BRD2 promotes antibody class switch recombination by facilitating DNA repair in collaboration with NIPBL

Abstract Efficient repair of DNA double-strand breaks in the Ig heavy chain gene locus is crucial for B-cell antibody class switch recombination (CSR). The regulatory dynamics of the repair pathway direct CSR preferentially through nonhomologous end joining (NHEJ) over alternative end joining (AEJ). Here, we demonstrate that the histone acetyl reader BRD2 suppresses AEJ and aberrant recombination as well as random genomic sequence capture at the CSR junctions. BRD2 deficiency impairs switch (S) region synapse, optimal DNA damage response (DDR), and increases DNA break end resection. Unlike BRD4, a similar bromodomain protein involved in NHEJ and CSR, BRD2 loss does not elevate RPA phosphorylation and R-loop formation in the S region. As BRD2 stabilizes the cohesion loader protein NIPBL in the S regions, the loss of BRD2 or NIPBL shows comparable deregulation of S-S synapsis, DDR, and DNA repair pathway choice during CSR. This finding extends beyond CSR, as NIPBL and BRD4 have been linked to Cornelia de Lange syndrome, a developmental disorder exhibiting defective NHEJ and Ig isotype switching. The interplay between these proteins sheds light on the intricate mechanisms governing DNA repair and immune system functionality.

Supplementary Figure S3.The sequence of Brd2 expression construct used in CSR complementation assay.The wild-type mouse BRD2 expression construct used in the CSR complementation assay (Fig. 1G) illustrates the arrangement of nucleotides encoding various functional domains and elements.EGFP was fused at the N-terminus, and Myc-FLAG (shaded in pink) was tagged at the C-terminus.The sequences colored in red have been mutated to prevent the degradation of the transcribed mRNA by siBrd2 and denoted as Brd2 R .The BD1 and BD2 domains (shaded in light blue) and the ET domain (shaded in light purple) are highlighted within the sequence.The nuclear localization signal (NLS) motif is positioned between the BD2 and ET domains.All the constructs used have an intact NLS (Fig. 1G and Fig. 5B)                        Forward Reverse Forward Reverse

