Phospho switch triggers Brd4 chromatin binding and activator recruitment for gene-specific targeting

Abstract

Bromodomain-containing protein 4 (Brd4) is an epigenetic reader and transcriptional regulator recently identified as a cancer therapeutic target for acute myeloid leukemia, multiple myeloma, and Burkitt's lymphoma. Although chromatin targeting is a crucial function of Brd4, there is little understanding of how bromodomains that bind acetylated histones are regulated, nor how the gene-specific activity of Brd4 is determined. Via interaction screen and domain mapping, we identified p53 as a functional partner of Brd4. Interestingly, Brd4 association with p53 is modulated by casein kinase II (CK2)-mediated phosphorylation of a conserved acidic region in Brd4 that selectively contacts either a juxtaposed bromodomain or an adjacent basic region to dictate the ability of Brd4 binding to chromatin and also the recruitment of p53 to regulated promoters. The unmasking of bromodomains and activator recruitment, concurrently triggered by the CK2 phospho switch, provide an intriguing mechanism for gene-specific targeting by a universal epigenetic reader.

Figure 1. Brd4 Interacts with Select Chromatin Modifiers and Transcription Factors

(A) Candidate screen of Brd4-interacting proteins by solution pulldown with purified protein (I, input) incubated with (+) or without (-) f:Brd4. IP with α-Brd4 antibody, followed by IB with anti-test protein antibody. (B) Brd4 interacts with p53 in 293 nuclear extract. (C) BID and PDID interact directly and independently with GST:p53, not GST. Proteins purified from bacteria or Sf9 as indicated. (D) p53 interacts with Brd4 FL, BID and PDID in vivo by BiFC live-cell imaging. Venus-N-p53 and Venus-C-Brd4 containing FL, PDID, BID, PDID-BID, or aa 149-284 were co-expressed in HCT116 p53 -/- cells and imaged by confocal microscopy. (E) f:PDID (purified from Sf9) and f:BID (purified from bacteria) interact strongly with REG (thick solid line) but weakly with DBD (thin dashed line) of p53. GST pulldown was performed by incubating f:PDID or f:BID with GST or each fusion. Bound f:PDID and f:BID detected by α-FLAG antibody with GST derivatives visualized by CBB staining. (F) p53 DNA-binding activity inhibited by BID but not PDID. EMSA performed by incubating f:p53, with or without f:PDID or f:BID, with ³²P-labeled DNA containing human p21 p53-binding site.

Figure 2. Phosphorylation-Induced Intramolecular Contact Switch in Brd4 Modulates PDID Interaction with p53 and Bromodomain Access to Acetylated Chromatin

(A) GST pulldown with f:PDID purified from Sf9 or bacteria (Bac) with (+) or without CIP or CK2 treatment. (B) GST pulldown with NPS- or CPS-containing fragment purified from Sf9 or Bac. (C) GST pulldown in 293 transfected with GST-tagged plasmid without (for endogenous p53 detection) or with 3f:BID co-expression (for 3f:BID detection). (D) GST pulldown with CK2-treated WT or mutant f:PDID purified from bacteria. Alanine-substituted S/T is indicated in red for mutants a, b, c, and d. (E) Model for p53 interaction with BID or PDID. (F) Drawing of protein fragments with domains retained (solid line) or deleted (dashed line). (G and H) GST pulldown with GST-fusions untreated or treated (+) with CK2 and ATP, prior to incubation with f:BD2 (G) or acetylated chromatin (H). (I) Model for phosphorylation-enhanced NPS-BD2 contact further inhibiting chromatin binding. (J) GST pulldown with GST:BD2 or GST:BID incubated with f:NPS (100 ng), with (+) or without (-) prior CK2 phosphorylation, in the absence (-) or presence (+) of f:BID or f:BD2 competitor (250 ng). (K) Model for unmasking BD2 binding to acetylated chromatin via NPS-BID contact. (L) GST pulldown with GST-fusions treated (+) or untreated (-) with CK2 prior to incubation with acetylated chromatin.

