Drosophila Polycomb Group Gene Research Articles

YY1 Positively Regulates Transcription by Targeting Promoters and Super-Enhancers through the BAF Complex in Embryonic Stem Cells.

SummaryYin Yang 1 (YY1) regulates early embryogenesis and adult tissue formation. However, the role of YY1 in stem cell regulation remains unclear. YY1 has a Polycomb group (PcG) protein-dependent role in mammalian cells. The PcG-independent functions of YY1 are also reported, although their underlying mechanism is still undefined. This paper reports the role of YY1 and BAF complex in the OCT4-mediated pluripotency network in mouse embryonic stem cells (mESCs). The interaction between YY1 and BAF complex promotes mESC proliferation and pluripotency. Knockdown of Yy1 or Smarca4, the core component of the BAF complex, downregulates pluripotency markers and upregulates several differentiation markers. Moreover, YY1 enriches at both promoter and super-enhancer regions to stimulate transcription. Thus, this study elucidates the role of YY1 in regulating pluripotency through its interaction with OCT4 and the BAF complex and the role of BAF complex in integrating YY1 into the core pluripotency network.

Mutations in the polycomb group gene polyhomeotic lead to epithelial instability in both the ovary and wing imaginal disc in Drosophila.

BackgroundMost human cancers originate from epithelial tissues and cell polarity and adhesion defects can lead to metastasis. The Polycomb-Group of chromatin factors were first characterized in Drosophila as repressors of homeotic genes during development, while studies in mammals indicate a conserved role in body plan organization, as well as an implication in other processes such as stem cell maintenance, cell proliferation, and tumorigenesis. We have analyzed the function of the Drosophila Polycomb-Group gene polyhomeotic in epithelial cells of two different organs, the ovary and the wing imaginal disc.ResultsClonal analysis of loss and gain of function of polyhomeotic resulted in segregation between mutant and wild-type cells in both the follicular and wing imaginal disc epithelia, without excessive cell proliferation. Both basal and apical expulsion of mutant cells was observed, the former characterized by specific reorganization of cell adhesion and polarity proteins, the latter by complete cytoplasmic diffusion of these proteins. Among several candidate target genes tested, only the homeotic gene Abdominal-B was a target of PH in both ovarian and wing disc cells. Although overexpression of Abdominal-B was sufficient to cause cell segregation in the wing disc, epistatic analysis indicated that the presence of Abdominal-B is not necessary for expulsion of polyhomeotic mutant epithelial cells suggesting that additional POLYHOMEOTIC targets are implicated in this phenomenon.ConclusionOur results indicate that polyhomeotic mutations have a direct effect on epithelial integrity that can be uncoupled from overproliferation. We show that cells in an epithelium expressing different levels of POLYHOMEOTIC sort out indicating differential adhesive properties between the cell populations. Interestingly, we found distinct modalities between apical and basal expulsion of ph mutant cells and further studies of this phenomenon should allow parallels to be made with the modified adhesive and polarity properties of different types of epithelial tumors.

Recruitment of Polycomb Complexes: a Role for SCM

Recruitment of Polycomb Complexes: a Role for SCM

Histone H2A deubiquitinase activity of the Polycomb repressive complex PR-DUB

Polycomb group (PcG) proteins are transcriptional repressors that control processes ranging from the maintenance of cell fate decisions and stem cell pluripotency in animals to the control of flowering time in plants. In Drosophila, genetic studies identified more than 15 different PcG proteins that are required to repress homeotic (HOX) and other developmental regulator genes in cells where they must stay inactive. Biochemical analyses established that these PcG proteins exist in distinct multiprotein complexes that bind to and modify chromatin of target genes. Among those, Polycomb repressive complex 1 (PRC1) and the related dRing-associated factors (dRAF) complex contain an E3 ligase activity for monoubiquitination of histone H2A (refs 1-4). Here we show that the uncharacterized Drosophila PcG gene calypso encodes the ubiquitin carboxy-terminal hydrolase BAP1. Biochemically purified Calypso exists in a complex with the PcG protein ASX, and this complex, named Polycomb repressive deubiquitinase (PR-DUB), is bound at PcG target genes in Drosophila. Reconstituted recombinant Drosophila and human PR-DUB complexes remove monoubiquitin from H2A but not from H2B in nucleosomes. Drosophila mutants lacking PR-DUB show a strong increase in the levels of monoubiquitinated H2A. A mutation that disrupts the catalytic activity of Calypso, or absence of the ASX subunit abolishes H2A deubiquitination in vitro and HOX gene repression in vivo. Polycomb gene silencing may thus entail a dynamic balance between H2A ubiquitination by PRC1 and dRAF, and H2A deubiquitination by PR-DUB.

