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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, Cañamero 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.

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BRD4 Inhibition Impairs the Transcriptional Elongation of SE-Associated Stem Cell Genes(A–C) ChIP analysis of the elongation factor CDK9 at SEs and promoters of OCT4 (A), PRDM14 (B), and c-MYC enhancer and promoter (C) following compound treatment. IgG was used as control. Bar graphs represent the mean enrichment relative to input, and error bars reflect SD of a representative experiment run in triplicate.(D–F) ChIP analysis of the elongation marks H3K36me3 and Pol II-pS2 on gene bodies (GBs) of SE-associated genes, OCT4 (D) and PRDM14 (E), and non-SE-associated gene, c-MYC (F), following compound treatment.(G–I) ChIP analysis of the elongation marks H3K36me3 on GB of OCT4 (G), PRDM14 (H), and c-MYC (I) in BRD4-depleted cells compared to NTC-transduced hESCs.(J) Traveling ratio distribution across SE- or non-SE-associated transcripts of similar expression before treatment and similarly downregulated after treatment. The initiating regions are defined as ±300 bp around the TSS, and the elongating regions are defined as +300 bp from the TSS to 3,000 bp after the transcript ends.(K) Distribution of fold changes of Pol II densities (normalized by total number of reads per treatment) at TSSs following compound treatment in SE- and non-SE-downregulated transcripts.(L) Distribution of fold changes of Pol II densities (normalized by total number of reads per treatment) at GBs following compound treatment in SE- and non-SE genes, downregulated following MS417 treatment. Dashed lines indicate the fraction of genes with reduced Pol II occupancy at gene bodies in the two gene categories.ns, not significant; ***p ≤ 0.001. See also Figure S6.
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Figure 6: BRD4 Inhibition Impairs the Transcriptional Elongation of SE-Associated Stem Cell Genes(A–C) ChIP analysis of the elongation factor CDK9 at SEs and promoters of OCT4 (A), PRDM14 (B), and c-MYC enhancer and promoter (C) following compound treatment. IgG was used as control. Bar graphs represent the mean enrichment relative to input, and error bars reflect SD of a representative experiment run in triplicate.(D–F) ChIP analysis of the elongation marks H3K36me3 and Pol II-pS2 on gene bodies (GBs) of SE-associated genes, OCT4 (D) and PRDM14 (E), and non-SE-associated gene, c-MYC (F), following compound treatment.(G–I) ChIP analysis of the elongation marks H3K36me3 on GB of OCT4 (G), PRDM14 (H), and c-MYC (I) in BRD4-depleted cells compared to NTC-transduced hESCs.(J) Traveling ratio distribution across SE- or non-SE-associated transcripts of similar expression before treatment and similarly downregulated after treatment. The initiating regions are defined as ±300 bp around the TSS, and the elongating regions are defined as +300 bp from the TSS to 3,000 bp after the transcript ends.(K) Distribution of fold changes of Pol II densities (normalized by total number of reads per treatment) at TSSs following compound treatment in SE- and non-SE-downregulated transcripts.(L) Distribution of fold changes of Pol II densities (normalized by total number of reads per treatment) at GBs following compound treatment in SE- and non-SE genes, downregulated following MS417 treatment. Dashed lines indicate the fraction of genes with reduced Pol II occupancy at gene bodies in the two gene categories.ns, not significant; ***p ≤ 0.001. See also Figure S6.

Mentions: In addition to Mediator, BRD4 has been found to interact with CDK9, the catalytic subunit of the P-TEFb transcriptional elongation complex (Bisgrove et al., 2007; Dawson et al., 2011). To study whether BRD4 regulates the expression of SE-associated stem cell genes via productive transcriptional elongation, we performed ChIP assays for CDK9, as well as two elongation marks, the phosphorylated Pol II at Ser2 (pS2) and H3K36me3. We found that together with BRD4, CDK9 is more associated to SE of stem cell genes (e.g., OCT4 and PRDM14; Figures 6A and 6B) than to genes bearing regular enhancer elements (e.g., c-MYC; Figure 6C). BRD4 inhibition resulted in dramatic loss of CDK9 from the SEs and promoters (Figures 6A and 6B). Next, we investigated whether loss of BRD4 and CDK9 from SEs results in reduced levels of elongation marks in gene bodies (GBs) of SE-associated stem cell genes. H3K36me3 and Pol II pS2 were reduced following compound treatment at the gene bodies of OCT4 and PRDM14 (Figures 6D and 6E). Conversely, c-MYC, a non-SE-associated gene, did not display any loss of elongation marks (Figure 6F), consistent with its unreduced expression levels following BRD4 inhibition (Figure S3G). BRD4 silencing was sufficient to recapitulate the effects of BET inhibition on the transcriptional elongation of SE-associated stem cell genes and did not affect the levels of elongation marks at c-MYC gene body (Figures 6G–6I). The levels of active transcription and initiation marks, such as H3K4me3 and Pol II pS5, were mostly unaffected by BET inhibition at both SE and non-SE-associated genes (Figures S6A–S6C). Furthermore, levels of H4K5 acetylation, one of the docking sites for BET binding to chromatin (Filippakopoulos et al., 2012), remained unchanged upon compound treatment (Figures S6D–S6F). These findings suggest that BRD4 controls SE-associated stem cell gene expres sion by regulating their transcriptional elongation rather than transcriptional initiation or histone acetylation.


