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• W2000965208 abstract "•OCT4 interacts with BRD4 in embryonic stem cells (ESCs)•BRD4 occupies the regulatory regions of pluripotent genes•BRD4 occupies and controls the lncRNAs in X chromosome inactivation•BET inhibition or depletion of BRD4 in ESCs shifts cell fate away from pluripotency Embryonic stem cell (ESC) pluripotency is controlled by defined transcription factors. During cellular differentiation, ESCs undergo a global epigenetic reprogramming. Female ESCs exemplify this process as one of the two X-chromosomes is globally silenced during X chromosome inactivation (XCI) to balance the X-linked gene disparity with XY males. The pluripotent factor OCT4 regulates XCI by triggering X chromosome pairing and counting. OCT4 directly binds Xite and Tsix, which encode two long noncoding RNAs (lncRNAs) that suppress the silencer lncRNA, Xist. To control its activity as a master regulator in pluripotency and XCI, OCT4 must have chromatin protein partners. Here we show that BRD4, a member of the BET protein subfamily, interacts with OCT4. BRD4 occupies the regulatory regions of pluripotent genes and the lncRNAs of XCI. BET inhibition or depletion of BRD4 reduces the expression of many pluripotent genes and shifts cellular fate showing that BRD4 is pivotal for transcription in ESCs. Embryonic stem cell (ESC) pluripotency is controlled by defined transcription factors. During cellular differentiation, ESCs undergo a global epigenetic reprogramming. Female ESCs exemplify this process as one of the two X-chromosomes is globally silenced during X chromosome inactivation (XCI) to balance the X-linked gene disparity with XY males. The pluripotent factor OCT4 regulates XCI by triggering X chromosome pairing and counting. OCT4 directly binds Xite and Tsix, which encode two long noncoding RNAs (lncRNAs) that suppress the silencer lncRNA, Xist. To control its activity as a master regulator in pluripotency and XCI, OCT4 must have chromatin protein partners. Here we show that BRD4, a member of the BET protein subfamily, interacts with OCT4. BRD4 occupies the regulatory regions of pluripotent genes and the lncRNAs of XCI. BET inhibition or depletion of BRD4 reduces the expression of many pluripotent genes and shifts cellular fate showing that BRD4 is pivotal for transcription in ESCs. Master transcription regulators control the pluripotent gene expression in embryonic stem cells (ESCs) (Avilion et al., 2003Avilion A.A. Nicolis S.K. Pevny L.H. Perez L. Vivian N. Lovell-Badge R. Multipotent cell lineages in early mouse development depend on SOX2 function.Genes Dev. 2003; 17: 126-140Crossref PubMed Scopus (1757) Google Scholar, Chambers et al., 2003Chambers I. Colby D. Robertson M. Nichols J. Lee S. Tweedie S. Smith A. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells.Cell. 2003; 113: 643-655Abstract Full Text Full Text PDF PubMed Scopus (2595) Google Scholar, Mitsui et al., 2003Mitsui K. Tokuzawa Y. Itoh H. Segawa K. Murakami M. Takahashi K. Maruyama M. Maeda M. Yamanaka S. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells.Cell. 2003; 113: 631-642Abstract Full Text Full Text PDF PubMed Scopus (2521) Google Scholar, Nichols et al., 1998Nichols J. Zevnik B. Anastassiadis K. Niwa H. Klewe-Nebenius D. Chambers I. Schöler H. Smith A. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4.Cell. 1998; 95: 379-391Abstract Full Text Full Text PDF PubMed Scopus (2627) Google Scholar). X chromosome inactivation (XCI) is a crucial epigenetic process that silences one of the two female X chromosomes to ensure equal X-linked gene expression with XY males (Payer and Lee, 2008Payer B. Lee J.T. X chromosome dosage compensation: how mammals keep the balance.Annu. Rev. Genet. 