Enhancer of Polycomb1, a Novel Homeodomain Only Protein-binding Partner, Induces Skeletal Muscle Differentiation

Homeodomain only protein, Hop, is an unusual small protein that modulates target gene transcription without direct binding to DNA. Here we show that Hop interacts with Enhancer of Polycomb1 (Epc1), a homolog of a Drosophila polycomb group gene product that regulates transcription, to induce the skeletal muscle differentiation. Yeast two-hybrid assay with the human adult heart cDNA library revealed that Hop can associate with Epc1. The amino-terminal domain of Epc1 as well as full Epc1 physically interacted with Hop in mammalian cells and in yeast. Epc1 is highly expressed in the embryonic heart and adult skeletal muscles. Serum deprivation induced differentiation of H9c2, a myoblast cell line, into skeletal myocytes, and Epc1 was up-regulated. Differentiation of H9c2 was induced by Epc1 overexpression, although it was severely impaired in Epc1-knockdown cells. Co-transfection of Hop potentiated Epc1-induced transactivation of myogenin and myotube formation. Hop knock-out mice elicited a decrease in myosin heavy chain and myogenin expressions in skeletal muscle and showed delay in hamstring muscle healing after injury. Differentiation was impaired in skeletal myoblasts from Hop knock-out mice. These results suggest that Epc1 plays a role in the initiation of skeletal muscle differentiation, and its interaction with Hop is required for the full activity.

Hyped‐up for Hox in Hyderabad

The EMBO Workshop on Upstream and Downstream of Hox Genes took place in Hyderabad, India, between 14 and 17 December 2005, and was organized by R. Mishra and F. Karch. ![][1] For many years, EMBO workshops dealing with new insights into the regulation and function of Hox genes have been held in Switzerland. However, last year, the EMBO World Programme, together with the Centre for Cellular and Molecular Biology (CCMB), held a workshop entitled Upstream and Downstream of Hox Genes in the CCMB's hometown of Hyderabad, India. Imagine beautiful gardens full of butterflies and palm trees, a delightful guest house neighbouring a state‐of‐the‐art research centre, all in a peaceful oasis surrounded by a pulsating and crowded Indian city. The question at the beginning of the meeting was whether the talks and discussions would match the stunning environment; it was answered rapidly and affirmatively. Hox genes have had an illustrious and curious history. Perhaps one of the most famous images in biology from the past century is the four‐winged mutant of the fruit fly Drosophila melanogaster caused by mutations in the Hox gene Ultrabithorax ( Ubx ). This fascinating mutant fly, discovered and characterized by Edward B. Lewis, has motivated many investigators to join the field to understand how these genes are responsible for such complex changes in morphology. Another highlight of Hox history was Matthew Scott's, William McGinnis's and Walter Gehring's discovery of the homeodomain—a 60‐amino‐acid DNA‐binding motif now known to be a key domain in hundreds of genes throughout the animal, fungal and plant kingdoms. Although a lot has been learned through experiments stemming from these important discoveries, there are still many open questions. Among the questions being debated in the field and at the meeting are: How is the regulation of the Hox genes initiated and maintained? How do Hox proteins … [1]: /embed/graphic-1.gif

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.

Isolation and expression analysis of petunia CURLY LEAF-like genes.

