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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 15, 2012 |
| Title |
Polycomb-like 3 is a PRC2 component that supports embryonic stem cell self-renewal |
| Organism |
Mus musculus |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
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.
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| Overall design |
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.
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| Contributor(s) |
Hunkapiller J, Shen Y, Diaz A, Cagney G, McCleary D, Ramalho-Santos M, Krogan N, Ren B, Song JS, Reiter JF |
| Citation(s) |
22438827 |
| Submission date |
Apr 01, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Jun S Song |
| Organization name |
UCSF
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| Department |
Institute for Human Genetics
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| Street address |
513 Parnassus Avenue, Box 0794
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| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94143-0794 |
| Country |
USA |
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| Platforms (1) |
| GPL9250 |
Illumina Genome Analyzer II (Mus musculus) |
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| Samples (10)
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| Relations |
| SRA |
SRP006224 |
| BioProject |
PRJNA139349 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE28325_RAW.tar |
4.6 Gb |
(http)(custom) |
TAR (of BAM) |
| GSE28325_Skellam_H3K27_depleted_sites.bed.gz |
8.4 Kb |
(ftp)(http) |
BED |
| GSE28325_Skellam_PCL3_binding_sites.bed.gz |
59.9 Kb |
(ftp)(http) |
BED |
| GSE28325_Skellam_Suz12_binding_sites.bed.gz |
99.5 Kb |
(ftp)(http) |
BED |
| GSE28325_Skellam_Suz12_depleted_sites.bed.gz |
68.3 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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