Volume 55, Issue 1, Pages 97-110 (July 2014) Chromatin Reader Brd4 Functions in Ig Class Switching as a Repair Complex Adaptor of Nonhomologous End-Joining  Andre Stanlie, Ashraf S. Yousif, Hideo Akiyama, Tasuku Honjo, Nasim A. Begum  Molecular Cell  Volume 55, Issue 1, Pages 97-110 (July 2014) DOI: 10.1016/j.molcel.2014.05.018 Copyright © 2014 Elsevier Inc. Terms and Conditions

Molecular Cell 2014 55, 97-110DOI: (10.1016/j.molcel.2014.05.018) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 Brd4 Is Required for CSR (A) Representative FACS profiles of IgA switching in CH12F3-2A cells. Cont., control. (B) CSR complementation assay using siRNA-resistant hBrd4. (C) Confirmation of knockdown efficiency of endogenous mouse Brd4 by immunoblot. (D) Effect of various concentrations of JQ1 on IgA switching, IgM expression, and viability. (E–G) CSR assay, percent live cells, and live cell number are summarized from three independent experiments. (H) Immunoblot specifically confirms the knockdown of Brd4. (I) Quantitative RT-PCR (qRT-PCR) of Brd4 transcript, μGLT, and αGLT from the indicated samples. The values are the mean of three independent experiments, and the error bars represent the SD. (J and K) Top: representative FACS profiles of effect of JQ1 on IgG1 or IgG3 switching in spleen B cells 48 hr after stimulation. The number in each FACS profile indicates the percent (%) IgG1+/3+ B220+ cells. Bottom view shows the compilation of IgG1 and IgG3 CSR assays from three independent experiments. See also Figures S1 and S7 and Tables S1 and S6. Molecular Cell 2014 55, 97-110DOI: (10.1016/j.molcel.2014.05.018) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 AID-Induced DNA Break Formation Is Independent of Brd4 (A) Top: schematic diagram of the position of the ChIP assay PCR products. Bottom view shows a Brd4 and (pan)H4Ac ChIP assay derived from the indicated samples. Background values from controls with no antibody were subtracted. (B) DNA DSB assay as determined by γH2AX ChIP was derived from the indicated samples. (C) Top: schematic diagram of the position of the ChIP assay PCR products specific to V region and MYC. Bottom view shows Brd4 status in the V region and MYC as determined by ChIP assay derived from the indicated samples. (D) Mutation frequency (freq.) of the V region and MYC after 24 hr of JP8Bdel activation. (E) Brd4 status in the V region, MYC, TUBA1B, and SH3KBP1 as determined by ChIP assay derived from the indicated samples. Inset shows qRT-PCR analysis of the transcripts as indicated. ChIP data were normalized to the input DNA signals, and the maximum value in each data set was set as 100%. In all data sets, SD values were derived from three independent experiments. See also Figure S2 and Tables S2–S5, and S6. Molecular Cell 2014 55, 97-110DOI: (10.1016/j.molcel.2014.05.018) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 Brd4 Plays a Role in the Recombination Phase of CSR (A) Analysis of Sμ-Sα recombination junctions from CIT-stimulated CH12F3-2A cells derived from control (n = 81), siBrd4 (n = 60), and JQ1-treated samples (n = 47). Sequences with small insertions (1–5 bp) at the junction were scored as zero microhomology. (B) Pie chart of short (0–3 bp) versus long (>3 bp) microhomology of Sμ-Sα junction derived from control, Brd4 knockdown, and JQ1-treated cells. The data were compiled from (A). Ave., average. (C) Top: PCR amplification scheme of Igh/c-myc chromosomal translocations. Triangles represent the positions of primers used to amplify the rearranged regions. Probes used in the Southern blot hybridization are shown as horizontal black and white bars. Bottom view shows representative agarose gels following hybridization with either myc- or Igh-specific probe. See also Figure S6 and Table S6. Molecular Cell 2014 55, 97-110DOI: (10.1016/j.molcel.2014.05.018) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 53BP1-Dependent NHEJ Relies on Brd4 (A) Top: schematic diagram of the I-SceI-induced NHEJ repair substrate. Triangles indicate the positions of primers used to amplify the cut and repaired fragment. Bottom view is a summary of EGFP-positive cell analysis by FACS 48 hr after cotransfection of I-SceI expression plasmids and siBrd4 into H1299dA3-1 cells. Rel. GFP, relative GFP. (B) Analysis of C-NHEJ by PCR of the genomic DNA isolated from the indicated samples. (C) Confirmation of Brd4 knockdown efficiency. IB, immunoblot. (D) qRT-PCR analysis of I-SceI transcript derived from the indicated samples. Rel. to GAPDH, relative to GAPDH; ND, not detected. (E) DNA DSB assay was determined by γH2AX ChIP from the indicated samples. (F) Brd4 and 53BP1 ChIP assay in H1299dA3-1 cells as indicated. (G and H) IR (γ-ray) sensitivity assay as determined by cell viability (G) and propidium iodide staining (H) normalized to the untreated cells from the same knockdown samples. In all data, SD values were derived from three independent experiments. ChIP data were normalized as stated in Figure 2. Rel., relative. See also Tables S5 and S6. Molecular Cell 2014 55, 97-110DOI: (10.1016/j.molcel.2014.05.018) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 53BP1 and UNG Regulation through Brd4 on the S Region Chromatin (A) Schematic diagram of the position of the ChIP assay PCR products. (B–F) 53BP1 (B), Msh2 (C), Mlh1 (D), Rad51 (E), and UNG (F) ChIP assays derived from the indicated samples under CIT-stimulated condition. (G and H) Brd4 ChIP assay derived from wild-type, UNG−/− (G), or 53BP1−/− (H) spleen B cells stimulated by LPS and IL-4. ChIP data were normalized as in Figure 2. See also Figures S3 and S4 and Table S6. Molecular Cell 2014 55, 97-110DOI: (10.1016/j.molcel.2014.05.018) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 Brd4 Tethers Repair Components to the Chromatin Marked by H4Ac and γH2AX (A–H) Sequential ChIP analysis in CIT-stimulated CH12F3-2A cells. Eluted DNA obtained from the first round of ChIP is subjected to either IgG control or the indicated second antibody. The values were calculated as in Figure 2. (I) coIP analysis in irradiated 293T cells transfected with Flag-Brd4 and GFP-UNG. The antibodies used for IPs or IB (immunoblot) are indicated. See also Table S6. Molecular Cell 2014 55, 97-110DOI: (10.1016/j.molcel.2014.05.018) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 7 Domain Analysis of Brd4 and Its Role in CSR (A) Schematic representation of the Brd4 constructs used for coIP and CSR complementation assay. (B) coIP analysis from irradiated 293T cells transfected with the indicated Brd4 construct. The IP was done using anti-Flag antibody and probed with the indicated antibodies. (C) Representative FACS profiles of CSR complementation assay by cotransfection of siBrd4 and the indicated hBrd4 construct. See also Figure S5 and Table S6. Molecular Cell 2014 55, 97-110DOI: (10.1016/j.molcel.2014.05.018) Copyright © 2014 Elsevier Inc. Terms and Conditions