this scs fragment, unlike scs(min), interferes in an orientation-dependent manner with the output of a regulatory region covering 80 kb of DNA (from iab-4 to iab-8). At the core of this orientation-dependent phenotype is a promoter located immediately adjacent to the scs insulator. In one orientation, the promoter traps the activity of the iab-3 through iab-5 cis-regulatory domains, diverting them from the abd-A gene. In the opposite orientation, the promoter is transcribing the iab-7 cis-regulatory domain, resulting in ectopic activation of the latter. Our data suggest that transcription through a Polycomb-Response Element (PRE) interferes with the maintenance of a Polycomb repression complex.

Animals↗

PubMed2002

Zeste maintains repression of Ubx transgenes: support for a new model of Polycomb repression.

During late embryogenesis, the expression domains of homeotic genes are maintained by two groups of ubiquitously expressed regulators: the Polycomb repressors and the Trithorax activators. It is not known how the activities of the two maintenance systems are initially targeted to the correct genes. Zeste and GAGA are sequence-specific DNA-binding proteins previously shown to be Trithorax group activators of the homeotic gene Ultrabithorax (Ubx). We demonstrate that Zeste and GAGA DNA-binding sites at the proximal promoter are also required to maintain, but not to initiate, repression of UBX: Furthermore, the repression mediated by Zeste DNA-binding site is abolished in zeste null embryos. These data imply that Zeste and probably GAGA mediate Polycomb repression. We present a model in which the dual transcriptional activities of Zeste and GAGA are an essential component of the mechanism that chooses which maintenance system is to be targeted to a given promoter.

Animals↗

PubMed2006

The Polycomb group protein EED is dispensable for the initiation of random X-chromosome inactivation.

The Polycomb group (PcG) proteins are thought to silence gene expression by modifying chromatin. The Polycomb repressive complex 2 (PRC2) plays an essential role in mammalian X-chromosome inactivation (XCI), a model system to investigate heritable gene silencing. In the mouse, two different forms of XCI occur. In the preimplantation embryo, all cells undergo imprinted inactivation of the paternal X-chromosome (Xp). During the peri-implantation period, cells destined to give rise to the embryo proper erase the imprint and randomly inactivate either the maternal X-chromosome or the Xp; extraembryonic cells, on the other hand, maintain imprinted XCI of the Xp. PRC2 proteins are enriched on the inactive-X during early stages of both imprinted and random XCI. It is therefore thought that PRC2 contributes to the initiation of XCI. Mouse embryos lacking the essential PRC2 component EED harbor defects in the maintenance of imprinted XCI in differentiating trophoblast cells. Assessment of PRC2 requirement in the initiation of XCI, however, has been hindered by the presence of maternally derived proteins in the early embryo. Here we show that Eed-/- embryos initiate and maintain random XCI despite lacking any functional EED protein prior to the initiation of random XCI. Thus, despite being enriched on the inactive X-chromosome, PcGs appear to be dispensable for the initiation and maintenance of random XCI. These results highlight the lineage- and differentiation state-specific requirements for PcGs in XCI and argue against PcG function in the formation of the facultative heterochromatin of the inactive X-chromosome.

Alleles↗

PubMed2003

The interactions of LIM protein KyoT with polycomb proteins.

LIM domain protein KyoT interacts with transcription factor RBP-J and modulates Notch signaling pathway. To study the function of KyoT, yeast two-hybrid was performed and proteins interacting with KyoT2 including Ring1 and hPc2 (human polycomb 2) that belong to PcG(polycomb group) were analyzed. The interactions between Ring1, hPc2, and KyoT2 were confirmed by changing vectors in two-hybrid analysis. Their interactions in vitro were confirmed by GST-pull down assay. Two-hybrid assay showed that the LIM domain of KyoT was responsible for interacting with the full-length Ring1 and hPc2. Moreover, the LIM domain also interacted with the C-terminus of hPc2. Interactions of KyoT2 with Ring1 and hPc2 suggested that PcG family might be involved in Notch signaling pathway.

Binding Sites↗

PubMed1996

Probes of chromatin accessibility in the Drosophila bithorax complex respond differently to Polycomb-mediated repression.

The Polycomb group (PcG) of genes are required for maintenance of the repressed state of the homeotic genes in Drosophila. There are similarities between the PcG repression and mating-type silencing in yeast or heterochromatic position effect in Drosophila, which suggest that PcG repression may involve a highly compacted chromatin structure. To test for such a structure, heterologous DNA- binding proteins were used as probes for DNA accessibility in Drosophila embryos. Binding sites for the yeast transcriptional activator GAL4 and for bacteriophage T7 RNA polymerase were inserted into the bithorax (bx) regulatory region of the endogenous Ultrabithorax (Ubx) gene, which is regulated by the PcG. Ubiquitously expressed GAL4 protein directs transcription through its binding sites only in the posterior segments where the bx region is active. The block to GAL4 activation in the more anterior segments is dependent on Polycomb (Pc) function. In contrast, T7 RNA polymerase can transcribe from its target promoter in all segments of the embryo. Thus, Pc-mediated repression blocks activated polymerase II transcription, but does not simply exclude all proteins.

Animals↗

PubMed2006

Polycomb, trithorax and the decision to differentiate.

For stem cells, life is full of potential: they have a high capacity to proliferate and a wide choice of future identities. When they differentiate, cells leave behind this freedom and become ever more committed to a single fate. Intriguingly, the Polycomb and Trithorax groups of proteins are vital to the very different natures of both stem cells and differentiated cells, but little is known about how they make the transition from one cell type to the other. A recent paper(1) throws light on this mystery, showing that the Polycomb proteins dramatically change their behaviour at a crucial moment of differentiation.

Animals↗

PubMed1995

Assignment of a Polycomb-like chromobox gene (CBX2) to human chromosome 17q25.

A human clone corresponding to the homologue of the murine Polycomb-like gene M33 has been used to map this gene (CBX2) to human chromosomes. Both somatic cell hybrid panels and FISH on metaphase chromosomes have been used. These techniques gave a consistent localization, at the tip of the long arm of chromosome 17 (17q25). This localization, as well as the potential role of a mammalian Polycomb-like protein, suggests a potential involvement in two different pathologies: the campomelic syndrome, an inherited disorder, and neoplastic disorders linked to allele loss already described in this region.

Amino Acid Sequence↗

PubMed1993

The Polycomb gene is differentially regulated during oogenesis and embryogenesis of Drosophila melanogaster.

Homeotic genes are responsible for determining the identity of body structures along the anterior-posterior axis. In Drosophila the early patterning system defines the differential expression pattern of the homeotic genes. In laterstages the Polycomb-group (Pc-G) genes were found to keep homeotic genes stably repressed in those domains where they have to be inactive. At the molecular level the Pc-G is supposed to exert its repressory role by influencing the higher order structure of chromatin. Here we show that during oogenesis the Polycomb (Pc) protein is localized in the polytene nuclei of the nurse cells. In addition, in late stages we observe overlapping gradients of expression in the somatic follicle cells, suggesting also an important function of Pc on the determinants involved in egg formation. During embryogenesis Pc is found in all tissues, though in later stages it preferentially accumulates in the CNS. Interestingly, we have identified a feedback-type regulation: the Ultrabithorax gene, a homeotic target gene of Pc, in its own domain of expression is down-regulating Pc.

Animals↗