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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 ESC Identity(A) Representative bright field images (upper panel) of hESCs treated for 3 days with BET inhibitors compared to FGF- or vehicle-treated cells. Alkaline Phosphatase (AP) staining following treatment is shown in bottom panel.(B) Quantification of AP-positive hESC colonies following FGF, vehicle, or compound treatment.(C) Bright field images (upper panel) and AP staining (bottom panel) of mESCs treated with BET inhibitors for 3 days in the presence of LIF.(D) Quantification of AP-positive mESC colonies following 3 days of compound treatment.(E–H) Bright field images of hESC (E) and mESC (G) colonies following BRD2, BRD3, and BRD4 suppression. Percentage of undifferentiated hESCs and mESC colonies is shown in (F) and (H), respectively. NTC, nontargeting control.(I) Cell-cycle analysis of hESCs following BET inhibitors treatment at the indicated time points as determined by FACS analysis.(J) Self-renewal capacity of vehicle- and compound-treated hESCs passaged on mitotically inactivated MEFs in the presence of FGF as measured by AP staining. AP, alkaline phosphatase; ND, not detected. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. The scale bar represents 100 μm. Error bars represent SD. See also Figures S1 and S2.
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Figure 1: BRD4 Regulates ESC Identity(A) Representative bright field images (upper panel) of hESCs treated for 3 days with BET inhibitors compared to FGF- or vehicle-treated cells. Alkaline Phosphatase (AP) staining following treatment is shown in bottom panel.(B) Quantification of AP-positive hESC colonies following FGF, vehicle, or compound treatment.(C) Bright field images (upper panel) and AP staining (bottom panel) of mESCs treated with BET inhibitors for 3 days in the presence of LIF.(D) Quantification of AP-positive mESC colonies following 3 days of compound treatment.(E–H) Bright field images of hESC (E) and mESC (G) colonies following BRD2, BRD3, and BRD4 suppression. Percentage of undifferentiated hESCs and mESC colonies is shown in (F) and (H), respectively. NTC, nontargeting control.(I) Cell-cycle analysis of hESCs following BET inhibitors treatment at the indicated time points as determined by FACS analysis.(J) Self-renewal capacity of vehicle- and compound-treated hESCs passaged on mitotically inactivated MEFs in the presence of FGF as measured by AP staining. AP, alkaline phosphatase; ND, not detected. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. The scale bar represents 100 μm. Error bars represent SD. See also Figures S1 and S2.

Mentions: To investigate the role of BET proteins in the maintenance of ESC identity, we inhibited BET activity by pharmacological and genetic means. We used highly specific, cell-permeable small molecules to effectively block acetylated lysine binding by BET proteins (Borah et al., 2011; Zhang et al., 2012). MS436 is a broad-range diazobenzene compound with high affinity for some BrD-containing proteins (Figure S1A; Zhang et al., 2013). MS417 is a thienotriazolodiazepine BrD inhibitor with high affinity and specificity for the BrDs of BET proteins (Figure S1A and S1B) and is structurally related to previously reported BET inhibitors, JQ1 (Filippakopoulos et al., 2010) and GSK525762A (Nicodeme et al., 2010). Treatment of human ESCs (hESCs) with either MS436 or MS417 altered colony integrity and reduced alkaline phosphatase (AP) activity, compared to vehicle- or fibroblast growth factor (FGF)-treated cells (Figures 1A and 1B). Similar results were obtained in murine ESCs (mESCs) treated with BET inhibitors in the presence of leukemia inhibitory factor (LIF) (Figures 1C and 1D). To dissect the relative contributions of individual BET proteins to the observed morphological changes of ESCs following BET inhibition, we independently silenced BRD2, BRD3, and BRD4 by short hairpin RNA (shRNA)-mediated transduction in both hESCs and mESCs (Figures S2A and S2B). Depletion of BRD2 and BRD3 did not perturb ESC colony formation (Figures 1E–1H). In contrast, BRD4 silencing disrupted both mouse and human ESC colony integrity, yielding a flattened and dispersed morphology similar to that observed with BET inhibitors and characteristic of differentiating ESCs (Figures 1E– 1H). Similar results were obtained with an independent small interfering RNA (siRNA)-mediated knockdown of BRD4 in hESCs (Figures S2C–S2E). Importantly, BET inhibition and BRD4 suppression impaired ESC colony formation without overt induction of apoptosis (Figure S2F; data not shown), indicating that chemical or genetic inhibition of BRD4 is not detrimental to ESC viability. We also observed that prolonged compound treatment resulted in accumulation of ESCs in the G1 phase of the cell cycle at the expense of S phase and cell proliferation (Figures 1I, S2G, and S2H), which is consistent with the acquisition of a more-differentiated phenotype. Finally, BET inhibition reduced the ability of both human and murine ESCs to form colonies after repeated passaging (Figures 1J and S2I); compound-treated colonies that did form were only weakly positive for AP (Figure S2J). Collectively, these results demonstrate that BRD4 is a key regulator of ESC maintenance.


