Project description:Native ChIP on chip for H3K27me3 in murine ES cells comparing WT and Ring1B-/- cells. Paper Abstract: How polycomb group proteins repress gene expression in vivo is not known. Whilst histone modifying activities of the polycomb repressive complexes have been studied extensively, in vitro data has suggested a direct activity of the PRC1 complex in compacting chromatin. Here, we investigate higher-order chromatin compaction of polycomb targets in vivo. We show that polycomb repressive complexes are required to maintain a compact chromatin state at Hox loci in embryonic stem (ES) cells. There is specific decompaction in the absence of PRC2 or PRC1. This is due to PRC1, since decompaction occurs in Ring1B null cells that still have PRC2-mediated H3K27 methylation. Moreover, we show that the ability of Ring1B to restore a compact chromatin state, and to repress Hox gene expression in ES cells, is not dependent on its histone ubiquitination activity. We suggest that Ring1B-mediated chromatin compaction acts to directly limit transcription in vivo. Biological replicates: 3 independently grown, harvested,preplated, micrococcal nuclease digested and ChIP for H3K27me3. 5 Technical replicates.
Project description:ChIP on chip for H3K27me3 in murine ES cells comparing Undifferentiated and Day 3 differentiated. Paper Abstract: How polycomb group proteins repress gene expression in vivo is not known. Whilst histone modifying activities of the polycomb repressive complexes have been studied extensively, in vitro data has suggested a direct activity of the PRC1 complex in compacting chromatin. Here, we investigate higher-order chromatin compaction of polycomb targets in vivo. We show that polycomb repressive complexes are required to maintain a compact chromatin state at Hox loci in embryonic stem (ES) cells. There is specific decompaction in the absence of PRC2 or PRC1. This is due to PRC1, since decompaction occurs in Ring1B null cells that still have PRC2-mediated H3K27 methylation. Moreover, we show that the ability of Ring1B to restore a compact chromatin state, and to repress Hox gene expression in ES cells, is not dependent on its histone ubiquitination activity. We suggest that Ring1B-mediated chromatin compaction acts to directly limit transcription in vivo. Biological replicates: 3 independently grown, harvested, micrococcal nuclease digested and ChIP for H3K27me3. 6 Technical replicates.
Project description:Native ChIP on chip for H3K27me3 in murine ES cells comparing WT and Ring1B-/- cells. Paper Abstract: How polycomb group proteins repress gene expression in vivo is not known. Whilst histone modifying activities of the polycomb repressive complexes have been studied extensively, in vitro data has suggested a direct activity of the PRC1 complex in compacting chromatin. Here, we investigate higher-order chromatin compaction of polycomb targets in vivo. We show that polycomb repressive complexes are required to maintain a compact chromatin state at Hox loci in embryonic stem (ES) cells. There is specific decompaction in the absence of PRC2 or PRC1. This is due to PRC1, since decompaction occurs in Ring1B null cells that still have PRC2-mediated H3K27 methylation. Moreover, we show that the ability of Ring1B to restore a compact chromatin state, and to repress Hox gene expression in ES cells, is not dependent on its histone ubiquitination activity. We suggest that Ring1B-mediated chromatin compaction acts to directly limit transcription in vivo.
Project description:Anterior-posterior differences in H3K27me3 and Ring1B enrichment over the 5 prime Hoxd genes in E10.5 murine distal forelimbs. Chromatin immunoprecipitation (ChIP) of H3K27me3 together with Ring1B and by ChIP-on-chip analysis demonstrated that over the 5 prime HoxD locus H3K27me3 enrichment is decreased and Ring1B enrichment is sparse in limb cells derived from the distal posterior forelimb bud of E10.5 mouse embryos.
Project description:Anterior-posterior differences in H3K27me3 and Ring1B enrichment over the 5 prime Hoxd genes in E10.5 murine distal forelimbs. Chromatin immunoprecipitation (ChIP) of H3K27me3 together with Ring1B and by ChIP-on-chip analysis demonstrated that over the 5 prime HoxD locus H3K27me3 enrichment is decreased and Ring1B enrichment is sparse in limb cells derived from the distal posterior forelimb bud of E10.5 mouse embryos. Array design includes 2 biological replicates for H3K27me3 in the cell lines and Ring1B in the limb tissue, and 2 biological replicates and 2 dye swap replicates for H3K27me3 in the limb tissue.
Project description:ChIP on chip for H3K27me3 in murine ES cells comparing Undifferentiated and Day 3 differentiated. Paper Abstract: How polycomb group proteins repress gene expression in vivo is not known. Whilst histone modifying activities of the polycomb repressive complexes have been studied extensively, in vitro data has suggested a direct activity of the PRC1 complex in compacting chromatin. Here, we investigate higher-order chromatin compaction of polycomb targets in vivo. We show that polycomb repressive complexes are required to maintain a compact chromatin state at Hox loci in embryonic stem (ES) cells. There is specific decompaction in the absence of PRC2 or PRC1. This is due to PRC1, since decompaction occurs in Ring1B null cells that still have PRC2-mediated H3K27 methylation. Moreover, we show that the ability of Ring1B to restore a compact chromatin state, and to repress Hox gene expression in ES cells, is not dependent on its histone ubiquitination activity. We suggest that Ring1B-mediated chromatin compaction acts to directly limit transcription in vivo.