The Arabidopsis CURLY LEAF (CLF) gene is required to repress transcription of the class C gene AGAMOUS (AG) in whorls 1 and 2 of flowers and also in vegetative organs. CLF encodes a protein with homology to the product of the Drosophila Polycomb-group gene Enhancer of zeste [E(z)], which is involved in embyogenesis. In this study, we isolated three petunia CLF-like genes (PhCLF1, PhCLF2 and PhCLF3) based on the sequence homology between CLF and E(Z). Sequence analysis suggests that PhCLF1 and PhCLF2 are orthologs of CLF, whereas PhCLF3 is an ortholog of the Arabidopsis gene EZA1. We identified several conserved domains among products of PhCLF genes and related genes. PhCLF1 and PhCLF2 were expressed in all floral organs and leaves. The PhCLF1 transcripts were accumulated especially in corolla limbs, and contained several alternatively spliced RNA species. PhCLF1 and PhCLF2 do not appear to be the BLIND gene, which is required to repress transcription of the petunia class C gene, but their expression was affected by the homeotic conversion of organs in the blind flower. Our findings show that expression of PhCLF1 is regulated differently from that of PhCLF2, and suggest that the two petunia CLF orthologs function differently from each other.

The mouse Edr2 (Mph2) gene has two forms of mRNA encoding 90- and 36-kDa polypeptides

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. Here we have isolated a 2.5-kb cDNA for Edr2, a mouse homolog of the Drosophila PcG gene Ph, although it was originally identified as a 3.8-kb cDNA. However, little is known about molecular basis of the 3.8-kb cDNA. Genomic and RNA analyses have shown that Edr2 locates on Chromosome 4 as a single copy gene and is transcribed into at least two transcript isoforms about 3.0 and 4.4 kb in length, most likely corresponding to the 2.5- and 3.8-kb cDNAs, respectively. The largest open reading frames in the 2.5- and 3.8-kb cDNAs encode 36- and 90-kDa polypeptides, respectively. The 36-kDa protein is a truncated form lacking of the N-terminal region of the 90-kDa protein. Interestingly, it has been demonstrated that the 3.0-kb mRNA accumulates at a much higher level than the 3.8-kb mRNA in mouse embryos and mature tissues. Immunostaining assay of mammalian cells has shown that the 36-kDa form tagged with HA colocalizes with the other PcG protein Mel18 in nuclei, suggesting that the smaller protein is capable of forming maltimeric complex with other PcG proteins. Therefore, the 36-kDa protein might function generally as a PcG protein.

Su(z)12, a novel Drosophila Polycomb group gene that is conserved in vertebrates and plants.

In both Drosophila and vertebrates, spatially restricted expression of HOX genes is controlled by the Polycomb group (PcG) repressors. Here we characterize a novel Drosophila PcG gene, Suppressor of zeste 12 (Su(z)12). Su(z)12 mutants exhibit very strong homeotic transformations and Su(z)12 function is required throughout development to maintain the repressed state of HOX genes. Unlike most other PcG mutations, Su(z)12 mutations are strong suppressors of position-effect variegation (PEV), suggesting that Su(z)12 also functions in heterochromatin-mediated repression. Furthermore, Su(z)12 function is required for germ cell development. The Su(z)12 protein is highly conserved in vertebrates and is related to the Arabidopsis proteins EMF2, FIS2 and VRN2. Notably, EMF2 is a repressor of floral homeotic genes. These results suggest that at least some of the regulatory machinery that controls homeotic gene expression is conserved between animals and plants.

Loss- and gain-of-function mutations show a polycomb group function for Ring1A in mice.

The products of the Polycomb group (PcG) of genes act as transcriptional repressors involved in the maintenance of homeotic gene expression patterns throughout development, from flies to mice. Biochemical and molecular evidence suggests that the mouse Ring1A gene is a member of the PcG of genes. However, genetic evidence is needed to establish PcG function for Ring1A, since contrary to all other murine PcG genes, there is no known Drosophila PcG gene encoding a homolog of the Ring1A protein. To study Ring1A function we have generated a mouse line lacking Ring1A and mouse lines overexpressing Ring1A. Both Ring1A(-/-)and Ring1A(+/-) mice show anterior transformations and other abnormalities of the axial skeleton, which indicates an unusual sensitivity of axial skeleton patterning to Ring1A gene dosage. Ectopic expression of Ring1A also results in dose-dependent anterior transformations of vertebral identity, many of which, interestingly, are shared by Ring1A(-/-) mice. In contrast, the alterations of Hox gene expression observed in both type of mutant mice are subtle and involve a reduced number of Hox genes. Taken together, these results provide genetic evidence for a PcG function of the mouse Ring1A gene.

