Project description:Suz12(Bgal/Bgal) ESCs express a truncated form of Suz12 fused to Beta-galactosidase. These cells maintain a reduced level of H3K27me3 despite this mutation to a core component of PRC2, unlike Eed-/- ESCs whose H3K27me3 is ablated. Two ESC lines mutant in genes of core components of Polycomb Repressive Complex 2 were assessed for H3K27me3 by ChIP-seq, as compared to a wild type ESC line.
Project description:Suz12(Bgal/Bgal) ESCs express a truncated form of Suz12 fused to Beta-galactosidase. These cells maintain a reduced level of H3K27me3 despite this mutation to a core component of PRC2, unlike Eed-/- ESCs whose H3K27me3 is ablated. RNA-seq was performed in wild type and Suz12(Bgal/Bgal) ESCs, here used to demonstrate the coverage of the Suz12 gene in mRNA reads.
Project description:Suz12(Bgal/Bgal) ESCs express a truncated form of Suz12 fused to Beta-galactosidase. These cells maintain a reduced level of H3K27me3 despite this mutation to a core component of PRC2, unlike Eed-/- ESCs whose H3K27me3 is ablated.
Project description:Suz12(Bgal/Bgal) ESCs express a truncated form of Suz12 fused to Beta-galactosidase. These cells maintain a reduced level of H3K27me3 despite this mutation to a core component of PRC2, unlike Eed-/- ESCs whose H3K27me3 is ablated.
Project description:Suz12(Bgal/Bgal) ESCs express a truncated form of Suz12 fused to Beta-galactosidase. These cells maintain a reduced level of H3K27me3 despite this mutation to a core component of PRC2, unlike Eed-/- ESCs whose H3K27me3 is ablated. This data shows the concomitant changes in H3K4me3 levels in these cells. An ESC line mutant in Suz12, a core component of Polycomb Repressive Complex 2, was assessed for H3K4me3 by ChIP-seq, as compared to a wild type ESC line, as well as both lines subjected to in vitro differentiation down the Spinal Motor Neuron pathway.
Project description:Suz12(Bgal/Bgal) ESCs express a truncated form of Suz12 fused to Beta-galactosidase. These cells maintain a reduced level of H3K27me3 despite this mutation to a core component of PRC2, unlike Eed-/- ESCs whose H3K27me3 is ablated. This data shows the concomitant changes in H3K4me3 levels in these cells.
Project description:Polycomb repressive complex 2 (PRC2) trimethylates lysine 27 of histone H3 (H3K27me3), which regulates gene expression and controls diverse biological transitions in development, embryonic stem cell (ESC) differentiation, and cancer. Here we show that Polycomb-like 3 (Pcl3) is a component of PRC2 that promotes H3K27 trimethylation in ESCs. Chromatin immunoprecipitation and sequencing (ChIP-seq) revealed that Pcl3 co-localizes and recruits Suz12 to CpG islands. Depletion of Pcl3 decreased Suz12 binding at over 60% of PRC2 targets, including many bivalent genes. Pcl3 promotes ESC self-renewal as knockdown of Pcl3 increased spontaneous differentiation. However, Pcl3 does not affect ESC pluripotency as teratomas derived from Pcl3-depleted ESCs were able to form all three germ layers. Mutation of conserved residues within the Pcl3 TUDOR domain, a domain that recognizes methylated histones, compromises H3K27me3, suggesting that the TUDOR domain of Pcl3 is crucial for function. Thus, Pcl3 is a component of PRC2 critical for histone methylation and PRC2 recruitment. We reverted a Suz12 gene trap (Suz12Gt/+) allele generated in a mouse ESC line to produce an allele that re-expresses Suz12 but that contains a loxP targeting site (Suz12Rev/+). Using a modified Floxin shuttle vector, we inserted an exon encoding amino acids 277-741 of Suz12 fused to a carboxy-terminal 6xHis-3xFlag TAP tag. The resultant allele (Suz12Suz12TAP/+) expresses the full-length TAP-tagged Suz12 from the endogenous locus. We then reduced Pcl3 expression by siRNA or shRNA. Finally, we performed ChIP-Seq using the Flag tag of Suz12Suz12TAP/+ cells expressing either Pcl3 or control shRNA. To perform Pcl3 ChIP-seq, we created ESC clones expressing Pcl3-shRNAs and a TAP-tagged Pcl3 not recognized by the Pcl3-shRNAs.
Project description:Polycomb repressive complex 2 (PRC2) regulates gene expression during lineage specification through trimethylation of lysine 27 on histone H3 (H3K27me3). In Drosophila, polycomb binding sites are dynamic chromatin regions coupled to incorporation of the histone variant H3.3. Here we show in mouse embryonic stem cells (ESCs) that H3.3 is required for proper establishment of H3K27me3 at the promoters of developmentally regulated genes. These promoters show reduced dynamics as determined by deposition of de novo synthesized histones, associated with reduced PRC2 occupancy. H3.3-depleted ESCs show upregulation of extraembryonic trophectoderm, as well as misregulation of other developmental genes upon differentiation. Our data demonstrate the importance of H3.3 incorporation in ESCs and suggest that changes in chromatin dynamics in its absence lead to misregulation of gene expression during differentiation. Moreover, our findings lend support to the emerging notion that H3.3 has multiple functions in distinct genomic locations that are not always correlated with an “active” chromatin state. Native ChIP analysis of three histone post-translational modifications (H3K4me3, H3K27me3, H3K27ac) in two mouse embryonic stem cell (ESC) lines (control and H3.3-depleted). Inputs sequenced as control. Native ChIP analysis of H3.3B-HA in control and Suz12-/- ESCs. Crosslinking ChIP analysis of histone H3 using a general H3 antibody in two ESC lines (control and H3.3-depleted). Crosslinking ChIP analysis Hira, UTX, and Jmjd3 in wild type and H3.3 KO ESCs.