5'-TTCTGTGGAGGACTGCAGCATTTG 5'-AGTACTGTCTCACCAATCCACTGAT Hprt [ NM_013556]
Forward Reverse 5'-CTCGAAGTGTTGGATACAGG 5'-TGGCCTATAGGCTCATAGTG . BRD2 depletion does not affect RNA Pol II (RNAPII) distribution in the recombining S regions during CSR.(A) Left: Schematic representation of the IgH locus with the positions of the ChIP-qPCR amplicons numbered 1-8 (small bars).Right: The timeline of siRNA transfection, CSR induction, and cell harvest is the same as Fig.1 A-C.(B-D) The ChIP results of RNAPII (B), RNAPII S2P (C), and RNAPII S5P (D); the plot on the right of each panel displays the cumulative profile of respective RNAPII on Sµ and Sa.All graphs show mean values ± SD.The statistical significance was calculated using a two-tailed Student's t-test of unequal variance (ns, non-significant).
. Impaired NHEJ-mediated repair of the I-SceI-induced DSBs upon the absence of BRD2.(A) Sequence analysis of I-SceI-induced Double-Strand Break (DSB) end-joining events in BRD2 depleted NHEJ reporter cell line.Genomic DNA was isolated from cells treated with siControl and siBrd2, followed by PCR amplification of the repaired junctions using primers flanking the two DSB sites.The two recognition sequences for I-SceI are 18 nucleotides long, colored in green and cyan, and bolded.The I-SceI cutting site is indicated by a bar, and microhomologies (MH) are in red.The nucleotide insertions (In) are black, and long deletions (D) are purple.(B) Pie charts comparing the junction patterns characterized by zero to short (0-3 nt) MH and longer (≥4 nt) MH along with nucleotide insertions.The number of junctions analyzed is indicated in the center of each chart.(C) The percentage of repaired junctions with the indicated length of MH or insertions was calculated and grouped under C-NHEJ or A-EJ.Statistical significance was calculated with a two-tailed Fisher's exact test (*p ≤ 0.05, ****p ≤ 0.0001).For this study, pooled genomic DNA from three experiments was used.
Depletion of BRD2 does not impede DNA repair by homologous recombination (HR).(A) Schematic representation of the HR assay utilizing DR-GFP reporter.(B) CH12F3-2A cells, with stably integrated DR-GFP were transfected with indicated siRNAs along with I-SceI (+) or empty vector (-).After 24 hours of transfection, GFP-positive cells were quantified by flow cytometry, and a representative result from four experiments is shown.(C) Top: Summarized result of GFP-positive cells from four independent HR assays.The error bars represent the standard deviation from the mean (**p ≤ 0.01).Bottom: Representative results of immunoblots of whole cell extracts from the transfected cells with control siRNA or siRNA targeting Brd2. .BRD2 depletion affects KU80, RAD51, and EXO1 occupancy at I-SceIinduced DSB sites.(A) The schematic of the NHEJ reporter construct displays two I-SceI-induced DSB sites (DSB-1 and DSB-2).Repair of these DSBs results in the loss of the intervening Thymidine Kinase (TK) gene and subsequent EGFP expression.The red bars adjacent to each DSB site indicate the positions of PCR amplicons analyzed in the ChIP assay (B-D).Analysis of DNA damage response and resection-associated proteins in the NHEJ reporter cell line transfected with siRNA Control or siBrd2, both in the presence and absence of I-SceI expression (B-E).(B-D) ChIP analyses show the occupancy of indicated proteins around DSB1 and DSB2 sites.Statistical significance was determined using a two-tailed Student's t-test of unequal variance (n=3 ±SD; *p ≤ 0.05; ** p ≤0.01; ***p ≤0.001; ns, non-significant).(E) A representative Western blot analysis demonstrates BRD2 depletion and expression of the proteins analyzed by ChIP (B-E).
Knockdown of Nipbl did not perturb RNA Pol II (RNAPII) distribution in the S regions.(A) Left: Schematic representation of the IgH locus highlighting Sµ and Sa.The small bars below the diagram indicate the positions of the ChIP-qPCR amplicons, numbered 1-8.Right: The assay timeline is the same as in Figure 5C-D.(B) RT-qPCR result shows the Nipbl knockdown efficiency and the status of µGLT and aGLT under identical conditions.(C-E) ChIP analysis of RNAPII (C), RNAPII S2P (D), and RNAPII S5P (E) occupancy.The plot on the right of each panel displays the cumulative profile of respective RNAPII surrounding Sµ and Sa.(B-E) The data represents mean ± SD (n= 3-4).Statistical significance was determined by a two-tailed Student's t-test of unequal variance.*p≤0.05;***p≤0.001;ns, non-significant.Knockdown of Brd4 but not Brd2 or Nipbl elevates S region R-loop.The R-loop or the DNA: RNA hybrid immunoprecipitation (DRIP) was performed using the S9.6 antibody in CH12F3-2A cells.(A) Schematic representation of IgH locus centering Sµ and Sa.The small bars below the diagram, denoted by a-f, show the positions of qPCR amplicons.(B) DRIP assay was conducted using CH12F3-2A cells transfected with indicated siRNAs and activated for CSR, as in Fig.6A-H.All genomic DNA samples were prepared with RNase A treatment, and each sample was divided into two halves for RNaseH (+) and without RNaseH (-) treatments.Both groups were then subjected to IP using the S9.6 antibody, and the background signals from the control IgG antibody were subtracted.To calculate the DRIP percentage, the values were normalized to the total input DNA Data show means ± SD (n=3).Statistical analysis was performed by Student's t-test (* p ≤ 0.05; ** p ≤0.01; ns, non-significant) and two-way ANOVA (**** p ≤ 0.0001).
. BRD2 prevents DSB-end resection in the S region during CSR.(A) Occupancy of the end resection components in CSR-activated CH12F3-2A cells transfected with siControl or siBrd2.A representative immunoblot using whole cell extracts of the transfected cells shows Brd2 knockdown efficiency.ChIP assays were performed using indicated antibodies; bar graphs represent the ChIP qPCR result of CtIP, EXO1, and BLM occupancy in the recombining S regions.(B) An illustration of quantitative DNA end resection assay.If the DSB is resected, the ssDNA will not be cut by the chosen restriction enzyme, and therefore, PCR products will be produced (right panel).On the other hand, if there is no DSB end processing, the RES will be intact and cut by the enzyme, resulting in no PCR product (left panel).RES, Restriction Endonuclease Site (yellow bar); dotted line, DNA cleavage by RE; ds, double strand; ss, single strand; arrows, primer positions; black crescent, indicates an exonucleolytic processing of the break-end.(C) Top: Schematic representation of the recombining S regions in CH12F3-2A cells where AIDinduced DSB occurs at Sµ and Sa (Chr 12).The Cd3e loci on Chr 9 is used as no DSB negative control.The positions of qPCR amplicons, each harboring a PvuII site (dotted line), are denoted by p1-p5 with small bars above.Bottom: Assessment of DSB end resection in CH12F3-2A cells transfected with siControl or siBrd2.Genomic DNA isolated from siRNA-treated and CITstimulated cells was digested or mock-digested with PvuII.DNA end resection adjacent to core Sm and Sa DSB (5'Sµ and 3'Sa) and the unrelated no DSB site (Cd3e) were measured by qPCR as described in the 'Materials and Methods' section.(D) Assessment of S region DSB end resection in Shld2 and Brd2 and Shld2 double knockdown in CH12F3-2A cells.In the bottom panel, the knockdown efficiency of Brd2 and Shld2 in the transfected cells was evaluated by immunoblotting whole cell lysate (left) and qRT-PCR of the total RNA (right), respectively.(A-D) Error bars represent the standard deviation from the mean (n=3; ± SD).Statistical significance was measured by a two-tailed Student's t-test of unequal variance (*p≤0.05;**p≤0.01;***p≤0.001;ns, non-significant).
-Sa recombination junctions from CH12F3-2A cells transfected with siControl.The bar (|) indicates a direct or blunt Sµ-Sa junction, the boldface shows microhomology or nucleotide overlap at the junction, and the underline indicates nucleotide insertion.Control (n = 41) -Sa recombination junctions from CH12F3-2A cells transfected with siBrd2.The bar (|) indicates a direct or blunt Sµ-Sa junction, the boldface shows microhomology or nucleotide overlap at the junction, and the underline indicates nucleotide insertion.-Sa recombination junctions from CH12F3-2A cells transfected with siNipbl.The bar (|) indicates a direct or blunt Sµ-Sa junction, the boldface shows microhomology or nucleotide overlap at the junction, and the underline indicates nucleotide insertion.