2008; 42: 733-772Crossref PubMed Scopus (365) Google Scholar, Lee and Bartolomei, 2013Lee J.T. Bartolomei M.S. X-inactivation, imprinting, and long noncoding RNAs in health and disease.Cell. 2013; 152: 1308-1323Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar). XCI is tightly linked with pluripotency, as this epigenetic silencing occurs upon cellular differentiation and conversion of female somatic cells to the induced stem-ness state is accompanied by a global epigenetic reprogramming and reactivation of the silenced X (Maherali et al., 2007Maherali N. Sridharan R. Xie W. Utikal J. Eminli S. Arnold K. Stadtfeld M. Yachechko R. Tchieu J. Jaenisch R. et al.Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution.Cell Stem Cell. 2007; 1: 55-70Abstract Full Text Full Text PDF PubMed Scopus (1388) Google Scholar, Navarro et al., 2008Navarro P. Chambers I. Karwacki-Neisius V. Chureau C. Morey C. Rougeulle C. Avner P. Molecular coupling of Xist regulation and pluripotency.Science. 2008; 321: 1693-1695Crossref PubMed Scopus (261) Google Scholar). The transcription factor OCT4 lies at the top of the XCI hierarchy regulating the pluripotent-associated long noncoding RNAs (lncRNAs): Xite (the enhancer for Tsix) and Tsix (the anti-sense repressor of Xist) (Donohoe et al., 2009Donohoe M.E. Silva S.S. Pinter S.F. Xu N. Lee J.T. The pluripotency factor Oct4 interacts with Ctcf and also controls X-chromosome pairing and counting.Nature. 2009; 460: 128-132Crossref PubMed Scopus (222) Google Scholar). Together Xite and Tsix mediate X-X homologous pairing and inhibit the silencer Xist prior to X chromosome choice (Xu et al., 2006Xu N. Tsai C.L. Lee J.T. Transient homologous chromosome pairing marks the onset of X inactivation.Science. 2006; 311: 1149-1152Crossref PubMed Scopus (322) Google Scholar). In addition to their roles in the study of pluripotency and cellular differentiation, mouse ESCs are established as ex vivo models of XCI, faithfully recapitulating XCI in the embryo (Clerc and Avner, 1998Clerc P. Avner P. Role of the region 3′ to Xist exon 6 in the counting process of X-chromosome inactivation.Nat. Genet. 1998; 19: 249-253Crossref PubMed Scopus (144) Google Scholar, Lee and Jaenisch, 1997Lee J.T. Jaenisch R. Long-range cis effects of ectopic X-inactivation centres on a mouse autosome.Nature. 1997; 386: 275-279Crossref PubMed Scopus (222) Google Scholar, Lee and Lu, 1999Lee J.T. Lu N. Targeted mutagenesis of Tsix leads to nonrandom X inactivation.Cell. 1999; 99: 47-57Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar, Penny et al., 1996Penny G.D. Kay G.F. Sheardown S.A. Rastan S. Brockdorff N. Requirement for Xist in X chromosome inactivation.Nature. 1996; 379: 131-137Crossref PubMed Scopus (991) Google Scholar, Rastan and Robertson, 1985Rastan S. Robertson E.J. X-chromosome deletions in embryo-derived (EK) cell lines associated with lack of X-chromosome inactivation.J. Embryol. Exp. Morphol. 