RYBP, a new repressor protein that interacts with components of the mammalian Polycomb complex, and with the transcription factor YY1.

The products of the Polycomb group (PcG) of genes are necessary for the maintenance of transcriptional repression of a number of important developmental genes, including the homeotic genes. A two-hybrid screen was used to search for putative new members of the PcG of genes in mammals. We have identified a new Zn finger protein, RYBP, which interacts directly with both Ring1 proteins (Ring1A and Ring1B) and with M33, two mutually interacting sets of proteins of the mammalian Polycomb complex. Ring1 binds RYBP and M33 through the same C-terminal domain, whereas the RYBP-M33 interaction takes place through an M33 domain not involved in Ring1 binding. RYBP also interacts directly with YY1, a transcription factor partially related to the product of the Drosophila pleiohomeotic gene. In addition, we show here that RYBP acts as a transcriptional repressor in transiently transfected cells. Finally, RYBP shows a dynamic expression pattern during embryogenesis which initially overlaps partially that of Ring1A in the central nervous system, and later becomes ubiquitous. Taken together, these data suggest that RYBP may play a relevant role in PcG function in mammals.

The Drosophila Polycomb Group Gene pleiohomeotic Encodes a DNA Binding Protein with Homology to the Transcription Factor YY1

Genes of the Polycomb group (PcG) of Drosophila encode proteins necessary for the maintenance of transcriptional repression of homeotic genes. PcG proteins are thought to act by binding as multiprotein complexes to DNA through Polycomb group response elements (PREs); however, specific DNA binding has not been demonstrated for any of the PcG proteins. We have identified a sequence-specific DNA binding protein that interacts with a PRE from the Drosophila engrailed gene. This protein (PHO) is a homolog of the ubiquitous mammalian transcription factor Yin Yang-1 and is encoded by pleiohomeotic, a known member of the PcG. We propose that PHO acts to anchor PcG protein complexes to DNA.

Characterization of interactions between the mammalian polycomb-group proteins Enx1/EZH2 and EED suggests the existence of different mammalian polycomb-group protein complexes.

In Drosophila melanogaster, the Polycomb-group (PcG) and trithorax-group (trxG) genes have been identified as repressors and activators, respectively, of gene expression. Both groups of genes are required for the stable transmission of gene expression patterns to progeny cells throughout development. Several lines of evidence suggest a functional interaction between the PcG and trxG proteins. For example, genetic evidence indicates that the enhancer of zeste [E(z)] gene can be considered both a PcG and a trxG gene. To better understand the molecular interactions in which the E(z) protein is involved, we performed a two-hybrid screen with Enx1/EZH2, a mammalian homolog of E(z), as the target. We report the identification of the human EED protein, which interacts with Enx1/EZH2. EED is the human homolog of eed, a murine PcG gene which has extensive homology with the Drosophila PcG gene extra sex combs (esc). Enx1/EZH2 and EED coimmunoprecipitate, indicating that they also interact in vivo. However, Enx1/EZH2 and EED do not coimmunoprecipitate with other human PcG proteins, such as HPC2 and BMI1. Furthermore, unlike HPC2 and BMI1, which colocalize in nuclear domains of U-2 OS osteosarcoma cells, Enx1/EZH2 and EED do not colocalize with HPC2 or BMI1. Our findings indicate that Enx1/EZH2 and EED are members of a class of PcG proteins that is distinct from previously described human PcG proteins.

Positional cloning of a global regulator of anterior–posterior patterning in mice

Anterior-posterior (A-P) patterning is of fundamental importance throughout vertebrate embryonic development. Murine members of the trithorax (trx) and Polycomb group (Pc-G) of genes regulate A-P patterning of segmented axial structures, demonstrating conserved upstream regulation of homeotic pathways between Drosophila and mouse. Here we report the positional cloning of a classical mouse mutation, eed (for embryonic ectoderm development), which is the highly conserved homologue of the Drosophila Pc-G gene esc (for extra sex combs), a long-term repressor of homeotic genes. Mutants homozygous for a null allele of eed display disrupted A-P patterning of the primitive streak during gastrulation. Mutant embryos lack a node, notochord and somites, and there is no neural induction. In contrast to absence of anterior structures, extra-embryonic mesoderm is abundant. Mice carrying a hypomorphic eed mutation exhibit posterior transformations along the axial skeleton. These results indicate that eed is required globally for A-P patterning throughout embryogenesis.

