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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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Mediator Is Required for BRD4-Dependent Regulation of SE-Associated Genes(A–F) ChIP analysis of MED1 and MED12 binding at enhancer regions and promoters (prom) of SE-associated stem cell genes [OCT4 (A and B) and PRDM14 (C and D) and a non-SE-associated gene, c-MYC (E and F)] in hESCs following BET inhibition. Bar graphs represent the mean enrichment relative to input, and error bars reflect SD of a representative experiment run in triplicate.(G) Expression levels of MED1 and MED12 by qRT-PCR following BET inhibition in hESCs and mESCs.(H) Expression of the indicated genes in shMed1 and shMed12 mESCs compared to NTC-transduced cells as detected by qRT-PCR.See also Figure S6H for primers design.
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Figure 5: Mediator Is Required for BRD4-Dependent Regulation of SE-Associated Genes(A–F) ChIP analysis of MED1 and MED12 binding at enhancer regions and promoters (prom) of SE-associated stem cell genes [OCT4 (A and B) and PRDM14 (C and D) and a non-SE-associated gene, c-MYC (E and F)] in hESCs following BET inhibition. Bar graphs represent the mean enrichment relative to input, and error bars reflect SD of a representative experiment run in triplicate.(G) Expression levels of MED1 and MED12 by qRT-PCR following BET inhibition in hESCs and mESCs.(H) Expression of the indicated genes in shMed1 and shMed12 mESCs compared to NTC-transduced cells as detected by qRT-PCR.See also Figure S6H for primers design.

Mentions: The Mediator complex is a transcriptional activator of the pluripotency gene network that represents a potentially important player in BRD4-mediated transcriptional regulation of SE-associated stem cell genes. We found that MED1 (Core Mediator) and MED12 (CDK8-module) subunits localize at both SEs and promoters of stem cell genes in hESCs (i.e., OCT4 and PRDM14; Figures 5A–5D). Notably, the binding of MED1 and MED12 at SEs, but not at promoters, was significantly reduced by BRD4 inhibition (Figures 5A–5D), suggesting that BRD4 may act as a docking site for the Mediator complex at SEs of stem cell genes, thus regulating their expression. Consistent with the observation that c-MYC was not downregulated following BRD4 inhibition (Figure S3G), we did not find a SE associated with c-MYC in hESCs. In this non-SE-associated gene, Mediator occupancy was not altered by compound treatment (Figures 5E and 5F). Importantly, displacement of Mediator from SEs upon compound treatment was not due to reduced levels of MED1 and MED12 following BRD4 inhibition in hESCs and mESCs (Figure 5G). Silencing of Mediator subunits resulted in suppression of stem cell genes, thus recapitulating the effects of BRD4 inhibition on the ESC transcriptional network (Figure 5H). The expression of c-Myc was also inhibited following Med1 and Med12 depletion (Figure 5H), suggesting that Mediator is a positive transcriptional regulator of c-MYC expression but independently of BRD4. Altogether, these data indicate that the expression of SE-associated stem cell genes relies on BRD4-dependent binding of Mediator-containing complexes at SEs.


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)

Mediator Is Required for BRD4-Dependent Regulation of SE-Associated Genes(A–F) ChIP analysis of MED1 and MED12 binding at enhancer regions and promoters (prom) of SE-associated stem cell genes [OCT4 (A and B) and PRDM14 (C and D) and a non-SE-associated gene, c-MYC (E and F)] in hESCs following BET inhibition. Bar graphs represent the mean enrichment relative to input, and error bars reflect SD of a representative experiment run in triplicate.(G) Expression levels of MED1 and MED12 by qRT-PCR following BET inhibition in hESCs and mESCs.(H) Expression of the indicated genes in shMed1 and shMed12 mESCs compared to NTC-transduced cells as detected by qRT-PCR.See also Figure S6H for primers design.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4317728&req=5

Figure 5: Mediator Is Required for BRD4-Dependent Regulation of SE-Associated Genes(A–F) ChIP analysis of MED1 and MED12 binding at enhancer regions and promoters (prom) of SE-associated stem cell genes [OCT4 (A and B) and PRDM14 (C and D) and a non-SE-associated gene, c-MYC (E and F)] in hESCs following BET inhibition. Bar graphs represent the mean enrichment relative to input, and error bars reflect SD of a representative experiment run in triplicate.(G) Expression levels of MED1 and MED12 by qRT-PCR following BET inhibition in hESCs and mESCs.(H) Expression of the indicated genes in shMed1 and shMed12 mESCs compared to NTC-transduced cells as detected by qRT-PCR.See also Figure S6H for primers design.
Mentions: The Mediator complex is a transcriptional activator of the pluripotency gene network that represents a potentially important player in BRD4-mediated transcriptional regulation of SE-associated stem cell genes. We found that MED1 (Core Mediator) and MED12 (CDK8-module) subunits localize at both SEs and promoters of stem cell genes in hESCs (i.e., OCT4 and PRDM14; Figures 5A–5D). Notably, the binding of MED1 and MED12 at SEs, but not at promoters, was significantly reduced by BRD4 inhibition (Figures 5A–5D), suggesting that BRD4 may act as a docking site for the Mediator complex at SEs of stem cell genes, thus regulating their expression. Consistent with the observation that c-MYC was not downregulated following BRD4 inhibition (Figure S3G), we did not find a SE associated with c-MYC in hESCs. In this non-SE-associated gene, Mediator occupancy was not altered by compound treatment (Figures 5E and 5F). Importantly, displacement of Mediator from SEs upon compound treatment was not due to reduced levels of MED1 and MED12 following BRD4 inhibition in hESCs and mESCs (Figure 5G). Silencing of Mediator subunits resulted in suppression of stem cell genes, thus recapitulating the effects of BRD4 inhibition on the ESC transcriptional network (Figure 5H). The expression of c-Myc was also inhibited following Med1 and Med12 depletion (Figure 5H), suggesting that Mediator is a positive transcriptional regulator of c-MYC expression but independently of BRD4. Altogether, these data indicate that the expression of SE-associated stem cell genes relies on BRD4-dependent binding of Mediator-containing complexes at SEs.

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