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, Cañamero M, Ohlmeyer M, Aifantis I, Zhou MM, Tsirigos A, Hernando E - Cell Rep (2014)

BRD4 Inhibition Impairs the Transcriptional Elongation of SE-Associated Stem Cell Genes(A–C) ChIP analysis of the elongation factor CDK9 at SEs and promoters of OCT4 (A), PRDM14 (B), and c-MYC enhancer and promoter (C) following compound treatment. IgG was used as control. Bar graphs represent the mean enrichment relative to input, and error bars reflect SD of a representative experiment run in triplicate.(D–F) ChIP analysis of the elongation marks H3K36me3 and Pol II-pS2 on gene bodies (GBs) of SE-associated genes, OCT4 (D) and PRDM14 (E), and non-SE-associated gene, c-MYC (F), following compound treatment.(G–I) ChIP analysis of the elongation marks H3K36me3 on GB of OCT4 (G), PRDM14 (H), and c-MYC (I) in BRD4-depleted cells compared to NTC-transduced hESCs.(J) Traveling ratio distribution across SE- or non-SE-associated transcripts of similar expression before treatment and similarly downregulated after treatment. The initiating regions are defined as ±300 bp around the TSS, and the elongating regions are defined as +300 bp from the TSS to 3,000 bp after the transcript ends.(K) Distribution of fold changes of Pol II densities (normalized by total number of reads per treatment) at TSSs following compound treatment in SE- and non-SE-downregulated transcripts.(L) Distribution of fold changes of Pol II densities (normalized by total number of reads per treatment) at GBs following compound treatment in SE- and non-SE genes, downregulated following MS417 treatment. Dashed lines indicate the fraction of genes with reduced Pol II occupancy at gene bodies in the two gene categories.ns, not significant; ***p ≤ 0.001. See also Figure S6.
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Figure 6: BRD4 Inhibition Impairs the Transcriptional Elongation of SE-Associated Stem Cell Genes(A–C) ChIP analysis of the elongation factor CDK9 at SEs and promoters of OCT4 (A), PRDM14 (B), and c-MYC enhancer and promoter (C) following compound treatment. IgG was used as control. Bar graphs represent the mean enrichment relative to input, and error bars reflect SD of a representative experiment run in triplicate.(D–F) ChIP analysis of the elongation marks H3K36me3 and Pol II-pS2 on gene bodies (GBs) of SE-associated genes, OCT4 (D) and PRDM14 (E), and non-SE-associated gene, c-MYC (F), following compound treatment.(G–I) ChIP analysis of the elongation marks H3K36me3 on GB of OCT4 (G), PRDM14 (H), and c-MYC (I) in BRD4-depleted cells compared to NTC-transduced hESCs.(J) Traveling ratio distribution across SE- or non-SE-associated transcripts of similar expression before treatment and similarly downregulated after treatment. The initiating regions are defined as ±300 bp around the TSS, and the elongating regions are defined as +300 bp from the TSS to 3,000 bp after the transcript ends.(K) Distribution of fold changes of Pol II densities (normalized by total number of reads per treatment) at TSSs following compound treatment in SE- and non-SE-downregulated transcripts.(L) Distribution of fold changes of Pol II densities (normalized by total number of reads per treatment) at GBs following compound treatment in SE- and non-SE genes, downregulated following MS417 treatment. Dashed lines indicate the fraction of genes with reduced Pol II occupancy at gene bodies in the two gene categories.ns, not significant; ***p ≤ 0.001. See also Figure S6.
Mentions: In addition to Mediator, BRD4 has been found to interact with CDK9, the catalytic subunit of the P-TEFb transcriptional elongation complex (Bisgrove et al., 2007; Dawson et al., 2011). To study whether BRD4 regulates the expression of SE-associated stem cell genes via productive transcriptional elongation, we performed ChIP assays for CDK9, as well as two elongation marks, the phosphorylated Pol II at Ser2 (pS2) and H3K36me3. We found that together with BRD4, CDK9 is more associated to SE of stem cell genes (e.g., OCT4 and PRDM14; Figures 6A and 6B) than to genes bearing regular enhancer elements (e.g., c-MYC; Figure 6C). BRD4 inhibition resulted in dramatic loss of CDK9 from the SEs and promoters (Figures 6A and 6B). Next, we investigated whether loss of BRD4 and CDK9 from SEs results in reduced levels of elongation marks in gene bodies (GBs) of SE-associated stem cell genes. H3K36me3 and Pol II pS2 were reduced following compound treatment at the gene bodies of OCT4 and PRDM14 (Figures 6D and 6E). Conversely, c-MYC, a non-SE-associated gene, did not display any loss of elongation marks (Figure 6F), consistent with its unreduced expression levels following BRD4 inhibition (Figure S3G). BRD4 silencing was sufficient to recapitulate the effects of BET inhibition on the transcriptional elongation of SE-associated stem cell genes and did not affect the levels of elongation marks at c-MYC gene body (Figures 6G–6I). The levels of active transcription and initiation marks, such as H3K4me3 and Pol II pS5, were mostly unaffected by BET inhibition at both SE and non-SE-associated genes (Figures S6A–S6C). Furthermore, levels of H4K5 acetylation, one of the docking sites for BET binding to chromatin (Filippakopoulos et al., 2012), remained unchanged upon compound treatment (Figures S6D–S6F). These findings suggest that BRD4 controls SE-associated stem cell gene expres sion by regulating their transcriptional elongation rather than transcriptional initiation or histone acetylation.

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.

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Related in: MedlinePlus