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. 2021 Jan 4;131(1):e133090. doi: 10.1172/JCI133090

Figure 4. BRD3 acts as a cofactor to maintain BCL6 autoregulation.

Figure 4

(A) Knockdown of BRD2 or BRD3 upregulated BCL6 expression. A549 cells transfected with 20 nM siRNAs targeting BRDs were collected 48 hours after transfection. BRD2, BRD3, BRD4, and BCL6 expression were detected by immunoblot analysis. (B) BRD4-specific inhibitors did not affect BCL6 expression. A549 and H292 cells were treated with BETi (OTX015 and JQ1), BRD4-specific inhibitors (DC-1 and DC-2), or a negative control compound (DC-control) for 6 hours. (C and D) Endogenous interaction of BCL6 and BRD3. A549 cell lysates were subjected to immunoprecipitation experiments using an anti-BCL6 or anti-BRD3 antibody. Immunoprecipitates were analyzed with antibodies against BRD2, BRD3, BRD4, and BCL6. Ten percent of total lysates was used as the input control. The anti-IgG antibody was used as a negative control. (E) Knockdown of BRD3 increased BCL6 mRNA levels. Data represent the mean ± SEM of biological triplicates. P values were analyzed by comparing siBRD3 groups with the control group. *P < 0.05 and ***P < 0.001, by unpaired, 2-tailed Student’s t test. (F) ChIP and re-ChIP were performed to test the cobinding of the BCL6 and BRD3 complex at the loci on the BCL6 promoter regions mentioned in Figure 3A. The first ChIP was performed using anti-BCL6 and anti-IgG antibodies. The second ChIP was performed using anti-BRD3 and anti-IgG antibodies to analyze the first ChIP (anti-BCL6) components. Data represent the mean ± SEM of biological triplicates. P values were analyzed by Student’s t test analysis for the first and second ChIPs. (G) BRD3 binding at the 3 indicated regions analyzed by ChIP-qPCR. Data represent the mean ± SEM of 3 independent experiments. P values were determined by unpaired, 2-tailed Student’s t test. (H) BRD3 maintained the BCL6 autoregulatory circuit. A549 cells were cotransfected with P1-Fluc, together with an equivalent amount of pcDNA3.1-BRD3, pcDNA3.1-BCL6, and/or pcDNA3.1-Ctrl. Cells were harvested for a luciferase assay 48 hours after transfection. Western blotting was subsequently conducted using specific antibodies against BCL6, BRD3, and GAPDH. Data represent the mean ± SD of biological triplicates. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA. Immunoblots in A, C, and D were contemporaneous and run in parallel from the same biological replicate.