Dataset Information


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 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

INSTRUMENT(S): Agilent-014868 Whole Mouse Genome Microarray 4x44K G4122F (Feature Number version)

ORGANISM(S): Mus musculus  

SUBMITTER: Jason T Huff