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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 Regulates the Expression of Stem Cell Genes(A) Scatter plots of gene expression at 24 hr and 120 hr in hESCs following MS436 treatment. Each point corresponds to a differentially expressed gene in compound-treated versus FGF-treated hESCs. Red dots indicate genes in the ESC gene categories (Table S1). Black dots represent stem cell genes validated throughout the manuscript. q value cutoff used throughout the analysis = 0.01.(B) Gene set enrichment analysis of downregulated genes at 24 hr and 120 hr following BET inhibition, according to MSigDB.(C and D) Time course expression of stem cell genes in hESCs (C) and mESCs (D), cultured in the presence of FGF (C) or LIF (D), upon compound treatment relative to vehicle-treated cells (dashed line), as detected by qRT-PCR.(E) Effect of BET inhibitor treatment (3 days) on OCT4 protein levels as detected by immunoblotting. Tubulin is used as a loading control.(F) Effect of compound treatment on hESC colony morphology (DAPI) and OCT4 protein expression as detected by immunostaining. The scale bar represents 100 μm.(G and H) Relative mRNA expression of the indicated stem cell and housekeeping genes (HK) in hESCs (G) and mESCs (H) after BRD4 genetic depletion, as detected by qRT-PCR. Dashed lines represent nontargeting control (NTC)-transduced cells.Error bars represent SD. See also Tables S1 and S2 and Figure S3.
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Figure 2: BRD4 Regulates the Expression of Stem Cell Genes(A) Scatter plots of gene expression at 24 hr and 120 hr in hESCs following MS436 treatment. Each point corresponds to a differentially expressed gene in compound-treated versus FGF-treated hESCs. Red dots indicate genes in the ESC gene categories (Table S1). Black dots represent stem cell genes validated throughout the manuscript. q value cutoff used throughout the analysis = 0.01.(B) Gene set enrichment analysis of downregulated genes at 24 hr and 120 hr following BET inhibition, according to MSigDB.(C and D) Time course expression of stem cell genes in hESCs (C) and mESCs (D), cultured in the presence of FGF (C) or LIF (D), upon compound treatment relative to vehicle-treated cells (dashed line), as detected by qRT-PCR.(E) Effect of BET inhibitor treatment (3 days) on OCT4 protein levels as detected by immunoblotting. Tubulin is used as a loading control.(F) Effect of compound treatment on hESC colony morphology (DAPI) and OCT4 protein expression as detected by immunostaining. The scale bar represents 100 μm.(G and H) Relative mRNA expression of the indicated stem cell and housekeeping genes (HK) in hESCs (G) and mESCs (H) after BRD4 genetic depletion, as detected by qRT-PCR. Dashed lines represent nontargeting control (NTC)-transduced cells.Error bars represent SD. See also Tables S1 and S2 and Figure S3.

Mentions: In deducing the mechanism by which BRD4 maintains the ESC state, we reasoned that BRD4 was likely to regulate the expression of genes required for ESC identity. Global gene-expression profiling by RNA sequencing of compound- versus FGF-treated hESCs confirmed the suppression of pluripotency transcripts (Table S1) at day 1 post-compound treatment, even more pronounced by day 5 (Figure 2A; Table S2). Gene set enrichment analysis of downregulated genes revealed enrichment in multiple ESC-related gene categories following compound treatment, indicating that the stem cell gene network is significantly perturbed by BET inhibition (Figure 2B). The expression of key determinants of ESC identity, including OCT4, NANOG, and PRDM14, was rapidly suppressed following compound treatment in human and mouse ESCs cultured even in the presence of FGF (hESCs; Figure 2C) or LIF (mESCs; Figure 2D), indicating that BET inhibition can counteract the extracellular signals that help to maintain ESCs in an undifferentiated state in vitro. Consistently, we found that OCT4 protein levels were reduced in hESCs following BET inhibition (Figures 2E and 2F). The rapid loss of expression of stem cell factors upon BET inhibition preceded the defects in cell cycle progression associated with the acquisition of a more differentiated phenotype (Figures 1I, S2G, and S2H), indicating that the former is not a result of the latter. Similar results were obtained in an established human induced pluripo-tent stem cell (iPSC) line (Papapetrou et al., 2009; Figure S3A) and in a pluripotent embryonic carcinoma cell line (NTERA-2; Figure S3B), indicating that the effects of BET inhibition on pluripotency gene expression are not restricted to ESC lines. Treatment of hESCs with the BET inhibitor JQ1 (Filippakopoulos et al., 2010) yielded comparable suppression of stem cell genes to MS417 treatment (Figure S3C). Moreover, RNAi-mediated BRD4 silencing led to reduced expression of key determinants of stem cell identity (e.g., OCT4, PRDM14, and NANOG) in human and mouse ESCs (Figures 2G and 2H), mimicking the effects of compound treatment. Interestingly, the expression of housekeeping genes was mostly unaffected upon BRD4 silencing (Figures 2G and 2H), suggesting a specific transcriptional regulation of stem cell genes by BRD4. The expression of stem cell genes was also suppressed in hESC, established iPSC, and NTERA lines following siRNA-mediated BRD4 depletion (Figures S3D–S3F). Our findings indicate that BRD4 specifically contributes to the transcriptional program that maintains the expression of many stem cell genes and ESC identity.


