Control of embryonic stem cell identity by BRD4-dependent transcriptional elongation of super-enhancer-associated pluripotency genes.
Bottom Line: Transcription factors and chromatin-remodeling complexes are key determinants of embryonic stem cell (ESC) identity.BRD4 maintains transcription of core stem cell genes such as OCT4 and PRDM14 by occupying their super-enhancers (SEs), large clusters of regulatory elements, and recruiting to them Mediator and CDK9, the catalytic subunit of the positive transcription elongation factor b (P-TEFb), to allow Pol-II-dependent productive elongation.Our study describes a mechanism of regulation of ESC identity that could be applied to improve the efficiency of ESC differentiation.
Affiliation: Department of Pathology, New York University School of Medicine, and Perlmutter Cancer Center, New York, NY 10016, USA; Helen L. and Martin S. Kimmel Center for Stem Cell Biology, NYU Langone Medical Center, New York, NY 10016, USA. Electronic address: email@example.com.Show MeSH
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Mentions: In order to dissect the mechanisms by which BRD4 regulates pluripotency gene expression, we performed chromatin immunoprecipitation sequencing (ChIP-seq) for BRD4 in vehicle- or compound-treated hESCs grown in the presence of FGF in two independent, highly reproducible (R2 = 0.815) biological replicates. Using stringent parameters (see Experimental Procedures section), we identified 4,026 BRD4-binding peaks that were common to both biological replicates. We first examined the distribution of BRD4 protein occupancy at defined regions relative to gene elements in vehicle-treated hESCs by classifying the identified peaks into (1) 1 kb transcription start site (TSS)-flanking regions of known transcripts; (2) gene body regions, excluding any overlapping regions with (1); or (3) upstream regions of 10 kb to 100 kb, excluding any overlapping regions with either (1) or (2). The remaining genomic loci were classified as intergenic regions. In vehicle-treated cells, BRD4 occupied gene bodies and TSSs (31.9% and 31.1%; Figure 4A) and was also present at upstream regions (31.1%; Figure 4A), suggesting a possible binding to regulatory elements in the genome in addition to promoters and gene bodies. BRD4 occupancy in hESCs was analyzed 6 hr after treatment to identify early compound-responsive elements. BET inhibition consistently resulted in robust and global displacement (defined as absence of peak in treatment; see Experimental Procedures) of BRD4 from the vast majority of the occupied genomic loci common to the two biological replicates (Figures 4B and 4C), indicating that our small-molecule compounds are potent displacers of BRD4 from chromatin. Gene set enrichment analysis of genes from which BRD4 was displaced following treatment revealed a significant enrichment in stem cell gene categories, enforcing the concept that BRD4 regulates the stem cell gene transcriptional network (Figure 4D; Table S4).
Affiliation: Department of Pathology, New York University School of Medicine, and Perlmutter Cancer Center, New York, NY 10016, USA; Helen L. and Martin S. Kimmel Center for Stem Cell Biology, NYU Langone Medical Center, New York, NY 10016, USA. Electronic address: firstname.lastname@example.org.