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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 Overexpression Induces while Brd4 Depletion Causes a Reduction in Pluripotent Gene Expression(A) Undifferentiated (d0) male ESCs were transfected with Myc-tagged BRD4 or Myc-only control and harvested at d2. Western blot analysis shows that the full-length Myc-BRD4 is expressed.(B) qRT-PCR of the c-Myc, Oct4, Nanog, Xist, and Tsix genes following the overexpression of myc-BRD4 versus myc-only empty vector.(C) Western blot analysis confirms knockdown in WT d0 male ESCs with scramble (Ctl) and Brd4 siRNA. RT-qPCR shows the expression of Brd4, Oct4, Nanog, Tsix, N-cadherin (N-cad), Nodal, and Gata6 genes.(D) Western blot confirms BRD4-knockdown in d6 WT male ESCs. RT-qPCR shows expression levels of the Brd4, Cyclin D1 (CycD1), cMyc, Tcl1, Oct4, Nanog, Xist, Tsix, and Xite genes.(E) Brd4 knockdown is confirmed in WT d6 female ESCs. Gene expression analysis (qRT-PCR) of the Brd4, Cyclin D1 (CycD1), cMyc, Tcl1, Oct4, Nanog, Xist, Tsix, and Xite genes.Graphs indicate three independent biological replicates. Error bars represent 1 SD from the mean.
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fig6: Brd4 Overexpression Induces while Brd4 Depletion Causes a Reduction in Pluripotent Gene Expression(A) Undifferentiated (d0) male ESCs were transfected with Myc-tagged BRD4 or Myc-only control and harvested at d2. Western blot analysis shows that the full-length Myc-BRD4 is expressed.(B) qRT-PCR of the c-Myc, Oct4, Nanog, Xist, and Tsix genes following the overexpression of myc-BRD4 versus myc-only empty vector.(C) Western blot analysis confirms knockdown in WT d0 male ESCs with scramble (Ctl) and Brd4 siRNA. RT-qPCR shows the expression of Brd4, Oct4, Nanog, Tsix, N-cadherin (N-cad), Nodal, and Gata6 genes.(D) Western blot confirms BRD4-knockdown in d6 WT male ESCs. RT-qPCR shows expression levels of the Brd4, Cyclin D1 (CycD1), cMyc, Tcl1, Oct4, Nanog, Xist, Tsix, and Xite genes.(E) Brd4 knockdown is confirmed in WT d6 female ESCs. Gene expression analysis (qRT-PCR) of the Brd4, Cyclin D1 (CycD1), cMyc, Tcl1, Oct4, Nanog, Xist, Tsix, and Xite genes.Graphs indicate three independent biological replicates. Error bars represent 1 SD from the mean.

Mentions: Approximately 50% of cellular P-TEFb is sequestered in an inactive complex consisting of HEXIM1 and 7sk snRNA (Yang et al., 2005). The active P-TEFb complex is associated with BRD4. Because inhibition of BRD4’s BET domain markedly reduced pluripotent mRNAs, we asked whether forced expression of BRD4 would alter P-TEFb’s stoichiometry and drive the expression of stem-ness genes. Full-length Myc-tagged BRD4 was transfected into d0 male ESCs. Whole-cell extracts (WCEs) and mRNA were harvested after 48 hr (d2). Western blot shows that the Myc-fused BRD4 protein (180 kDa) is overexpressed as compared with the Myc-only control (Figure 6A). Forced expression of BRD4 induced cMyc, Oct4, Nanog, Xist, and Tsix expression (Figure 6B). These results suggest that BRD4 induction can activate the pluripotent and XCI genes in ESCs.


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 Overexpression Induces while Brd4 Depletion Causes a Reduction in Pluripotent Gene Expression(A) Undifferentiated (d0) male ESCs were transfected with Myc-tagged BRD4 or Myc-only control and harvested at d2. Western blot analysis shows that the full-length Myc-BRD4 is expressed.(B) qRT-PCR of the c-Myc, Oct4, Nanog, Xist, and Tsix genes following the overexpression of myc-BRD4 versus myc-only empty vector.(C) Western blot analysis confirms knockdown in WT d0 male ESCs with scramble (Ctl) and Brd4 siRNA. RT-qPCR shows the expression of Brd4, Oct4, Nanog, Tsix, N-cadherin (N-cad), Nodal, and Gata6 genes.(D) Western blot confirms BRD4-knockdown in d6 WT male ESCs. RT-qPCR shows expression levels of the Brd4, Cyclin D1 (CycD1), cMyc, Tcl1, Oct4, Nanog, Xist, Tsix, and Xite genes.(E) Brd4 knockdown is confirmed in WT d6 female ESCs. Gene expression analysis (qRT-PCR) of the Brd4, Cyclin D1 (CycD1), cMyc, Tcl1, Oct4, Nanog, Xist, Tsix, and Xite genes.Graphs indicate three independent biological replicates. Error bars represent 1 SD from the mean.
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fig6: Brd4 Overexpression Induces while Brd4 Depletion Causes a Reduction in Pluripotent Gene Expression(A) Undifferentiated (d0) male ESCs were transfected with Myc-tagged BRD4 or Myc-only control and harvested at d2. Western blot analysis shows that the full-length Myc-BRD4 is expressed.(B) qRT-PCR of the c-Myc, Oct4, Nanog, Xist, and Tsix genes following the overexpression of myc-BRD4 versus myc-only empty vector.(C) Western blot analysis confirms knockdown in WT d0 male ESCs with scramble (Ctl) and Brd4 siRNA. RT-qPCR shows the expression of Brd4, Oct4, Nanog, Tsix, N-cadherin (N-cad), Nodal, and Gata6 genes.(D) Western blot confirms BRD4-knockdown in d6 WT male ESCs. RT-qPCR shows expression levels of the Brd4, Cyclin D1 (CycD1), cMyc, Tcl1, Oct4, Nanog, Xist, Tsix, and Xite genes.(E) Brd4 knockdown is confirmed in WT d6 female ESCs. Gene expression analysis (qRT-PCR) of the Brd4, Cyclin D1 (CycD1), cMyc, Tcl1, Oct4, Nanog, Xist, Tsix, and Xite genes.Graphs indicate three independent biological replicates. Error bars represent 1 SD from the mean.
Mentions: Approximately 50% of cellular P-TEFb is sequestered in an inactive complex consisting of HEXIM1 and 7sk snRNA (Yang et al., 2005). The active P-TEFb complex is associated with BRD4. Because inhibition of BRD4’s BET domain markedly reduced pluripotent mRNAs, we asked whether forced expression of BRD4 would alter P-TEFb’s stoichiometry and drive the expression of stem-ness genes. Full-length Myc-tagged BRD4 was transfected into d0 male ESCs. Whole-cell extracts (WCEs) and mRNA were harvested after 48 hr (d2). Western blot shows that the Myc-fused BRD4 protein (180 kDa) is overexpressed as compared with the Myc-only control (Figure 6A). Forced expression of BRD4 induced cMyc, Oct4, Nanog, Xist, and Tsix expression (Figure 6B). These results suggest that BRD4 induction can activate the pluripotent and XCI genes in ESCs.

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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Related in: MedlinePlus