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 Regulates the Expression of Stem Cell Genes(A) Scatter plots of gene expression at 24 hr and 120 hr in hESCs following MS436 treatment. Each point corresponds to a differentially expressed gene in compound-treated versus FGF-treated hESCs. Red dots indicate genes in the ESC gene categories (Table S1). Black dots represent stem cell genes validated throughout the manuscript. q value cutoff used throughout the analysis = 0.01.(B) Gene set enrichment analysis of downregulated genes at 24 hr and 120 hr following BET inhibition, according to MSigDB.(C and D) Time course expression of stem cell genes in hESCs (C) and mESCs (D), cultured in the presence of FGF (C) or LIF (D), upon compound treatment relative to vehicle-treated cells (dashed line), as detected by qRT-PCR.(E) Effect of BET inhibitor treatment (3 days) on OCT4 protein levels as detected by immunoblotting. Tubulin is used as a loading control.(F) Effect of compound treatment on hESC colony morphology (DAPI) and OCT4 protein expression as detected by immunostaining. The scale bar represents 100 μm.(G and H) Relative mRNA expression of the indicated stem cell and housekeeping genes (HK) in hESCs (G) and mESCs (H) after BRD4 genetic depletion, as detected by qRT-PCR. Dashed lines represent nontargeting control (NTC)-transduced cells.Error bars represent SD. See also Tables S1 and S2 and Figure S3.
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Related In: Results  -  Collection

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Figure 2: BRD4 Regulates the Expression of Stem Cell Genes(A) Scatter plots of gene expression at 24 hr and 120 hr in hESCs following MS436 treatment. Each point corresponds to a differentially expressed gene in compound-treated versus FGF-treated hESCs. Red dots indicate genes in the ESC gene categories (Table S1). Black dots represent stem cell genes validated throughout the manuscript. q value cutoff used throughout the analysis = 0.01.(B) Gene set enrichment analysis of downregulated genes at 24 hr and 120 hr following BET inhibition, according to MSigDB.(C and D) Time course expression of stem cell genes in hESCs (C) and mESCs (D), cultured in the presence of FGF (C) or LIF (D), upon compound treatment relative to vehicle-treated cells (dashed line), as detected by qRT-PCR.(E) Effect of BET inhibitor treatment (3 days) on OCT4 protein levels as detected by immunoblotting. Tubulin is used as a loading control.(F) Effect of compound treatment on hESC colony morphology (DAPI) and OCT4 protein expression as detected by immunostaining. The scale bar represents 100 μm.(G and H) Relative mRNA expression of the indicated stem cell and housekeeping genes (HK) in hESCs (G) and mESCs (H) after BRD4 genetic depletion, as detected by qRT-PCR. Dashed lines represent nontargeting control (NTC)-transduced cells.Error bars represent SD. See also Tables S1 and S2 and Figure S3.
Mentions: In deducing the mechanism by which BRD4 maintains the ESC state, we reasoned that BRD4 was likely to regulate the expression of genes required for ESC identity. Global gene-expression profiling by RNA sequencing of compound- versus FGF-treated hESCs confirmed the suppression of pluripotency transcripts (Table S1) at day 1 post-compound treatment, even more pronounced by day 5 (Figure 2A; Table S2). Gene set enrichment analysis of downregulated genes revealed enrichment in multiple ESC-related gene categories following compound treatment, indicating that the stem cell gene network is significantly perturbed by BET inhibition (Figure 2B). The expression of key determinants of ESC identity, including OCT4, NANOG, and PRDM14, was rapidly suppressed following compound treatment in human and mouse ESCs cultured even in the presence of FGF (hESCs; Figure 2C) or LIF (mESCs; Figure 2D), indicating that BET inhibition can counteract the extracellular signals that help to maintain ESCs in an undifferentiated state in vitro. Consistently, we found that OCT4 protein levels were reduced in hESCs following BET inhibition (Figures 2E and 2F). The rapid loss of expression of stem cell factors upon BET inhibition preceded the defects in cell cycle progression associated with the acquisition of a more differentiated phenotype (Figures 1I, S2G, and S2H), indicating that the former is not a result of the latter. Similar results were obtained in an established human induced pluripo-tent stem cell (iPSC) line (Papapetrou et al., 2009; Figure S3A) and in a pluripotent embryonic carcinoma cell line (NTERA-2; Figure S3B), indicating that the effects of BET inhibition on pluripotency gene expression are not restricted to ESC lines. Treatment of hESCs with the BET inhibitor JQ1 (Filippakopoulos et al., 2010) yielded comparable suppression of stem cell genes to MS417 treatment (Figure S3C). Moreover, RNAi-mediated BRD4 silencing led to reduced expression of key determinants of stem cell identity (e.g., OCT4, PRDM14, and NANOG) in human and mouse ESCs (Figures 2G and 2H), mimicking the effects of compound treatment. Interestingly, the expression of housekeeping genes was mostly unaffected upon BRD4 silencing (Figures 2G and 2H), suggesting a specific transcriptional regulation of stem cell genes by BRD4. The expression of stem cell genes was also suppressed in hESC, established iPSC, and NTERA lines following siRNA-mediated BRD4 depletion (Figures S3D–S3F). Our findings indicate that BRD4 specifically contributes to the transcriptional program that maintains the expression of many stem cell genes and ESC identity.

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