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An RNAi Screen of Chromatin Proteins Identifies Tip60-p400 as a Regulator of Embryonic Stem Cell Identity, Experiment A

ABSTRACT: Proper regulation of chromatin structure is necessary for the maintenance of cell type-specific gene expression patterns. The embryonic stem cell (ESC) expression pattern governs self-renewal and pluripotency. Here, we present an RNAi screen in mouse ESCs of 1008 loci encoding chromatin proteins. We identified 68 proteins that exhibit diverse phenotypes upon knockdown (KD), including seven subunits of the Tip60-p400 complex. Phenotypic analyses revealed that Tip60-p400 is necessary to maintain characteristic features of ESCs. We show that p400 localization to the promoters of both silent and active genes is dependent upon histone H3 lysine 4 trimethylation (H3K4me3). Furthermore, the Tip60-p400 KD gene expression profile is enriched for developmental regulators and significantly overlaps with that of the transcription factor Nanog. Depletion of Nanog reduces p400 binding to target promoters without affecting H3K4me3 levels. Together, these data indicate that Tip60-p400 integrates signals from Nanog and H3K4me3 to regulate gene expression in ESCs. Keywords: knockdown-gene expression profiling

ORGANISM(S): Mus musculus

PROVIDER: GSE11240 | GEO | 2008/07/11

SECONDARY ACCESSION(S): PRJNA109185

REPOSITORIES: GEO

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An RNAi Screen of Chromatin Proteins Identifies Tip60-p400 as a Regulator of Embryonic Stem Cell Identity, Experiment B

2008-07-11 | GSE11241 | GEO

An RNAi Screen of Chromatin Proteins Identifies Tip60-p400 as a Regulator of Embryonic Stem Cell Identity, Experiment B

Project description:Proper regulation of chromatin structure is necessary for the maintenance of cell type-specific gene expression patterns. The embryonic stem cell (ESC) expression pattern governs self-renewal and pluripotency. Here, we present an RNAi screen in mouse ESCs of 1008 loci encoding chromatin proteins. We identified 68 proteins that exhibit diverse phenotypes upon knockdown (KD), including seven subunits of the Tip60-p400 complex. Phenotypic analyses revealed that Tip60-p400 is necessary to maintain characteristic features of ESCs. We show that p400 localization to the promoters of both silent and active genes is dependent upon histone H3 lysine 4 trimethylation (H3K4me3). Furthermore, the Tip60-p400 KD gene expression profile is enriched for developmental regulators and significantly overlaps with that of the transcription factor Nanog. Depletion of Nanog reduces p400 binding to target promoters without affecting H3K4me3 levels. Together, these data indicate that Tip60-p400 integrates signals from Nanog and H3K4me3 to regulate gene expression in ESCs. Keywords: ChIP-chip We identified genes encoding subunits of the Tip60-p400 complex in an RNAi screen of chromatin proteins in mouse embryonic stem cells (ESCs), which upon depletion resulted in a dramatic phenotype. To investigate the role of this complex in gene expression in ESCs, we performed ChIP-chip location analysis using genome-wide promoter tiling arrays. We immunoprecipitated chromatin with an antibody to p400 and competitively hybridized it to input material on two arrays with probes interrogating essentially all mouse promoters with 100 bp tiling of -3250 bp to +750 bp around the transcription start sites.

2008-07-11 | E-GEOD-11241 | biostudies-arrayexpress

RNAi profiling of mouse chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity, experiment A

Project description:Proper regulation of chromatin structure is necessary for the maintenance of cell type-specific gene expression patterns. The embryonic stem cell (ESC) expression pattern governs self-renewal and pluripotency. Here, we present an RNAi screen in mouse ESCs of 1008 loci encoding chromatin proteins. We identified 68 proteins that exhibit diverse phenotypes upon knockdown (KD), including seven subunits of the Tip60-p400 complex. Phenotypic analyses revealed that Tip60-p400 is necessary to maintain characteristic features of ESCs. We show that p400 localization to the promoters of both silent and active genes is dependent upon histone H3 lysine 4 trimethylation (H3K4me3). Furthermore, the Tip60-p400 KD gene expression profile is enriched for developmental regulators and significantly overlaps with that of the transcription factor Nanog. Depletion of Nanog reduces p400 binding to target promoters without affecting H3K4me3 levels. Together, these data indicate that Tip60-p400 integrates signals from Nanog and H3K4me3 to regulate gene expression in ESCs. Experiment Overall Design: We identified genes encoding subunits of the Tip60-p400 complex in an RNAi screen of chromatin proteins in mouse embryonic stem cells (ESCs), which upon depletion resulted in a dramatic phenotype. To investigate the role of this complex in gene expression in ESCs, we performed expression profiling upon depletion of the catalytic subunits. We performed 4 biological replicates of p400 or Tip60 knockdown and compared them to biological replicates of control EGFP knockdown. The p400 (Ep400) experiment was performed as competitive two-color hybridizations on one 4x44K array with dye swaps and the Tip60 (Htatip) experiment was performed as single color hybridizations on two 4x44K arrays. Note: the Tip60 KD replicate 3 was excluded from downstream analysis of differential expression, because the intensity profile was an outlier in diagnostic analyses

