Limits...
Control of embryonic stem cell identity by BRD4-dependent transcriptional elongation of super-enhancer-associated pluripotency genes.

Di Micco R, Fontanals-Cirera B, Low V, Ntziachristos P, Yuen SK, Lovell CD, Dolgalev I, Yonekubo Y, Zhang G, Rusinova E, Gerona-Navarro G, CaƱamero M, Ohlmeyer M, Aifantis I, Zhou MM, Tsirigos A, Hernando E - Cell Rep (2014)

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.

View Article: PubMed Central - PubMed

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: raffaella.dimicco@nyumc.org.

Show MeSH

Related in: MedlinePlus

BRD4 Occupies and Regulates Super-Enhancer-Associated Genes(A) Genome-wide distribution of BRD4 in 1-kb-flanked TSS regions, gene bodies (excluding flanked TSS regions), upstream (up to 100 kb and excluding flanked TSSs and gene bodies), and intergenic regions (excluding all of the above) by ChIP-seq.(B) Number of BRD4-binding peaks common to two biological replicates after 6 hr of vehicle or MS417 treatment.(C) Heatmap representation of ChIP-seq binding for BRD4 peaks common to two independent biological replicates before and after BET inhibition, rank ordered form the most BRD4 to lowest BRD4. Immunoglobulin G (IgG) is shown as a negative control for enrichment.(D) Gene set enrichment analysis of BRD4 target genes (e.g., genes from which BRD4 is displaced following BET inhibition [top ten categories are shown]).(E and F) Genome browser representations of BRD4 and IgG ChIP-seq reads at stem cell genes [i.e., OCT4 (E) and PRDM14 (F)], in FGF-cultured hESCs treated with vehicle or MS417. Black bars indicate super-enhancers (SEs) (Hnisz et al., 2013). Arrows indicate direction of transcription. Insert represents a close up for BRD4 and IgG binding at PRDM14 SE before and after treatment. Chromosomal locations are indicated.(G) Read density representation of global BRD4 occupancy at typical enhancer (TE) and SE elements. The x axis shows the center of TE regions flanked by 5 kb as well as the start and end of SE regions flanked by 5 kb of adjacent sequence. The y axis indicates reads per kilobase of transcript per million reads mapped (RPKMs).(H) Table indicates the percentage and total number of BRD4-overlapping TEs and SEs at BRD4 peak signal distributions comparable between TEs and SEs. TEs were extended upstream and downstream from their centers such that their size equals the mean size of SEs. Control (ctrl) refers to the percent of peaks at SEs/TEs if the BRD4-displaced regions were randomly distributed along the genome.(I) Histogram indicates the percentage of BRD4 target genes that are downregulated, constant, and upregulated in SE- and non-SE-associated gene categories upon compound treatment.See also Tables S4 and S5 and Figure S7.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4317728&req=5

Figure 4: BRD4 Occupies and Regulates Super-Enhancer-Associated Genes(A) Genome-wide distribution of BRD4 in 1-kb-flanked TSS regions, gene bodies (excluding flanked TSS regions), upstream (up to 100 kb and excluding flanked TSSs and gene bodies), and intergenic regions (excluding all of the above) by ChIP-seq.(B) Number of BRD4-binding peaks common to two biological replicates after 6 hr of vehicle or MS417 treatment.(C) Heatmap representation of ChIP-seq binding for BRD4 peaks common to two independent biological replicates before and after BET inhibition, rank ordered form the most BRD4 to lowest BRD4. Immunoglobulin G (IgG) is shown as a negative control for enrichment.(D) Gene set enrichment analysis of BRD4 target genes (e.g., genes from which BRD4 is displaced following BET inhibition [top ten categories are shown]).(E and F) Genome browser representations of BRD4 and IgG ChIP-seq reads at stem cell genes [i.e., OCT4 (E) and PRDM14 (F)], in FGF-cultured hESCs treated with vehicle or MS417. Black bars indicate super-enhancers (SEs) (Hnisz et al., 2013). Arrows indicate direction of transcription. Insert represents a close up for BRD4 and IgG binding at PRDM14 SE before and after treatment. Chromosomal locations are indicated.(G) Read density representation of global BRD4 occupancy at typical enhancer (TE) and SE elements. The x axis shows the center of TE regions flanked by 5 kb as well as the start and end of SE regions flanked by 5 kb of adjacent sequence. The y axis indicates reads per kilobase of transcript per million reads mapped (RPKMs).(H) Table indicates the percentage and total number of BRD4-overlapping TEs and SEs at BRD4 peak signal distributions comparable between TEs and SEs. TEs were extended upstream and downstream from their centers such that their size equals the mean size of SEs. Control (ctrl) refers to the percent of peaks at SEs/TEs if the BRD4-displaced regions were randomly distributed along the genome.(I) Histogram indicates the percentage of BRD4 target genes that are downregulated, constant, and upregulated in SE- and non-SE-associated gene categories upon compound treatment.See also Tables S4 and S5 and Figure S7.

