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The BET family member BRD4 interacts with OCT4 and regulates pluripotency gene expression.

Wu T, Pinto HB, Kamikawa YF, Donohoe ME - Stem Cell Reports (2015)

Bottom Line: OCT4 directly binds Xite and Tsix, which encode two long noncoding RNAs (lncRNAs) that suppress the silencer lncRNA, Xist.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.

View Article: PubMed Central - PubMed

Affiliation: Burke Medical Research Institute, 785 Mamaroneck Avenue, White Plains, NY 10605, USA; Departments of Neuroscience and Cell and Developmental Biology, Weill Cornell Medical College, New York, NY 10065, USA.

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BRD4 Is Expressed in Differentiating Female and Male ESCs, Interacts with OCT4, and the Expression of Many Pluripotent Genes Rely on the BRD4 Protein Partner P-TEFb(A) Western blot analysis of BRD4 (top) and OCT4 (middle) in WT female and male ESCs whole cell extracts (WCEs) on differentiation d0, d4, and d8. Histone 3 (H3) (bottom) is used as a load control for protein expression.(B) HEK cells were co-transfected (Tf) with full-length Myc-tagged BRD4 and Flag-tagged OCT4. WCEs were immunoprecipitated (IP) with anti-Flag antibodies and western blot analysis with anti-Myc antibodies. The arrow denotes BRD4 binding OCT4 in the upper panel. The lower shows Flag-OCT4 expression detected by western blot.(C) Co-IP of BRD4 and OCT4 in ESCs. Immunoprecipitation with anti-OCT4 or control antibodies to test interaction with endogenous BRD4 in d0 female ESCs. Arrow marks BRD4 detected by anti-BRD4 western.(D) Reciprocal immunoprecipitation using anti-BRD4 or control antibodies to test interaction with OCT4 in female ESCs. Arrow marks OCT4 detected by anti-OCT4 western.(E) Endogenous BRD4 co-immunoprecipitates with endogenous OCT4 in d0 male ESCs. Arrow marks BRD4 following western with anti-BRD4.(F) Reciprocal immunoprecipitation using anti-BRD4 or control antibodies to test binding to OCT4 in male ESCs. Arrow marks OCT4 following anti-OCT4 western.
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fig1: BRD4 Is Expressed in Differentiating Female and Male ESCs, Interacts with OCT4, and the Expression of Many Pluripotent Genes Rely on the BRD4 Protein Partner P-TEFb(A) Western blot analysis of BRD4 (top) and OCT4 (middle) in WT female and male ESCs whole cell extracts (WCEs) on differentiation d0, d4, and d8. Histone 3 (H3) (bottom) is used as a load control for protein expression.(B) HEK cells were co-transfected (Tf) with full-length Myc-tagged BRD4 and Flag-tagged OCT4. WCEs were immunoprecipitated (IP) with anti-Flag antibodies and western blot analysis with anti-Myc antibodies. The arrow denotes BRD4 binding OCT4 in the upper panel. The lower shows Flag-OCT4 expression detected by western blot.(C) Co-IP of BRD4 and OCT4 in ESCs. Immunoprecipitation with anti-OCT4 or control antibodies to test interaction with endogenous BRD4 in d0 female ESCs. Arrow marks BRD4 detected by anti-BRD4 western.(D) Reciprocal immunoprecipitation using anti-BRD4 or control antibodies to test interaction with OCT4 in female ESCs. Arrow marks OCT4 detected by anti-OCT4 western.(E) Endogenous BRD4 co-immunoprecipitates with endogenous OCT4 in d0 male ESCs. Arrow marks BRD4 following western with anti-BRD4.(F) Reciprocal immunoprecipitation using anti-BRD4 or control antibodies to test binding to OCT4 in male ESCs. Arrow marks OCT4 following anti-OCT4 western.

Mentions: 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., 2007). 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).


The BET family member BRD4 interacts with OCT4 and regulates pluripotency gene expression.

Wu T, Pinto HB, Kamikawa YF, Donohoe ME - Stem Cell Reports (2015)

BRD4 Is Expressed in Differentiating Female and Male ESCs, Interacts with OCT4, and the Expression of Many Pluripotent Genes Rely on the BRD4 Protein Partner P-TEFb(A) Western blot analysis of BRD4 (top) and OCT4 (middle) in WT female and male ESCs whole cell extracts (WCEs) on differentiation d0, d4, and d8. Histone 3 (H3) (bottom) is used as a load control for protein expression.(B) HEK cells were co-transfected (Tf) with full-length Myc-tagged BRD4 and Flag-tagged OCT4. WCEs were immunoprecipitated (IP) with anti-Flag antibodies and western blot analysis with anti-Myc antibodies. The arrow denotes BRD4 binding OCT4 in the upper panel. The lower shows Flag-OCT4 expression detected by western blot.(C) Co-IP of BRD4 and OCT4 in ESCs. Immunoprecipitation with anti-OCT4 or control antibodies to test interaction with endogenous BRD4 in d0 female ESCs. Arrow marks BRD4 detected by anti-BRD4 western.(D) Reciprocal immunoprecipitation using anti-BRD4 or control antibodies to test interaction with OCT4 in female ESCs. Arrow marks OCT4 detected by anti-OCT4 western.(E) Endogenous BRD4 co-immunoprecipitates with endogenous OCT4 in d0 male ESCs. Arrow marks BRD4 following western with anti-BRD4.(F) Reciprocal immunoprecipitation using anti-BRD4 or control antibodies to test binding to OCT4 in male ESCs. Arrow marks OCT4 following anti-OCT4 western.
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fig1: BRD4 Is Expressed in Differentiating Female and Male ESCs, Interacts with OCT4, and the Expression of Many Pluripotent Genes Rely on the BRD4 Protein Partner P-TEFb(A) Western blot analysis of BRD4 (top) and OCT4 (middle) in WT female and male ESCs whole cell extracts (WCEs) on differentiation d0, d4, and d8. Histone 3 (H3) (bottom) is used as a load control for protein expression.(B) HEK cells were co-transfected (Tf) with full-length Myc-tagged BRD4 and Flag-tagged OCT4. WCEs were immunoprecipitated (IP) with anti-Flag antibodies and western blot analysis with anti-Myc antibodies. The arrow denotes BRD4 binding OCT4 in the upper panel. The lower shows Flag-OCT4 expression detected by western blot.(C) Co-IP of BRD4 and OCT4 in ESCs. Immunoprecipitation with anti-OCT4 or control antibodies to test interaction with endogenous BRD4 in d0 female ESCs. Arrow marks BRD4 detected by anti-BRD4 western.(D) Reciprocal immunoprecipitation using anti-BRD4 or control antibodies to test interaction with OCT4 in female ESCs. Arrow marks OCT4 detected by anti-OCT4 western.(E) Endogenous BRD4 co-immunoprecipitates with endogenous OCT4 in d0 male ESCs. Arrow marks BRD4 following western with anti-BRD4.(F) Reciprocal immunoprecipitation using anti-BRD4 or control antibodies to test binding to OCT4 in male ESCs. Arrow marks OCT4 following anti-OCT4 western.
Mentions: 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., 2007). 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).

Bottom Line: OCT4 directly binds Xite and Tsix, which encode two long noncoding RNAs (lncRNAs) that suppress the silencer lncRNA, Xist.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.

View Article: PubMed Central - PubMed

Affiliation: Burke Medical Research Institute, 785 Mamaroneck Avenue, White Plains, NY 10605, USA; Departments of Neuroscience and Cell and Developmental Biology, Weill Cornell Medical College, New York, NY 10065, USA.

Show MeSH
Related in: MedlinePlus