1985; 90: 379-388PubMed Google Scholar). In undifferentiated ESCs, the single male X and both female X chromosomes are active. The lncRNAs Xite, Tsix, and Xist are all expressed on these active X chromosomes in the pluripotent state. ESCs can be differentiated by suspension culture for 4 days without leukemia inhibitory factor (LIF) and maintained thereafter under adherent conditions (Martin and Evans, 1975Martin G.R. Evans M.J. Differentiation of clonal lines of teratocarcinoma cells: formation of embryoid bodies in vitro.Proc. Natl. Acad. Sci. USA. 1975; 72: 1441-1445Crossref PubMed Scopus (527) Google Scholar). Following differentiation, the male X chromosome loses expression of these lncRNAs to retain activity of the single X, whereas the female ESCs have a choice of active versus inactive X. On the future active X, Xite and Tsix expression persists to keep Xist levels low. In contrast, on the future inactive X, Xite and Tsix are extinguished, and Xist levels are greatly upregulated. OCT4 partners with the chromatin insulator CTCF, specifying the early decisions of XCI (counting, X-X pairing, and choice) (Xu et al., 2006Xu N. Tsai C.L. Lee J.T. Transient homologous chromosome pairing marks the onset of X inactivation.Science. 2006; 311: 1149-1152Crossref PubMed Scopus (322) Google Scholar, Xu et al., 2007Xu N. Donohoe M.E. Silva S.S. Lee J.T. Evidence that homologous X-chromosome pairing requires transcription and Ctcf protein.Nat. Genet. 2007; 39: 1390-1396Crossref PubMed Scopus (157) Google Scholar, Donohoe et al., 2009Donohoe M.E. Silva S.S. Pinter S.F. Xu N. Lee J.T. The pluripotency factor Oct4 interacts with Ctcf and also controls X-chromosome pairing and counting.Nature. 2009; 460: 128-132Crossref PubMed Scopus (222) Google Scholar). During differentiation, ESC chromatin shifts from a transcriptionally permission, euchromatic, to a more heterochromatic state (Azuara et al., 2006Azuara V. Perry P. Sauer S. Spivakov M. Jørgensen H.F. John R.M. Gouti M. Casanova M. Warnes G. Merkenschlager M. Fisher A.G. Chromatin signatures of pluripotent cell lines.Nat. Cell Biol. 2006; 8: 532-538Crossref PubMed Scopus (1037) Google Scholar, Meshorer and Misteli, 2006Meshorer E. Misteli T. Chromatin in pluripotent embryonic stem cells and differentiation.Nat. Rev. Mol. Cell Biol. 2006; 7: 540-546Crossref PubMed Scopus (544) Google Scholar, Niwa, 2007Niwa H. Open conformation chromatin and pluripotency.Genes Dev. 2007; 21: 2671-2676Crossref PubMed Scopus (79) Google Scholar). These changes in chromatin packaging are accompanied by alterations in histone post-translational modifications (PTMs) crucial for modulation of chromatin structure and gene expression (Bernstein et al., 2006Bernstein B.E. Mikkelsen T.S. Xie X. Kamal M. Huebert D.J. Cuff J. Fry B. Meissner A. Wernig M. Plath K. et al.A bivalent chromatin structure marks key developmental genes in embryonic stem cells.Cell. 2006; 125: 315-326Abstract Full Text Full Text PDF PubMed Scopus (3956) Google Scholar). Histone PTM writers such as the Polycomb group proteins (Boyer et al., 2006Boyer L.A. Plath K. Zeitlinger J. Brambrink T. Medeiros L.A. Lee T.I. Levine S.S. Wernig M. Tajonar A. Ray M.K. et al.Polycomb complexes repress developmental regulators in murine embryonic stem cells.Nature. 2006; 441: 349-353Crossref PubMed Scopus (1999) Google Scholar) and erasers such as the demethylases (Adamo et al., 2011Adamo A. Sesé B. Boue S. Castaño J. Paramonov I. Barrero M.J. Izpisua Belmonte J.C. LSD1 regulates the balance between self-renewal and differentiation in human embryonic stem cells.Nat. Cell Biol. 2011; 13: 652-659Crossref PubMed Scopus (236) Google Scholar, Loh et al., 2007Loh Y.H. Zhang W. Chen X. George J. Ng H.H. Jmjd1a and Jmjd2c histone H3 Lys 9 demethylases regulate self-renewal in embryonic stem cells.Genes Dev. 2007; 21: 2545-2557Crossref PubMed Scopus (404) Google Scholar, Mansour et al., 2012Mansour A.A. Gafni O. Weinberger L. Zviran A. Ayyash M. Rais Y. Krupalnik V. Zerbib M. Amann-Zalcenstein D. Maza I. et al.The H3K27 demethylase Utx regulates somatic and germ cell epigenetic reprogramming.Nature. 