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PubMed2004

YY1 DNA binding and PcG recruitment requires CtBP.

We found that mammalian Polycomb group (PcG) protein YY1 can bind to Polycomb response elements in Drosophila embryos and can recruit other PcG proteins to DNA. PcG recruitment results in deacetylation and methylation of histone H3. In a CtBP mutant background, recruitment of PcG proteins and concomitant histone modifications do not occur. Surprisingly, YY1 DNA binding in vivo is also ablated. CtBP mutation does not result in YY1 degradation or transport from the nucleus, suggesting a mechanism whereby YY1 DNA binding ability is masked. These results reveal a new role for CtBP in controlling YY1 DNA binding and recruitment of PcG proteins to DNA.

Alcohol Oxidoreductases↗
PubMed1995

Gene trap capture of a novel mouse gene, jumonji, required for neural tube formation.

A mouse mutation, termed jumonji (jmj), was generated by a gene trap strategy. Expression of the trapped gene (jmj gene), as monitored by X-gal staining, was detected predominantly at the midbrain-hindbrain boundary and in the cerebellum, depending on the stage of development. All embryos homozygous for the jmj mutation died before embryonic day 15.5. Some, but not all, of the homozygotes developed an abnormal groove in a region just anterior to the midbrain-hindbrain boundary on the neural plate at embryonic day 8-8.5 and showed a defect in neural tube closure in the midbrain region. Analyses of jmj cDNA revealed that the jmj gene is novel, conserved among vertebrates, and disrupted by vector insertion in the jmj homozygotes. The amino acid sequence deduced from the cDNA shared a portion of significant homology with human retinoblastoma-binding protein RBP-2 and with a putative protein encoded by human gene XE169 that escapes X-chromosome inactivation. These results suggest that jmj gene is essential for normal morphogenesis of the neural tube.

Amino Acid Sequence↗
PubMed2003

Role of histone H3 lysine 27 methylation in X inactivation.

The Polycomb group (PcG) protein Eed is implicated in regulation of imprinted X-chromosome inactivation in extraembryonic cells but not of random X inactivation in embryonic cells. The Drosophila homolog of the Eed-Ezh2 PcG protein complex achieves gene silencing through methylation of histone H3 on lysine 27 (H3-K27), which suggests a role for H3-K27 methylation in imprinted X inactivation. Here we demonstrate that transient recruitment of the Eed-Ezh2 complex to the inactive X chromosome (Xi) occurs during initiation of X inactivation in both extraembryonic and embryonic cells and is accompanied by H3-K27 methylation. Recruitment of the complex and methylation on the Xi depend on Xist RNA but are independent of its silencing function. Together, our results suggest a role for Eed-Ezh2-mediated H3-K27 methylation during initiation of both imprinted and random X inactivation and demonstrate that H3-K27 methylation is not sufficient for silencing of the Xi.

Animals↗
PubMed2004

Reactivation of the paternal X chromosome in early mouse embryos.

It is generally accepted that paternally imprinted X inactivation occurs exclusively in extraembryonic lineages of mouse embryos, whereas cells of the embryo proper, derived from the inner cell mass (ICM), undergo only random X inactivation. Here we show that imprinted X inactivation, in fact, occurs in all cells of early embryos and that the paternal X is then selectively reactivated in cells allocated to the ICM. This contrasts with more differentiated cell types where X inactivation is highly stable and generally irreversible. Our observations illustrate that an important component of genome plasticity in early development is the capacity to reverse heritable gene silencing decisions.

Acetylation↗
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↗
PubMed2003

The human polycomb group EED protein interacts with the integrase of human immunodeficiency virus type 1.

Human EED, a member of the superfamily of WD-40 repeat proteins and of the Polycomb group proteins, has been identified as a cellular partner of the human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein (R. Peytavi et al., J. Biol. Chem. 274:1635-1645, 1999). In the present study, EED was found to interact with HIV-1 integrase (IN) both in vitro and in vivo in yeast. In vitro, data from mutagenesis studies, pull-down assays, and phage biopanning suggested that EED-binding site(s) are located in the C-terminal domain of IN, between residues 212 and 264. In EED, two putative discrete IN-binding sites were mapped to its N-terminal moiety, at a distance from the MA-binding site, but EED-IN interaction also required the integrity of the EED last two WD repeats. EED showed an apparent positive effect on IN-mediated DNA integration reaction in vitro, in a dose-dependent manner. In situ analysis by immunoelectron microscopy (IEM) of cellular distribution of IN and EED in HIV-1-infected cells (HeLa CD4(+) cells or MT4 lymphoid cells) showed that IN and EED colocalized in the nucleus and near nuclear pores, with maximum colocalization events occurring at 6 h postinfection (p.i.). Triple colocalizations of IN, EED, and MA were also observed in the nucleoplasm of infected cells at 6 h p.i., suggesting the ocurrence of multiprotein complexes involving these three proteins at early steps of the HIV-1 virus life cycle. Such IEM patterns were not observed with a noninfectious, envelope deletion mutant of HIV-1.

