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Genome-wide identification of Polycomb-associated RNAs by RIP-seq

ABSTRACT: Large-scale analyses have revealed that 60-90% of the mammalian genome is transcriptionally active. Because <2% of sequences have protein-coding potential, why so much cellular energy is expended on RNA synthesis is a major question in the post-genomic era. The hypothesis that RNA may serve as recruiting platforms for chromatin modifiers has gained ground with discoveries linking long ncRNAs, such as RepA, Xist, and Tsix, to locus-specific targeting of Polycomb proteins to the X-chromosome. Long ncRNAs have also been associated with Polycomb proteins at human HOX loci. Here, we ask if RNA targeting may be a general feature of regulation for mouse Polycomb repressive complex 2 (PRC2). We develop a method to capture the 'PRC2 transcriptome' by combining RNA immunoprecipitation (RIP) with deep sequencing (seq). RIP-seq of mouse embryonic stem cells identifies >3000 RNAs in the PRC2 transcriptome. Approximately 14% of RNAs originate from previously described PRC2-binding sites and many are promoter-associated. The transcriptome includes a large number of unannotated noncoding and antisense RNAs, with the X-chromosome exhibiting a high density of PRC2 RNAs. Imprinted genes and other disease genes, including those involved in cancer, are also well-represented. Functional validation of select candidates confirms RNA-PRC2 interactions in vivo and recruitment of Polycomb proteins in cis. Thus, RNA cofactors may be one general mechanism, among others, for targeting mammalian PRC2. Given PRC2's essential roles during stem cell renewal, development, and cancer, the PRC2 transcriptome described herein will provide a valuable resource for regenerative medicine and cancer biology.

ORGANISM(S): Mus musculus

PROVIDER: GSE17064 | GEO | 2010/12/22

SECONDARY ACCESSION(S): PRJNA119843

REPOSITORIES: GEO

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Project description:We recently showed that the mammalian genome encodes more than a thousand large intergenic non-coding RNAs (lincRNAs) that are clearly conserved across mammals and thus functional. Gene expression patterns have implicated these lincRNAs in diverse biological processes including cell cycle regulation, immune surveillance, and embryonic stem cell pluripotency. However, the mechanism by which these lincRNAs function is unknown. Here, we expand the catalog of human lincRNAs to ~3300 by analyzing chromatin-state maps of various human cell types. Inspired by the observation that the well-characterized lincRNA HOTAIR bind the Polycomb Repressive Complex 2 (PRC2), we tested whether many lincRNAs are physically associated with PRC2. Remarkably, we observe that ~20% of lincRNAs expressed in various cell types are bound by PRC2, and that additional lincRNAs are bound by other chromatin-modifying complexes. Moreover, we show that siRNA-mediated depletion of certain lincRNAs associated with PRC2 leads to changes in gene expression and that the upregulated genes are enriched for those normally silenced by PRC2. We propose a model in which some lincRNAs guide chromatin–modifying complexes to specific genomic loci to regulate gene expression. siRNA-mediated lincRNA knockdown siRNAs targeting specific lincRNAs were transected into human fibroblasts. Non-targeting siRNAs were used as controls. Both of these experiment types were hybridized to Affymetrix gene expression arrays. PRC2 RIP-Chip The PRC2 complex was immunoprecipitated using antibodies targetting Suz12 and the associated RNA was hybridized to Nimblegen tiling arrays representing lincRNAs. In parallel an IgG IP was performed and hybridized to the same NG arrays. RIP was performed for PRC2 and hybridized using a Cy3 label. In parallel, RIP was performed using IGG and labelled with Cy5 PRC2 RIP-Chip (dye swap) The PRC2 complex was immunoprecipitated using antibodies targetting Suz12 and the associated RNA was hybridized to Nimblegen tiling arrays representing lincRNAs. In parallel an IgG IP was performed and hybridized to the same NG arrays. RIP was performed for PRC2 and hybridized using a Cy5 label. In parallel, RIP was performed using IGG and labelled with Cy3 PRC2 RIP-Chip (no proteins) The PRC2 complex was immunoprecipitated using antibodies targetting Suz12 and the associated RNA was hybridized to Nimblegen tiling arrays representing lincRNAs. In parallel an IgG IP was performed and hybridized to the same NG arrays. RIP was performed for PRC2 and hybridized using a Cy3 label. In parallel, RIP was performed using IGG and labelled with Cy5 COREST RIP-Chip The COREST complex was immunoprecipitated using antibodies targetting COREST and the associated RNA was hybridized to Nimblegen tiling arrays representing lincRNAs. In parallel an IgG IP was performed and hybridized to the same NG arrays. RIP was performed for COREST and hybridized using a Cy3 label. In parallel, RIP was performed using IGG and labelled with Cy5 H3K27me3 and H3K4me2 modified histones RIP-Chip The H3K27me3 and H3K4me2 modified histones were immunoprecipitated using antibodies targetting COREST and the associated RNA was hybridized to Nimbelgen tiling arrays representing lincRNAs. In parallel an IgG IP was performed and hybridized to the same NG arrays. RIP was performed for COREST and hybridized using a Cy3 label. In parallel, RIP was performed using IGG and labelled with Cy5 Human lincRNA expression Human lincRNAs were profiled in HeLa, Lung FB, and Foot FB. In addition, nuclear and cytoplasmic fractions were taken in HeLa cells Total RNA was extracted from HeLa, Foot, and Lung cells and hybridized to NG tiling array. In addition, the nucleus was fractionated and RNA was hybridized to an array.

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Genome-wide identification of Polycomb-associated RNAs by RIP-seq

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