2012; 488: 409-413Crossref PubMed Scopus (272) Google Scholar, Wang et al., 2011Wang T. Chen K. Zeng X. Yang J. Wu Y. Shi X. Qin B. Zeng L. Esteban M.A. Pan G. Pei D. The histone demethylases Jhdm1a/1b enhance somatic cell reprogramming in a vitamin-C-dependent manner.Cell Stem Cell. 2011; 9: 575-587Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar) play important roles in early development. We postulate that histone readers together with OCT4 play a role in the transcriptional control of the XCI lncRNAs as well as pluripotent genes. One candidate is the chromatin reader, BRD4. BRD4 is a member of the BET (bromodomain and extraterminal domain) family of tandem bromodomain-containing proteins that can bind acetylated histones H3 and H4 and influence transcription (Chiang, 2009Chiang C.M. Brd4 engagement from chromatin targeting to transcriptional regulation: selective contact with acetylated histone H3 and H4.F1000 Biol. Rep. 2009; 1: 98Crossref PubMed Google Scholar). BRD4 is an epigenetic reader originally identified as a mitotic chromosome-binding protein that remains associated with acetylated chromatin throughout the entire cell cycle and is thought to provide epigenetic bookmarking after cell division (Dey et al., 2000Dey A. Ellenberg J. Farina A. Coleman A.E. Maruyama T. Sciortino S. Lippincott-Schwartz J. Ozato K. A bromodomain protein, MCAP, associates with mitotic chromosomes and affects G(2)-to-M transition.Mol. Cell. Biol. 2000; 20: 6537-6549Crossref PubMed Scopus (225) Google Scholar, Dey et al., 2003Dey A. Chitsaz F. Abbasi A. Misteli T. Ozato K. The double bromodomain protein Brd4 binds to acetylated chromatin during interphase and mitosis.Proc. Natl. Acad. Sci. USA. 2003; 100: 8758-8763Crossref PubMed Scopus (480) Google Scholar). BRD4 has a direct role in transcription as it associates with positive transcription elongation factor b (P-TEFb) to enhance RNA polymerase II (RNAP II) and control productive mRNA synthesis (Yang et al., 2008Yang Z. He N. Zhou Q. Brd4 recruits P-TEFb to chromosomes at late mitosis to promote G1 gene expression and cell cycle progression.Mol. Cell. Biol. 2008; 28: 967-976Crossref PubMed Scopus (257) Google Scholar). At many developmental genes RNAP II stalls or pauses after transcribing a nascent transcript about 20–65 nucleotides in length (Adelman and Lis, 2012Adelman K. Lis J.T. Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans.Nat. Rev. Genet. 2012; 13: 720-731Crossref PubMed Scopus (735) Google Scholar). Nearly 30% of the genes in human ESCs commence transcription initiation but do not undergo transcriptional elongation (Guenther et al., 2007Guenther M.G. Levine S.S. Boyer L.A. Jaenisch R. Young R.A. A chromatin landmark and transcription initiation at most promoters in human cells.Cell. 2007; 130: 77-88Abstract Full Text Full Text PDF PubMed Scopus (1481) Google Scholar). This suggests that transcriptional pausing is an additional checkpoint control during development (Levine, 2011Levine M. Paused RNA polymerase II as a developmental checkpoint.Cell. 2011; 145: 502-511Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). The release from transcriptional pausing is associated with P-TEFb recruitment, the eviction of pause factors, the phosphorylation at serine 2 of the carboxyl-terminal domain (CTD) in RNAP II, and the production of elongated mRNAs. Although BRD4 is known to play crucial roles in the oncogenic and viral programs, very little is known about its function in early normal development. The loss of Brd4 in the mouse results in peri-implantation lethality, with an ablation of the inner cell mass the source for ESCs (Houzelstein