Table S1 .
List of antibodies

Table S2 .
List of primers and siRNAs

Supplementary Table S4. MiQE Checklist for RT-qPCR
MIQE checklist for authors, reviewers and editors.All essential information (E) must be submitted with the manuscript.Desirable information (D) should be submitted if available.If using primers obtained from RTPrimerDB, information on qPCR target, oligonucleotides, protocols and validation is available from that source.*: Assessing the absence of DNA using a no RT assay is essential when first extracting RNA.Once the sample has been validated as RDNA-free, inclusion of a no-RT control is desirable, but no longer essential.**: Disclosure of the probe sequence is highly desirable and strongly encouraged.However, since not all commercial pre-designed assay vendors provide this information, it cannot be an essential requirement.Use of such assays is advised against.

qPCR OLIGONUCLEOTIDES Supplementary Table S5. MiQE Checklist for qPCR related to ChIP, DRIP, and DNA end-resection assay
DNA was prepared following IP or without IP as may require.Name of kit and details of any modifications E ChIP assay: Active Motif ChIP Kit & following the instruction.DNA extraction by Pheno l: Chlorofom.Sample detection was within the values in the standard curve.Cq variation at lower limit E All assays were performed within the linear range of the standard curve.Confidence intervals throughout range D Evidence for limit of detection E All assays were performed within the linear range of the standard curve.If multiplex, efficiency and LOD of each assay.MIQE checklist for authors, reviewers and editors.All essential information (E) must be submitted with the manuscript.Desirable information (D) should be submitted if available.If using primers obtained from RTPrimerDB, information on qPCR target, oligonucleotides, protocols and validation is available from that source.*: Assessing the absence of DNA using a no RT assay is essential when first extracting RNA.Once the sample has been validated as RDNA-free, inclusion of a no-RT control is desirable, but no longer essential.**: Disclosure of the probe sequence is highly desirable and strongly encouraged.However, since not all commercial pre-designed assay vendors provide this information, it cannot be an essential requirement.Use of such assays is advised against.