Amino Acid Sequence↗
PubMed1993

The Drosophila Polycomb-group gene Enhancer of zeste contains a region with sequence similarity to trithorax.

As is typical of Polycomb-group loci, the Enhancer of zeste [E(z)] gene negatively regulates the segment identity genes of the Antennapedia (ANT-C) and Bithorax (BX-C) gene complexes. A second class of loci, collectively known as the trithorax group, plays an antagonistic role as positive regulators of the ANT-C and BX-C genes. Molecular analysis of the E(z) gene predicts a 760-amino-acid protein product. A region of 116 amino acids near the E(z) carboxy terminus is 41.2% identical (68.4% similar) with a carboxy-terminal region of the trithorax protein. This portion of the trithorax protein is part of a larger region previously shown to share extensive homology with a human protein (ALL-1/Hrx) that is implicated in acute leukemias. Over this same 116 amino acids, E(z) and ALL-1/Hrx are 43.9% identical (68.4% similar). Otherwise, E(z) is not significantly similar to any previously described proteins. As this region of sequence similarity is shared by two proteins with antagonistic functions, we suggest that it may comprise a domain that interacts with a common target, either nucleic acid or protein. Opposite effects on transcription might then be determined by other portions of the two proteins.

Alleles↗
PubMed1996

Interaction of Vav with ENX-1, a putative transcriptional regulator of homeobox gene expression.

The proto-oncogene product Vav plays a critical role in hematopoietic signal transduction. By using the yeast two-hybrid system, we identified a novel human protein, ENX-1, which interacts specifically with Vav both in vitro and in vivo. ENX-1 represents the human homolog of the Drosophila Enhancer of zeste gene, a member of the Polycomb group of genes, which are transcriptional regulators of homeobox gene expression. Interaction with ENX-1 suggests that Vav functions as an upstream element in the transcriptional regulation of homeobox genes, known to be important effectors in the hematopoietic system.

Amino Acid Sequence↗
PubMed1997

Evolutionary conservation and predicted structure of the Drosophila extra sex combs repressor protein.

The Drosophila extra sex combs (esc) protein, a member of the Polycomb group (PcG), is a transcriptional repressor of homeotic genes. Genetic studies have shown that esc protein is required in early embryos at about the time that other PcG proteins become engaged in homeotic gene repression. The esc protein consists primarily of multiple copies of the WD repeat, a motif that has been implicated in protein-protein interaction. To further investigate the domain organization of esc protein, we have isolated and characterized esc homologs from divergent insect species. We report that esc protein is highly conserved in housefly (72% identical to Drosophila esc), butterfly (55% identical), and grasshopper (56% identical). We show that the butterfly homolog provides esc function in Drosophila, indicating that the sequence similarities reflect functional conservation. Homology modeling using the crystal structure of another WD repeat protein, the G-protein beta-subunit, predicts that esc protein adopts a beta-propeller structure. The sequence comparisons and modeling suggest that there are seven WD repeats in esc protein which together form a seven-bladed beta-propeller. We locate the conserved regions in esc protein with respect to this predicted structure. Site-directed mutagenesis of specific loops, predicted to extend from the propeller surface, identifies conserved parts of esc protein required for function in vivo. We suggest that these regions might mediate physical interaction with esc partner proteins.

Amino Acid Sequence↗
PubMed1997

The product of the murine homolog of the Drosophila extra sex combs gene displays transcriptional repressor activity.