et al., 2002Houzelstein D. Bullock S.L. Lynch D.E. Grigorieva E.F. Wilson V.A. Beddington R.S. Growth and early postimplantation defects in mice deficient for the bromodomain-containing protein Brd4.Mol. Cell. Biol. 2002; 22: 3794-3802Crossref PubMed Scopus (218) Google Scholar), suggesting a role for this gene in the cell differentiation-linked processes of XCI and pluripotency. Here we investigate BRD4’s function in these crucial developmental processes. Our studies show that Brd4 interacts with the pluripotent factor OCT4 and is important for maintaining stem cell fate and the expression of the lncRNAs controlling XCI. We postulate that a co-activator such as BRD4 might play a role in epigenetic memory for binary cell fate (“stem-ness” versus differentiation) and XCI (active versus inactive X chromosome) status in ESCs. To explore this possibility, we examined the developmental expression pattern for the BRD4 protein in differentiating female and male ESCs. To differentiate the ESCs, we removed LIF and mouse embryonic feeders on nonadherent plates as previously described (Donohoe et al., 2007Donohoe M.E. Zhang L.F. Xu N. Shi Y. Lee J.T. Identification of a Ctcf cofactor, Yy1, for the X chromosome binary switch.Mol. Cell. 2007; 25: 43-56Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Our results show that the BRD4 protein is expressed at similar levels during differentiation day 0 (d0) (pre-XCI), day 4 (d4) (time of the establishment of XCI), and day 8 (d8) (post-XCI) in both female and male ESCs (Figure 1A). In contrast, the OCT4 protein is present at d0 and d4 and is greatly reduced by d8 in these cells. Because the loss of mouse Brd4 has a peri-implantation phenotype (at the time random XCI takes place in the epiblast) and given the tight linkage between XCI and differentiation, we questioned whether BRD4 might interact with pluripotent factors. Using a candidate approach, we tested BRD4 for partnering with “stem-ness”-associated transcription factors. Full-length Myc-tagged BRD4 and Flag-OCT4 were co-transfected into human embryonic kidney (HEK) cells and tested for their interaction. We observed a specific OCT4-BRD4 interaction following co-immunoprecipitation (co-IP) (Figure 1B). Next, we queried whether an endogenous partnering of BRD4 and OCT4 occurs in undifferentiated male and female ESCs. BRD4 co-IPs with a specific OCT4 antibody in female ESCs confirming in vivo BRD4-OCT4 interaction (Figure 1C). Reciprocal co-IP confirms the endogenous OCT4-BRD4 complex (Figure 1D). Similar results were observed in male ESCs (Figures 1E and 1F). Although there are three BRD4 isoforms, the antibody used here recognizes an epitope present only in the longest isoform, suggesting that this is the isoform that partners with Oct4. Taken together, the chromatin reader BRD4 is expressed throughout cellular differentiation in both male and female ESCs and partners in vitro and in vivo with OCT4, a key regulator of pluripotency and XCI. We determined that BRD4 interacts with OCT4. To further investigate the genes that it targets, we examined BRD4 occupancy in undifferentiated male ESCs using chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-seq). First, we validated the BRD4 and OCT4 occupancy at Oct4 and Nanog regulatory regions in d0 male ESCs using quantitative chromatin immunoprecipitation (qChIP) (Figure S1A). Using a stringent statistical criteria (p < 0.05) we identified significant enrichment of ChIP-seq peaks (Figure S1B). As shown in Figure 2A, BRD4 binds the gene body and regulatory regions of Nanog, Pou5f1/Oct4, Sox2, c-Myc, Fgf4, and the lncRNAs