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 ESC Identity(A) Representative bright field images (upper panel) of hESCs treated for 3 days with BET inhibitors compared to FGF- or vehicle-treated cells. Alkaline Phosphatase (AP) staining following treatment is shown in bottom panel.(B) Quantification of AP-positive hESC colonies following FGF, vehicle, or compound treatment.(C) Bright field images (upper panel) and AP staining (bottom panel) of mESCs treated with BET inhibitors for 3 days in the presence of LIF.(D) Quantification of AP-positive mESC colonies following 3 days of compound treatment.(E–H) Bright field images of hESC (E) and mESC (G) colonies following BRD2, BRD3, and BRD4 suppression. Percentage of undifferentiated hESCs and mESC colonies is shown in (F) and (H), respectively. NTC, nontargeting control.(I) Cell-cycle analysis of hESCs following BET inhibitors treatment at the indicated time points as determined by FACS analysis.(J) Self-renewal capacity of vehicle- and compound-treated hESCs passaged on mitotically inactivated MEFs in the presence of FGF as measured by AP staining. AP, alkaline phosphatase; ND, not detected. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. The scale bar represents 100 μm. Error bars represent SD. See also Figures S1 and S2.
© Copyright Policy - open-access
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Figure 1: BRD4 Regulates ESC Identity(A) Representative bright field images (upper panel) of hESCs treated for 3 days with BET inhibitors compared to FGF- or vehicle-treated cells. Alkaline Phosphatase (AP) staining following treatment is shown in bottom panel.(B) Quantification of AP-positive hESC colonies following FGF, vehicle, or compound treatment.(C) Bright field images (upper panel) and AP staining (bottom panel) of mESCs treated with BET inhibitors for 3 days in the presence of LIF.(D) Quantification of AP-positive mESC colonies following 3 days of compound treatment.(E–H) Bright field images of hESC (E) and mESC (G) colonies following BRD2, BRD3, and BRD4 suppression. Percentage of undifferentiated hESCs and mESC colonies is shown in (F) and (H), respectively. NTC, nontargeting control.(I) Cell-cycle analysis of hESCs following BET inhibitors treatment at the indicated time points as determined by FACS analysis.(J) Self-renewal capacity of vehicle- and compound-treated hESCs passaged on mitotically inactivated MEFs in the presence of FGF as measured by AP staining. AP, alkaline phosphatase; ND, not detected. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. The scale bar represents 100 μm. Error bars represent SD. See also Figures S1 and S2.
Mentions: To investigate the role of BET proteins in the maintenance of ESC identity, we inhibited BET activity by pharmacological and genetic means. We used highly specific, cell-permeable small molecules to effectively block acetylated lysine binding by BET proteins (Borah et al., 2011; Zhang et al., 2012). MS436 is a broad-range diazobenzene compound with high affinity for some BrD-containing proteins (Figure S1A; Zhang et al., 2013). MS417 is a thienotriazolodiazepine BrD inhibitor with high affinity and specificity for the BrDs of BET proteins (Figure S1A and S1B) and is structurally related to previously reported BET inhibitors, JQ1 (Filippakopoulos et al., 2010) and GSK525762A (Nicodeme et al., 2010). Treatment of human ESCs (hESCs) with either MS436 or MS417 altered colony integrity and reduced alkaline phosphatase (AP) activity, compared to vehicle- or fibroblast growth factor (FGF)-treated cells (Figures 1A and 1B). Similar results were obtained in murine ESCs (mESCs) treated with BET inhibitors in the presence of leukemia inhibitory factor (LIF) (Figures 1C and 1D). To dissect the relative contributions of individual BET proteins to the observed morphological changes of ESCs following BET inhibition, we independently silenced BRD2, BRD3, and BRD4 by short hairpin RNA (shRNA)-mediated transduction in both hESCs and mESCs (Figures S2A and S2B). Depletion of BRD2 and BRD3 did not perturb ESC colony formation (Figures 1E–1H). In contrast, BRD4 silencing disrupted both mouse and human ESC colony integrity, yielding a flattened and dispersed morphology similar to that observed with BET inhibitors and characteristic of differentiating ESCs (Figures 1E– 1H). Similar results were obtained with an independent small interfering RNA (siRNA)-mediated knockdown of BRD4 in hESCs (Figures S2C–S2E). Importantly, BET inhibition and BRD4 suppression impaired ESC colony formation without overt induction of apoptosis (Figure S2F; data not shown), indicating that chemical or genetic inhibition of BRD4 is not detrimental to ESC viability. We also observed that prolonged compound treatment resulted in accumulation of ESCs in the G1 phase of the cell cycle at the expense of S phase and cell proliferation (Figures 1I, S2G, and S2H), which is consistent with the acquisition of a more-differentiated phenotype. Finally, BET inhibition reduced the ability of both human and murine ESCs to form colonies after repeated passaging (Figures 1J and S2I); compound-treated colonies that did form were only weakly positive for AP (Figure S2J). Collectively, these results demonstrate that BRD4 is a key regulator of ESC maintenance.

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