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).


Control of embryonic stem cell identity by BRD4-dependent transcriptional elongation of super-enhancer-associated pluripotency genes.

Di Micco R, Fontanals-Cirera B, Low V, Ntziachristos P, Yuen SK, Lovell CD, Dolgalev I, Yonekubo Y, Zhang G, Rusinova E, Gerona-Navarro G, CaƱamero M, Ohlmeyer M, Aifantis I, Zhou MM, Tsirigos A, Hernando E - Cell Rep (2014)

BRD4 Occupies and Regulates Super-Enhancer-Associated Genes(A) Genome-wide distribution of BRD4 in 1-kb-flanked TSS regions, gene bodies (excluding flanked TSS regions), upstream (up to 100 kb and excluding flanked TSSs and gene bodies), and intergenic regions (excluding all of the above) by ChIP-seq.(B) Number of BRD4-binding peaks common to two biological replicates after 6 hr of vehicle or MS417 treatment.(C) Heatmap representation of ChIP-seq binding for BRD4 peaks common to two independent biological replicates before and after BET inhibition, rank ordered form the most BRD4 to lowest BRD4. Immunoglobulin G (IgG) is shown as a negative control for enrichment.(D) Gene set enrichment analysis of BRD4 target genes (e.g., genes from which BRD4 is displaced following BET inhibition [top ten categories are shown]).(E and F) Genome browser representations of BRD4 and IgG ChIP-seq reads at stem cell genes [i.e., OCT4 (E) and PRDM14 (F)], in FGF-cultured hESCs treated with vehicle or MS417. Black bars indicate super-enhancers (SEs) (Hnisz et al., 2013). Arrows indicate direction of transcription. Insert represents a close up for BRD4 and IgG binding at PRDM14 SE before and after treatment. Chromosomal locations are indicated.(G) Read density representation of global BRD4 occupancy at typical enhancer (TE) and SE elements. The x axis shows the center of TE regions flanked by 5 kb as well as the start and end of SE regions flanked by 5 kb of adjacent sequence. The y axis indicates reads per kilobase of transcript per million reads mapped (RPKMs).(H) Table indicates the percentage and total number of BRD4-overlapping TEs and SEs at BRD4 peak signal distributions comparable between TEs and SEs. TEs were extended upstream and downstream from their centers such that their size equals the mean size of SEs. Control (ctrl) refers to the percent of peaks at SEs/TEs if the BRD4-displaced regions were randomly distributed along the genome.(I) Histogram indicates the percentage of BRD4 target genes that are downregulated, constant, and upregulated in SE- and non-SE-associated gene categories upon compound treatment.See also Tables S4 and S5 and Figure S7.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4317728&req=5

Figure 4: BRD4 Occupies and Regulates Super-Enhancer-Associated Genes(A) Genome-wide distribution of BRD4 in 1-kb-flanked TSS regions, gene bodies (excluding flanked TSS regions), upstream (up to 100 kb and excluding flanked TSSs and gene bodies), and intergenic regions (excluding all of the above) by ChIP-seq.(B) Number of BRD4-binding peaks common to two biological replicates after 6 hr of vehicle or MS417 treatment.(C) Heatmap representation of ChIP-seq binding for BRD4 peaks common to two independent biological replicates before and after BET inhibition, rank ordered form the most BRD4 to lowest BRD4. Immunoglobulin G (IgG) is shown as a negative control for enrichment.(D) Gene set enrichment analysis of BRD4 target genes (e.g., genes from which BRD4 is displaced following BET inhibition [top ten categories are shown]).(E and F) Genome browser representations of BRD4 and IgG ChIP-seq reads at stem cell genes [i.e., OCT4 (E) and PRDM14 (F)], in FGF-cultured hESCs treated with vehicle or MS417. Black bars indicate super-enhancers (SEs) (Hnisz et al., 2013). Arrows indicate direction of transcription. Insert represents a close up for BRD4 and IgG binding at PRDM14 SE before and after treatment. Chromosomal locations are indicated.(G) Read density representation of global BRD4 occupancy at typical enhancer (TE) and SE elements. The x axis shows the center of TE regions flanked by 5 kb as well as the start and end of SE regions flanked by 5 kb of adjacent sequence. The y axis indicates reads per kilobase of transcript per million reads mapped (RPKMs).(H) Table indicates the percentage and total number of BRD4-overlapping TEs and SEs at BRD4 peak signal distributions comparable between TEs and SEs. TEs were extended upstream and downstream from their centers such that their size equals the mean size of SEs. Control (ctrl) refers to the percent of peaks at SEs/TEs if the BRD4-displaced regions were randomly distributed along the genome.(I) Histogram indicates the percentage of BRD4 target genes that are downregulated, constant, and upregulated in SE- and non-SE-associated gene categories upon compound treatment.See also Tables S4 and S5 and Figure S7.
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).

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.

View Article: PubMed Central - PubMed

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: raffaella.dimicco@nyumc.org.

Show MeSH
Related in: MedlinePlus