Tsix and Xist. An enhanced peak of BRD4 binding is situated over Sox2. To investigate whether BRD4 is involved in XCI, we examined BRD4 occupancy in male and female day 4 of differentiation (d4) ESCs using qChIP. We used the OCT4-binding sites within the pluripotency-associated Xite and Tsix lncRNAs (Xite enhancer, Tsix site D, and Xist intron 1B) as guides for potential BRD4 in vivo binding in the XCI locus (Donohoe et al., 2009Donohoe M.E. Silva S.S. Pinter S.F. Xu N. Lee J.T. The pluripotency factor Oct4 interacts with Ctcf and also controls X-chromosome pairing and counting.Nature. 2009; 460: 128-132Crossref PubMed Scopus (222) Google Scholar, Navarro et al., 2008Navarro P. Chambers I. Karwacki-Neisius V. Chureau C. Morey C. Rougeulle C. Avner P. Molecular coupling of Xist regulation and pluripotency.Science. 2008; 321: 1693-1695Crossref PubMed Scopus (261) Google Scholar, Navarro et al., 2010Navarro P. Oldfield A. Legoupi J. Festuccia N. Dubois A. Attia M. Schoorlemmer J. Rougeulle C. Chambers I. Avner P. Molecular coupling of Tsix regulation and pluripotency.Nature. 2010; 468: 457-460Crossref PubMed Scopus (145) Google Scholar), as well as several known OCT4-regulated promoters (Nanog and Sox2) (Chew et al., 2005Chew J.L. Loh Y.H. Zhang W. Chen X. Tam W.L. Yeap L.S. Li P. Ang Y.S. Lim B. Robson P. Ng H.H. Reciprocal transcriptional regulation of Pou5f1 and Sox2 via the Oct4/Sox2 complex in embryonic stem cells.Mol. Cell. Biol. 2005; 25: 6031-6046Crossref PubMed Scopus (540) Google Scholar, Rodda et al., 2005Rodda D.J. Chew J.L. Lim L.H. Loh Y.H. Wang B. Ng H.H. Robson P. Transcriptional regulation of nanog by OCT4 and SOX2.J. Biol. Chem. 2005; 280: 24731-24737Crossref PubMed Scopus (821) Google Scholar) and enhancers (Oct4) (Chew et al., 2005Chew J.L. Loh Y.H. Zhang W. Chen X. Tam W.L. Yeap L.S. Li P. Ang Y.S. Lim B. Robson P. Ng H.H. Reciprocal transcriptional regulation of Pou5f1 and Sox2 via the Oct4/Sox2 complex in embryonic stem cells.Mol. Cell. Biol. 2005; 25: 6031-6046Crossref PubMed Scopus (540) Google Scholar). In addition to BRD4, these loci were tested for OCT4 binding and the acetylated histone 4 (H4Ac), a PTM associated with gene activation and a mark that bromodomain-containing proteins can bind and read (Chiang, 2009Chiang C.M. Brd4 engagement from chromatin targeting to transcriptional regulation: selective contact with acetylated histone H3 and H4.F1000 Biol. Rep. 2009; 1: 98Crossref PubMed Google Scholar). Among the BET family members, BRD4’s bromodomains have the highest affinity for H4Ac (Jung et al., 2014Jung M. Philpott M. Müller S. Schulze J. Badock V. Ebersp Aumlcher U. Moosmayer D. Bader B. Schmees N. Fernández-Montalv A. et al.Affinity Map of BRD4 Interactions with the Histone H4 Tail and the Small Molecule Inhibitor JQ1.J Biol Chem. 2014; 289: 9304-9319Crossref PubMed Scopus (96) Google Scholar). Chromatin was prepared from d4 male ESCs and subjected to qChIP. BRD4 is enriched nearly 16-fold at Tsix site D chromatin (Figure 2B). Tsix site D resides in the repeat region DxPas34, the epigenetic switch/control region for XCI. Deletion of DxPas34 on one of the two female Xs nearly always results in this X chromosome to be chosen for inactivation (Lee and Lu, 1999Lee J.T. Lu N. Targeted mutagenesis of Tsix leads to nonrandom X inactivation.Cell. 1999; 99: 47-57Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). Xite and Xist intron 1B, a known strong OCT4 binding domain chromatin shows enhanced BRD4 and OCT4 occupancy over control at d4 in male ESCs. In contrast the immunity gene Toll-like receptor 9 (TLR9), a negative control for OCT4 