Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

ChIP-chip of siGFP- or siHOTAIR-treated foreskin fibroblasts with anti-LSD1 or anti-SUZ12 antibodies on human HG18 Nimblegen promoter arrays

ABSTRACT: Long intergenic noncoding RNAs (lincRNAs) regulate chromatin states and epigenetic inheritance. Here we show that the lincRNA HOTAIR serves as a scaffold for at least two distinct histone modification complexes. A 5’ domain of HOTAIR binds Polycomb Repressive Complex 2 (PRC2) while a 3’ domain of HOTAIR binds the LSD1/CoREST/REST complex. The ability to tether two distinct complexes enables RNA-mediated assembly of PRC2 and LSD1, and coordinates targeting of PRC2 and LSD1 to chromatin for coupled histone H3 lysine 27 methylation and lysine 4 demethylation. Our results suggest that lincRNAs may serve as scaffolds by providing binding surfaces to assemble select histone modification enzymes, and thereby specify the pattern of histone modifications on target genes. LSD1 and SUZ12 co-occupied on 721 genes in human foreskin fibroblasts. Coordinate loss of SUZ12 and LSD1 occupancy was caused by HOTAIR knockdown. Comparison occupancy of LSD1 and SUZ12 of siGFP and siHOTAIR foreskin fibroblasts on human HG18 promoter arrays. Human foreskin fibroblasts were transfected with siGFP or siHOTAIR. The cells were harvested and ChIP analysis with anti-LSD1 and anti-SUZ12 antibodies was performed.

ORGANISM(S): Homo sapiens

SUBMITTER: Miao-Chih Tsai

PROVIDER: E-GEOD-22344 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Long noncoding RNA as modular scaffold of histone modification complexes.

Tsai Miao-Chih MC Manor Ohad O Wan Yue Y Mosammaparast Nima N Wang Jordon K JK Lan Fei F Shi Yang Y Segal Eran E Chang Howard Y HY

Science (New York, N.Y.) 20100708 5992

Long intergenic noncoding RNAs (lincRNAs) regulate chromatin states and epigenetic inheritance. Here, we show that the lincRNA HOTAIR serves as a scaffold for at least two distinct histone modification complexes. A 5' domain of HOTAIR binds polycomb repressive complex 2 (PRC2), whereas a 3' domain of HOTAIR binds the LSD1/CoREST/REST complex. The ability to tether two distinct complexes enables RNA-mediated assembly of PRC2 and LSD1 and coordinates targeting of PRC2 and LSD1 to chromatin for cou ...[more]

PMID: 20616235

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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.

Dataset Information

ChIP-chip of siGFP- or siHOTAIR-treated foreskin fibroblasts with anti-LSD1 or anti-SUZ12 antibodies on human HG18 Nimblegen promoter arrays

Publications

Long noncoding RNA as modular scaffold of histone modification complexes.

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