The heterogeneous nuclear ribonucleoprotein K protein represents a novel class of proteins that may act as docking platforms that orchestrate cross-talk among molecules involved in signal transduction and gene expression. Using a fragment of K protein as bait in the yeast two-hybrid screen, we isolated a cDNA that encodes a protein whose primary structure has extensive similarity to the Drosophila melanogaster extra sex combs (esc) gene product, Esc, a putative silencer of homeotic genes. The cDNA that we isolated is identical to the cDNA of the recently positionally cloned mouse embryonic ectoderm development gene, eed. Like Esc, Eed contains six WD-40 repeats in the C-terminal half of the protein and is thought to repress homeotic gene expression during mouse embryogenesis. Eed binds to K protein through a domain in its N terminus, but interestingly, this domain is not found in the Drosophila Esc. Gal4-Eed fusion protein represses transcription of a reporter gene driven by a promoter that contains Gal4-binding DNA elements. Eed also represses transcription when recruited to a target promoter by Gal4-K protein. Point mutations within the eed gene that are responsible for severe embryonic development abnormalities abolished the transcriptional repressor activity of Eed. Results of this study suggest that Eed-restricted homeotic gene expression during embryogenesis reflects the action of Eed as a transcriptional repressor. The Eed-mediated transcriptional effects are likely to reflect the interaction of Eed with multiple molecular partners, including K protein.

Amino Acid Sequence↗
PubMed1998

The Drosophila esc and E(z) proteins are direct partners in polycomb group-mediated repression.

The extra sex combs (esc) and Enhancer of zeste [E(z)] proteins are members of the Drosophila Polycomb group (Pc-G) of transcriptional repressors. Here we present evidence for direct physical interaction between the esc and E(z) proteins using yeast two-hybrid and in vitro binding assays. In addition, coimmunoprecipitation from embryo extracts demonstrates association of esc and E(z) in vivo. We have delimited the esc-binding domain of E(z) to an N-terminal 33-amino-acid region. Furthermore, we demonstrate that site-directed mutations in the esc protein previously shown to impair esc function in vivo disrupt esc-E(z) interactions in vitro. We also show an in vitro interaction between the heed and EZH1 proteins, which are human homologs of esc and E(z), respectively. These results suggest that the esc-E(z) molecular partnership has been conserved in evolution. Previous studies suggested that esc is primarily involved in the early stages of Pc-G-mediated silencing during embryogenesis. However, E(z) is continuously required in order to maintain chromosome binding by other Pc-G proteins. In light of these earlier observations and the molecular data presented here, we discuss how esc-E(z) protein complexes may contribute to transcriptional silencing by the Pc-G.

Amino Acid Sequence↗
PubMed2001

The polycomb-group gene Ezh2 is required for early mouse development.

Polycomb-group (Pc-G) genes are required for the stable repression of the homeotic selector genes and other developmentally regulated genes, presumably through the modulation of chromatin domains. Among the Drosophila Pc-G genes, Enhancer of zeste [E(z)] merits special consideration since it represents one of the Pc-G genes most conserved through evolution. In addition, the E(Z) protein family contains the SET domain, which has recently been linked with histone methyltransferase (HMTase) activity. Although E(Z)-related proteins have not (yet) been directly associated with HMTase activity, mammalian Ezh2 is a member of a histone deacetylase complex. To investigate its in vivo function, we generated mice deficient for Ezh2. The Ezh2 null mutation results in lethality at early stages of mouse development. Ezh2 mutant mice either cease developing after implantation or initiate but fail to complete gastrulation. Moreover, Ezh2-deficient blastocysts display an impaired potential for outgrowth, preventing the establishment of Ezh2-null embryonic stem cells. Interestingly, Ezh2 is up-regulated upon fertilization and remains highly expressed at the preimplantation stages of mouse development. Together, these data suggest an essential role for Ezh2 during early mouse development and genetically link Ezh2 with eed and YY1, the only other early-acting Pc-G genes.

Animals↗
PubMed2003

A 1-megadalton ESC/E(Z) complex from Drosophila that contains polycomblike and RPD3.

Polycomb group (PcG) proteins are required to maintain stable repression of the homeotic genes and others throughout development. The PcG proteins ESC and E(Z) are present in a prominent 600-kDa complex as well as in a number of higher-molecular-mass complexes. Here we identify and characterize a 1-MDa ESC/E(Z) complex that is distinguished from the 600-kDa complex by the presence of the PcG protein Polycomblike (PCL) and the histone deacetylase RPD3. In addition, the 1-MDa complex shares with the 600-kDa complex the histone binding protein p55 and the PcG protein SU(Z)12. Coimmunoprecipitation assays performed on embryo extracts and gel filtration column fractions indicate that, during embryogenesis E(Z), SU(Z)12, and p55 are present in all ESC complexes, while PCL and RPD3 are associated with ESC, E(Z), SU(Z)12, and p55 only in the 1-MDa complex. Glutathione transferase pulldown assays demonstrate that RPD3 binds directly to PCL via the conserved PHD fingers of PCL and the N terminus of RPD3. PCL and E(Z) colocalize virtually completely on polytene chromosomes and are associated with a subset of RPD3 sites. As previously shown for E(Z) and RPD3, PCL and SU(Z)12 are also recruited to the insertion site of a minimal Ubx Polycomb response element transgene in vivo. Consistent with these biochemical and cytological results, Rpd3 mutations enhance the phenotypes of Pcl mutants, further indicating that RPD3 is required for PcG silencing and possibly for PCL function. These results suggest that there may be multiple ESC/E(Z) complexes with distinct functions in vivo.