occupancy (Mochizuki et al., 2008Mochizuki K. Nishiyama A. Jang M.K. Dey A. Ghosh A. Tamura T. Natsume H. Yao H. Ozato K. The bromodomain protein Brd4 stimulates G1 gene transcription and promotes progression to S phase.J. Biol. Chem. 2008; 283: 9040-9048Crossref PubMed Scopus (169) Google Scholar), is not enriched for either OCT4 or BRD4. The Oct4 enhancer chromatin shows a 10- and 25-fold enrichment of BRD4 and OCT4 binding, respectively. At d4 H4Ac PTM shows high levels at all pluripotency-associated regulatory chromatin. We next examined BRD4, OCT4, and H4Ac in vivo occupancy in differentiating d4 female ESCs. Here we observe similar dynamic changes. At d4, the time of XCI establishment in female ESCs, we see BRD4 and OCT4 enrichment compared with background at Xite, Xist intron 1B, Oct4, Nanog, and Sox2 (Figure 2D). The TLR9 regulatory region is an exception, showing a lack of BRD4 or OCT4 occupancy. All of these chromatin sites tested show enrichment of H4Ac PTM. Collectively, our data confirm that BRD4 binding to the OCT4-associated regulatory regions in both male and female ESCs. These results suggest that both BRD4 and OCT4 can occupy the same regulatory elements in XCI and pluripotency. Although BRD4 can directly recognize and bind acetylated histones, recent studies show that its interaction partners can stabilize its binding to chromatin (Chiang, 2009Chiang C.M. Brd4 engagement from chromatin targeting to transcriptional regulation: selective contact with acetylated histone H3 and H4.F1000 Biol. Rep. 2009; 1: 98Crossref PubMed Google Scholar, Stewart et al., 2013Stewart H.J. Horne G.A. Bastow S. Chevassut T.J. BRD4 associates with p53 in DNMT3A-mutated leukemia cells and is implicated in apoptosis by the bromodomain inhibitor JQ1.Cancer Med. 2013; 2: 826-835Crossref PubMed Scopus (64) Google Scholar). Therefore, we asked whether OCT4 could tether and enhance BRD4 binding to pluripotent regulatory regions facilitating active transcription. To address this, we utilized the OCT4-regulated ESC line, ZHBTc4 (Niwa et al., 2000Niwa H. Miyazaki J. Smith A.G. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells.Nat. Genet. 2000; 24: 372-376Crossref PubMed Scopus (2857) Google Scholar), which depletes OCT4 protein expression upon addition of doxycycline (Dox) (Figure 2D). Following exposure to Dox, ZHBTc4 ESCs ablate OCT4 protein, whereas BRD4 and ACTIN levels do not change (Figure 2E). We performed qChIP for OCT4 and BRD4 on these OCT4-regulated sites in the absence and presence of Dox. As shown in Figure 2F, we observe a reduction of OCT4 occupancy on all regulatory regions following OCT4 protein depletion. After OCT4 removal, the Nanog promoter shows a statistically significant reduction (∼50%) in BRD4 binding, as does the Tcl1 promoter (data not shown). In contrast, the Fgf4 enhancer, the Xist intron 1B, the Oct4 enhancer, Tsix site D, and the Xite enhancer do not show a significant change in BRD4 occupancy. Ablation of OCT4 protein reveals preferential changes in H4Ac levels at the Fgf4, Oct4, and Xite enhancers (Figure 2G). Our qChIP results suggest that BRD4 binding is dynamic and that OCT4 can recruit or stabilize BRD4 to particular pluripotent regulatory regions. The BET domain proteins can be inhibited by the small molecule JQ1, which selectively binds the tandem bromodomains displacing BRD4 and P-TEFb from acetylated chromatin leading to a decrease in RNAP II elongation at active genes (Filippakopoulos et al., 2010Filippakopoulos P. Qi J. Picaud S. Shen Y. Smith W.B. Fedorov O. Morse E.M. Keates T. Hickman T.T. Felletar I. et al.Selective inhibition of BET bromodomains.Nature. 