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. 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.
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.
Project description:Genome-wide analysis of Jarid2, Suz12 and H3K27me3 profiling in DCR-/- and DCRfl/fl ESCs performed by ChIP-seq and transcriptome analysis by RNA-seq
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.
