Project description:Genome organization influences transcriptional regulation by facilitating interactions between gene promoters and distal regulatory elements. To analyse distal promoter contacts mediated by the PRC1 complex we used Capture Hi-C (CHi-C) to enrich for promoter-interactions in a HiC library in Ring1a KO and Ring1a/b dKO mouse ES cells.
Project description:Nuclear RNA was isolated from all three cell types to enable differential expression analysis of both coding and non-coding RNA shortly after tamoxifen treatment, that resulted in conditional knock-out of Ring1b in the Ring1a -/- background.
Project description:H3K4me1 (ab8895 Abcam) and H3K27ac (ab4729 Abcam) antibodies were used for ChIP-seq in Ring1a-/- mouse ES cells and after 48h tamoxifen treatment in conditional knock-out of Ring1b in the Ring1a -/- background.
Project description:We used microarrays to investigate the restoration of repression of PRC1 target gene expression in Ring1A/B-dKO ES cells stably expressing either of mock, WT or mutant Ring1B construct.
Project description:The Polycomb group (PcG) gene products mediate heritable silencing of developmental regulators in metazoans, participating in one of two distinct multimeric protein complexes, the Polycomb repressive complexes-1 (PRC1) and -2 (PRC2)1-5. PRC2 catalyses trimethylation of histone H3 at lysine 27 (H3K27) which in turn is thought to provide a recruitment site for PRC13-7. Recent studies demonstrate that mono-ubiquitylation of histone H2A at lysine 119 is important in PcG mediated silencing with the core PRC1 component Ring1A/B functioning as the E3 ligase8. PRC2 has been shown to share target genes with the core transcription network to maintain embryonic stem (ES) cells including Oct4 and Nanog9. Here we identify an essential role for PRC1 in repressing developmental regulators in ES cells, and thereby in maintaining ES cell pluripotency. A significant proportion of the PRC1 target genes are also repressed by Oct4. We demonstrate that engagement of PRC1 and PRC2 at target genes is Oct4-dependent and moreover that Ring1B interacts with Oct4. Collectively these results show that PcG complexes are instrumental in Oct4-dependent repression required to maintain pluripotency of ES cells. This study provides a first functional link between a core ES cell regulator and global epigenetic regulation of the genome. Keywords: genetic modification
Project description:We used microarrays to investigate the restoration of repression of PRC1 target gene expression in Ring1A/B-dKO ES cells stably expressing either of mock, WT or mutant Ring1B construct. Total RNAs were extracted from the respective ES cells, and were subjected to microarray analysis using Affymetrix GeneChip Mouse Genome 430A 2.0 arrays
Project description:The Polycomb group (PcG) gene products mediate heritable silencing of developmental regulators in metazoans, participating in one of two distinct multimeric protein complexes, the Polycomb repressive complexes-1 (PRC1) and -2 (PRC2)1-5. PRC2 catalyses trimethylation of histone H3 at lysine 27 (H3K27) which in turn is thought to provide a recruitment site for PRC13-7. Recent studies demonstrate that mono-ubiquitylation of histone H2A at lysine 119 is important in PcG mediated silencing with the core PRC1 component Ring1A/B functioning as the E3 ligase8. PRC2 has been shown to share target genes with the core transcription network to maintain embryonic stem (ES) cells including Oct4 and Nanog9. Here we identify an essential role for PRC1 in repressing developmental regulators in ES cells, and thereby in maintaining ES cell pluripotency. A significant proportion of the PRC1 target genes are also repressed by Oct4. We demonstrate that engagement of PRC1 and PRC2 at target genes is Oct4-dependent and moreover that Ring1B interacts with Oct4. Collectively these results show that PcG complexes are instrumental in Oct4-dependent repression required to maintain pluripotency of ES cells. This study provides a first functional link between a core ES cell regulator and global epigenetic regulation of the genome. Experiment Overall Design: We observed gene expression of Ring1B single and Ring1A/B double KO cells using Affymetrix chip: MOE 430 2.0. Experiment Overall Design: Because constitutive Ring1A/B-dKO cells can not be maintained we generated conditional KO cells with OHT.
