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. Author manuscript; available in PMC: 2014 Mar 7.
Published in final edited form as: Mol Cell. 2013 Jan 11;49(5):843–857. doi: 10.1016/j.molcel.2012.12.006

Figure 7. Inhibition of CK2-Mediated NPS Phosphorylation Reduces Brd4 Chromatin Occupancy and Target Gene Transcription in Ker-CT Cells.

Figure 7

(A and B) Reduction of p21, HDM2, PUMA, and 14-3-3σ RNA (A) and Brd4 occupancy at p53-binding sites (B) in Ker-CT treated with TBB (vs. DMSO).

(C) IB of phosphorylated Brd4, S392-phosphorylated p53 (following IP with α-p53 DO-1 antibody), and total Brd4, CK2α, and p53 protein levels in Ker-CT treated with DMSO (-) or TBB (+).

(D) CK2-phosphorylated p53, but not p300-acetylated p53, fails to interact with Brd4. Purified p53 was mock-treated or modified by p300 acetylation or CK2 phosphorylation prior to incubation with Brd4, followed by IP with α-Brd4 and IB with various α-p53 antibodies (lanes 1-3). The specificity of antibodies against acetyl-K373/382 and phosphorylated S392 of p53, prepared from untreated (-) or p300- or CK2-modified p53 (+), was shown on the right (lanes 4-7).

(E) Reduction of c-Fos, c-Myc, CCND1, PIM2, and DCPS, but not RPL13A, RNA levels in TBB-treated Ker-CT (vs. DMSO treatment).

(F) ChIP of endogenous Brd4 occupancy at c-Fos and c-Myc genes in DMSO- and TBB-treated Ker-CT. Error bars, SD (n=2-6) in (A,B,E,F).

(G) Model for CK2-mediated phosphorylation of Brd4 leading to intramolecular contact switch, allowing BD2/BD1 binding to acetylated (Ac) chromatin and recruitment of p53 for gene-specific targeting and activation of transcription.