A role for mel-18, a Polycomb group-related vertebrate gene, during theanteroposterior specification of the axial skeleton.

Segment identity in both invertebrates and vertebrates is conferred by spatially restricted distribution of homeotic gene products. In Drosophila, the expression of Homeobox genes during embryogenesis is initially induced by segmentation gene products and then maintained by Polycomb group and Trithorax group gene products. Polycomb group gene homologs are conserved in vertebrates. Murine mel-18 and closely related bmi-1 are homologous to posterior sex combs and suppressor two of zeste. Mel-18 protein mediates a transcriptional repression via direct binding to specific DNA sequences. To gain further insight into the function of Mel-18, we have inactivated the mel-18 locus by homologous recombination. Mice lacking mel-18 survive to birth and die around 4 weeks after birth after exhibiting strong growth retardation. Similar to the Drosophila posterior sex combs mutant, posterior transformations of the axial skeleton were reproducibly observed in mel-18 mutants. The homeotic transformations were correlated with ectopic expression of Homeobox cluster genes along the anteroposterior axis in the developing paraxial mesoderm. Surprisingly, mel-18-deficient phenotypes are reminiscent of bmi-1 mutants. These results indicate that the vertebrate Polycomb group genes mel-18 and bmi-1, like Drosophila Polycomb group gene products, might play a crucial role in maintaining the silent state of Homeobox gene expression during paraxial mesoderm development.

Mel-18, a Polycomb group-related mammalian gene, encodes a transcriptional negative regulator with tumor suppressive activity.

The mammalian mel-18/bmi-1 gene products share an amino acid sequence and a secondary structure, including a RING-finger motif, with the Drosophila Polycomb group (PcG) gene products Psc and Su(z)2, implying that they represent a gene family with related functions. As Drosophila PcG gene products are thought to function as transcriptional repressors by modifying chromatin structure, Mel-18/Bmi-1 might be expected to have similar activities. Here we have analyzed the function of mel-18 and found that Mel-18 acts as a transcriptional repressor via its target DNA sequence, 5'-GACTNGACT-3'. Interestingly, this binding sequence is found within regulatory or non-coding regions of various genes, including the c-myc, bcl-2 and Hox genes, suggesting diverse functions of mel-18 as the mammalian homolog of the PcG gene. We also demonstrate that mel-18 has tumor suppressor activity, in contrast to bmi-1, which has been defined as a proto-oncogene.

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.

Mutations in polycombeotic, a Drosophila polycomb-group gene, cause a wide range of maternal and zygotic phenotypes.

The polycomb-group genes, a set of genes characterized by mutations that cause similar phenotypes and dosage-dependent interactions, are required for the normal expression of segment-specific homeotic loci. Here we report that polycombeotic (formerly 1(3)1902), originally identified by a lethal mutation that causes a small-disc phenotype, is also a member of this group of essential genes. Adults homozygous for temperature-sensitive pco alleles that were exposed to the restrictive temperature during larval life display the second and third leg to first leg transformation characteristic of polycomb-group mutants. Adult females homozygous for temperature-sensitive alleles exposed to the restrictive temperature during oogenesis produce embryos that show anterior segments with structures normally unique to the eighth abdominal segment, another transformation characteristic of polycomb-group mutants. Mutations in the polycombeotic gene also cause defects not reported for mutations in other polycomb-group genes. Females homozygous for the most extreme temperature-sensitive allele are sterile, and larvae homozygous for null alleles have small imaginal discs and reduced frequencies of mitotic figures in the brain. Dominant mutations originally identified as enhancers or suppressors of zeste are gain-of-function alleles of polycombeotic. The type and variety of defects displayed by different mutations in this gene indicate that the product might be involved in chromosome structure and/or function.