Animals↗
PubMed2004

Cross-regulation among the polycomb group genes in Drosophila melanogaster.

Genes of the Polycomb group in Drosophila melanogaster function as long-term transcriptional repressors. A few members of the group encode proteins found in two evolutionarily conserved chromatin complexes, Polycomb repressive complex 1 (PRC1) and the ESC-E(Z) complex. The majority of the group, lacking clear biochemical functions, might be indirect regulators. The transcript levels of seven Polycomb group genes were assayed in embryos mutant for various other genes in the family. Three Polycomb group genes were identified as upstream positive regulators of the core components of PRC1. There is also negative feedback regulation of some PRC1 core components by other PRC1 genes. Finally, there is positive regulation of PRC1 components by the ESC-E(Z) complex. These multiple pathways of cross-regulation help to explain the large size of the Polycomb group family of genes, but they complicate the genetic analysis of any single member.

Animals↗
PubMed2004

Jumonji represses atrial natriuretic factor gene expression by inhibiting transcriptional activities of cardiac transcription factors.

Mice with a homozygous knockout of the jumonji (jmj) gene showed abnormal heart development and defective regulation of cardiac-specific genes, including the atrial natriuretic factor (ANF). ANF is one of the earliest markers of cardiac differentiation and a hallmark for cardiac hypertrophy. Here, we show that JMJ represses ANF gene expression by inhibiting transcriptional activities of Nkx2.5 and GATA4. JMJ represses the Nkx2.5- or GATA4-dependent activation of the reporter genes containing the ANF promoter-enhancer or containing the Nkx2.5 or GATA4-binding consensus sequence. JMJ physically associates with Nkx2.5 and GATA4 in vitro and in vivo as determined by glutathione S-transferase pull-down and immunoprecipitation assays. Using mutational analyses, we mapped the protein-protein interaction domains in JMJ, Nkx2.5, and GATA4. We identified two DNA-binding sites of JMJ in the ANF enhancer by gel mobility shift assays. However, these JMJ-binding sites do not seem to mediate ANF repression by JMJ. Mutational analysis of JMJ indicates that the protein-protein interaction domain of JMJ mediates the repression of ANF gene expression. Therefore, JMJ may play important roles in the down-regulation of ANF gene expression and in heart development.

Amino Acid Motifs↗
PubMed2005

Subunit contributions to histone methyltransferase activities of fly and worm polycomb group complexes.

The ESC-E(Z) complex of Drosophila melanogaster Polycomb group (PcG) repressors is a histone H3 methyltransferase (HMTase). This complex silences fly Hox genes, and related HMTases control germ line development in worms, flowering in plants, and X inactivation in mammals. The fly complex contains a catalytic SET domain subunit, E(Z), plus three noncatalytic subunits, SU(Z)12, ESC, and NURF-55. The four-subunit complex is >1,000-fold more active than E(Z) alone. Here we show that ESC and SU(Z)12 play key roles in potentiating E(Z) HMTase activity. We also show that loss of ESC disrupts global methylation of histone H3-lysine 27 in fly embryos. Subunit mutations identify domains required for catalytic activity and/or binding to specific partners. We describe missense mutations in surface loops of ESC, in the CXC domain of E(Z), and in the conserved VEFS domain of SU(Z)12, which each disrupt HMTase activity but preserve complex assembly. Thus, the E(Z) SET domain requires multiple partner inputs to produce active HMTase. We also find that a recombinant worm complex containing the E(Z) homolog, MES-2, has robust HMTase activity, which depends upon both MES-6, an ESC homolog, and MES-3, a pioneer protein. Thus, although the fly and mammalian PcG complexes absolutely require SU(Z)12, the worm complex generates HMTase activity from a distinct partner set.

Animals↗