Project description:H3K27me3 is a chromatin modification depositied by Suz12, a component of the Polycomb Group 2 complex, and is associated with transcriptional repression. In contrast, H3K79me2 is a chromatin modification associated with active gene transcription. It is deposited by the histone methyltransferase Dot1L and generally is localized just downstream of the transcriptional start site and extends down the body of the gene. To gain insight into the transcriptional state of genes in hES cells, chromatin immunoprecipitation coupled with massive parallel sequencing (ChIP-seq) was performed to determine the genome-wide occupancy of the H3K27me3 and H3K79me2 chromatin modifications and genome-wide occupancy of the Suz12. DNA was enriched by chromatin immunoprecipitation (ChIP) and analyzed by Solexa sequencing A sample of whole cell extract was sequenced and used as the background to determine enrichment. ChIP was performed using an antibody against H3K27me3, H3K79me2, and Suz12 (Abcam).
Project description:H3K27me3 is a chromatin modification depositied by Suz12, a component of the Polycomb Group 2 complex, and is associated with transcriptional repression. In contrast, H3K79me2 is a chromatin modification associated with active gene transcription. It is deposited by the histone methyltransferase Dot1L and generally is localized just downstream of the transcriptional start site and extends down the body of the gene. To gain insight into the transcriptional state of genes in hES cells, chromatin immunoprecipitation coupled with massive parallel sequencing (ChIP-seq) was performed to determine the genome-wide occupancy of the H3K27me3 and H3K79me2 chromatin modifications and genome-wide occupancy of the Suz12.
Project description:LUM1_676443: human, purified fusion protein EZH1-SUZ12. For studying phosphorylation of serine 583 on human SUZ12.
LUM1_679396: mouse, antibody IP of mouse embryonic stem cells. For studying phosphorylation of serine 585 on mouse SUZ12.
Project description:Chromatin immunoprecipitation followed by Solexa sequencing for H3K27me3 and H3K79me2 in Fibroblasts, Embryonic stem cells, and fibroblast undergoing reprogramming
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:We used chromatin immunoprecipitation (ChIP) in combination with human promoter microarrays to idnetify SUZ12 and H3K27me3-occupied gene promoters in prostate cancer cells and tissues. We observed a common set of SUZ12 and H3K27me3-occupied genes in LNCaP cells, and metastatic prostate cancer tissues across individuals as well as tissue types of the metastatic sites. We also found significant overlap of cancer polycomb targets with previously published embryonic stem cell polycomb targets. In addition, a strong association between cancer polycomb targets with prostate cancer outcome was observed. We developed a cancer Polycomb Repression Signature, comprised direct targets of PRC2 silencing in cancer, that is predictive of cancer outcome in multiple expression profling datasets of tumors. Keywords: protein-DNA interaction