2010; 468: 1067-1073Crossref PubMed Scopus (2705) Google Scholar). Because BRD4 can associate with the regulatory regions of the pluripotent genes, we hypothesize that BET inhibition alters the expression of pluripotent-associated genes. To test this, we first performed a dose curve using JQ1 and DMSO control in both male and female d6 ESCs. We found that JQ1 treatment of male ESCs can inhibit the c-MYC protein expression in a dose-dependent manner without the accompaniment of cell death as reported (Filippakopoulos et al., 2010Filippakopoulos P. Qi J. Picaud S. Shen Y. Smith W.B. Fedorov O. Morse E.M. Keates T. Hickman T.T. Felletar I. et al.Selective inhibition of BET bromodomains.Nature. 2010; 468: 1067-1073Crossref PubMed Scopus (2705) Google Scholar) (Figure 3A). Intriguingly, the pluripotency trans-factor OCT4 is decreased as compared with BRD4 and ACTIN levels following BET inhibition in male ESCs (Figure 3A). We queried BRD4 and P-TEFb occupancy by qChIP following JQ1 BET inhibition. BRD4 and CDK9 occupancy are lost at the Oct4 enhancer, Sox2 promoter, and the Nanog promoter as well as Xite, Tsix Site D, Xist intron 1B, and the Xist promoter transcriptional start site (TSS) (Figures S2A and S2B). In contrast, the histone 4 acetylation (H4Ac) levels at these sites did not consistently show a diminution after JQ1 BET inhibition (Figure S2C). Next, we asked whether JQ1 treatment might alter BRD4’s ability to partner with OCT4. Indeed, BET inhibition diminishes BRD4 binding to OCT4 (Figure S3A). These results suggest that JQ1 exposure specifically displaces BRD4 binding to the pluripotent and XCI regulatory regions without altering the H4Ac levels and BET inhibition alters its partnering with OCT4. The XCI lncRNAs Xist and Tsix were greatly depressed after BET inhibition (Figure 3B). BRD4 recruits P-TEFb to chromosomes to promote G1-phase gene transcription and progression to S phase in fibroblast cell cycles (Yang et al., 2008Yang Z. He N. Zhou Q. Brd4 recruits P-TEFb to chromosomes at late mitosis to promote G1 gene expression and cell cycle progression.Mol. Cell. Biol. 2008; 28: 967-976Crossref PubMed Scopus (257) Google Scholar). Consistent with this, we find that the cell cycle regulator p21 (Figure 3B) and Cyclin D1 (Figure 4B) are diminished in male ESCs following BET treatment. Next we examined a panel of pluripotency genes levels to see whether they were altered by BET displacement. Undifferentiated ESCs show a great reduction of Oct4, Nanog, Tsix, and Xist after JQ1 exposure (Figure S3B). Consistent with a diminished protein level, Oct4 mRNA levels were vastly depressed following JQ1 treatment as well as the Nanog gene (Figure 3C). The endodermal marker Gata4 was repressed in male ESCs (Figure 3C). Sox2, another pluripotent factor, was not depressed. Nor was 7sk snRNA, a P-TEFb inhibitor (Yang et al., 2001Yang Z. Zhu Q. Luo K. Zhou Q. The 7SK small nuclear RNA inhibits the CDK9/cyclin T1 kinase to control transcription.Nature. 2001; 414: 317-322Crossref PubMed Scopus (538) Google Scholar, Nguyen et al., 2001Nguyen V.T. Kiss T. Michels A.A. 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Hay N. FoxOs inhibit mTORC1 and activate Akt by inducing the expression of Sestrin3 and Rictor.Dev. Cell. 2010; 18: 592-604Abstract Full Text Full Text" @default.
• W2000965208 created "2016-06-24" @default.
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• W2000965208 title "The BET Family Member BRD4 Interacts with OCT4 and Regulates Pluripotency Gene Expression" @default.
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