2008-10-20 | E-GEOD-11240 | biostudies-arrayexpress

Hdac6 regulates Tip60-p400 function in stem cells (sequencing)

Project description:The Tip60 (also known as Kat5) lysine acetyltransferase functions broadly as a transcriptional co-activator that acetylates histones. In contrast, Tip60 functions in embryonic stem cells (ESCs) both to silence genes that promote differentiation and to activate genes required for proliferation. The mechanism by which Tip60 functions as a repressor is unknown. Here we show that the class II histone deacetylase Hdac6 co-purifies with Tip60-p400 complex from ESCs and is necessary for complete silencing of most differentiation genes targeted by Tip60. In contrast to differentiated cells, where Hdac6 is mainly cytoplasmic and does not interact with Tip60, Hdac6 is largely nuclear in ESCs and neural stem cells (NSCs) and interacts with Tip60-p400 in both cell types. Hdac6 is enriched at promoters bound by Tip60-p400 in ESCs, but while Tip60 binds on both sides of transcription start sites (TSSs), Hdac6 binding overlaps with only the downstream Tip60 peak. Surprisingly, Hdac6 does not deacetylate histones at these sites, but rather is required for Tip60 binding. These data suggest that nuclear exclusion of Hdac6 during differentiation plays a major role in modulation of Tip60-p400 function. We determined the genome-wide localization of Tip60 and Hdac6 in mouse ES cells, and examined genomic binding profiles of Tip60 and Hdac6 upon indicated knockdown by ChIP-seq. We examined genomic binding profiles of p400 upon indicated knockdown by ChIP-seq.

2013-11-20 | E-GEOD-42209 | biostudies-arrayexpress

Hdac6 regulates Tip60-p400 function in stem cells (sequencing)

2013-11-20 | GSE42209 | GEO

Expression analysis of HCT116 cells transfected using Tip60 or p400-targetting siRNA

Project description:Investigation of whole genome gene expression level changes in HCT116 cells upon knockdown of Tip60 or p400, compared to control siRNA-transfected cells. Two different siRNA directed against Tip60 were used and experiments were done in duplicate. Same for p400-targetting or control siRNA. Hybridization experiment using total RNA recovered from independent cell cultures of HCT116 transfected using control, Tip60-targetting or p400-targetting siRNA.

2013-11-22 | E-GEOD-45615 | biostudies-arrayexpress

Hdac6 is a stem-cell specific modulator of Tip60-p400 function (gene expression)

Project description:To identify the functional role of Hdac6 in embryonic stem cells, we examined gene expression changes in mouse ES cells upon Tip60, Hdac6 and double knockdown. Two biological replicates each for control (GFP), Tip60, Hdac6 and double KD in E14 mouse ESCs. Lentiviral-based shRNA was used and gene expression changes were determined 3 days after KD. The global gene expression profiles of Tip60, Hdac6 and double KD were compared to control KD cells.

2013-11-20 | E-GEOD-42208 | biostudies-arrayexpress

Expression analysis of HCT116 cells transfected using Tip60 or p400-targetting siRNA

2013-11-22 | GSE45615 | GEO

An RNAi Screen of Chromatin Proteins Identifies Tip60-p400 as a Regulator of Embryonic Stem Cell Identity

Project description:This SuperSeries is composed of the following subset Series: GSE11240: An RNAi Screen of Chromatin Proteins Identifies Tip60-p400 as a Regulator of Embryonic Stem Cell Identity, Experiment A GSE11241: An RNAi Screen of Chromatin Proteins Identifies Tip60-p400 as a Regulator of Embryonic Stem Cell Identity, Experiment B Keywords: SuperSeries Refer to individual Series

2008-07-11 | E-GEOD-11243 | biostudies-arrayexpress

Chip-chip from HCT116 cells with p400 antibody

Project description:The chromatin remodeller p400 has oncogenic properties that promote early steps of colon cancer. Chromatin immunoprecipitation (ChIP) of p400 together with chromatin profiling by ChIP-on-chip analysis demonstrated that p400 directly binds the chromatin at promoters of p400 target genes. Study of p400 binding on promoters of HCT116 cells

2013-11-22 | E-GEOD-45614 | biostudies-arrayexpress

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