Figure 3. Phosphorylated Brd4 Binding to Acetylated Chromatin Regulated by Intramolecular Contact Switch from NPS-BD2 to NPS-BID

(A) Short form (S) of f:Brd4 purified from bacteria binds acetylated chromatin after CK2/ATP treatment. (B) Untreated (UNT) FL f:Brd4 purified from Sf9 loses chromatin-binding activity after dephosphorylation. (C) Unphosphorylated Brd4 present in nucleoplasm (100 mM salt elution) and phosphorylated Brd4 bound to chromatin eluted at different salt concentrations (200-600 mM) from 293 nuclear extract. (D) Drawing of Brd4 domain-specific deletion mutants. (E and F) Chromatin-binding performed with f:Brd4 proteins purified from Sf9 (E) or transiently expressed in 293 cells (F). (G) Drawing of alanine-substituted mutations (in red) in full-length Brd4. (H and I) Chromatin-binding performed with mutants constructed in FL f:Brd4 purified from Sf9 (H) or transiently expressed in 293 cells (I).

Figure 4. Brd4-Regulated p21 Gene Expression Is p53-Dependent

(A) Knockdown of Brd4 in 293 by two separate siRNAs reduces Brd4 protein (bottom) and RNA derived from p21 but not RPL13A (bar graph). (B) Brd4 co-occupies with p53 at -2.3 kb of p21 gene in 293 detected by ChIP with primer pairs as indicated. Two known p53-binding sites (p53BS) are marked. (C and D) Occupancy of Brd4 and p53 at p21 -2.3 kb in 293 is reduced upon knockdown of Brd4 (C) or p53 (D). Reduced p53 protein level in p53 knockdown is shown. (E and F) Co-occupancy of p53 and Brd4 at p21 -2.3 kb detectable in p53 +/+ but not p53 -/-HCT116 cells (E) is further enhanced upon Dox (0.5 μM, 6 hr) treatment (F). (G) Dox-induced p21 RNA level seen in HCT116 p53 +/+, not p53 -/-, cells diminished upon Brd4 knockdown. Error bars, SD (n=2-3) in (A-G).

Figure 5. Unmasking BD2 Binding to Chromatin and Recruitment of p53 Are Both Necessary for Brd4-Regulated p21 Gene Transcription

(A) ChIP of p21 gene occupancy by FL f:Brd4, ΔNPS or ΔBID transiently expressed in 293. FL occupancy at -2.3 kb specified as 1.0. (B) Measurement of p21 RNA level changes in 293 (bar graph) transiently expressing different f:Brd4 proteins (bottom). (C and D) ChIP of p21 -2.3 kb bound by FL or domain-deleted f:Brd4 transiently expressed in 293 (C) or by endogenous p53 (D). (E) Measurement of p21 RNA level changes in 293 (bar graph) transiently expressing WT or NPS serine mutants (bottom) constructed in FL f:Brd4. (F) ChIP of p21 -2.3 kb bound by WT or NPS serine mutants. (G and H) Occupancy of Pol II (G) and CycT1 (H) at -2.3 kb, TSS, and +1.0 kb of p21 gene in 293 transiently expressing f:Brd4 FL, domain deletion, or S492/494A mutant. (I) Measurement of p21 RNA level (top) and G0/G1 population (bottom) in Dox-treated 293 transiently expressing f:Brd4 or vector. Cleavage of PARP-1, monitored by IB, is shown. (J and K) Measurement of p21 RNA level (J) and G0/G1 population (K) in 293 transiently expressing different f:Brd4 proteins (J, bottom) with or without Dox treatment (1 μM, 8 hr). Error bars, SD (n=2-3) in (A-K).

Figure 6. Brd4-Regulated c-Myc and c-Fos Gene Transcription in 293 Cells Also Modulated by Intramolecular Contact Switch in Brd4

(A and B) ChIP of endogenous Brd4 occupancy on c-Myc (A) and c-Fos (B) genes. (C) Reduction of c-Fos and c-Myc RNA correlates with decreased Brd4 occupancy on c-Fos and c-Myc genes upon Brd4 knockdown. (D) ChIP of c-Fos gene bound by FL f:Brd4, ΔNPS or ΔBID transiently expressed in 293. FL occupancy at +1.5 kb specified as 1.0. (E) ChIP of c-Myc gene at five different regions bound by FL or domain-deleted f:Brd4 transiently expressed in 293. FL occupancy at +0.4 kb specified as 1.0. (F) Measurement of c-Fos and c-Myc RNA level changes in 293 transiently expressing different f:Brd4 proteins. Error bars, SD (n=2-3) in (A-F).

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

(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.