Brd4 modulate plasma cell differentiation through targeting BLIMP1
BLIMP1 (encoded by prdm1 ) is a critical transcription factor that controls the terminal differentiation of B cells into plasma cells (Angelin-Duclos, Cattoretti et al., 2000; Radbruch et al., 2006). Therefore, to investigate how Brd4 controls differentiation of B cells into plasma cells, we firstly determined the effect of Brd4 inhibition on BLIMP1 expression in active CD19+ B cells. Purified CD19+ B cells were incubated with anti-IgM, rCD40L, CpG ODN2006, rIL-10 and rIL-15 for 7 days. We observed that treatment with PFI-1 or JQ1 reduced the costimulation-induced expression of BLIMP1 (Fig. 4A). We further evaluated the in vivo effect of Brd4 deficiency on the expression of BLIMPT1 in CD19+ B cells from Brd4-CKO mice. Isolated splenic B cells were stimulated with LPS (10 μg/mL) and IL-4 (10 ng/mL) for 48 h. We found that the reduced expression of BLIMP1, but not prdm2, xbp-1 and CD69 in Brd4-CKO mice compared with WT mice (Fig. 4B). These data suggest that Brd4 controls the terminal differentiation of B cells into plasma cells by targeting BLIMP1.
Previous reports indicate that Brd4 can bind directly to transcription factors, therefore, to investigate how Brd4 modulates BLIMP1 expression, we determined whether Brd4 binds to and coordinately activates the BLIMP1 promoter. Because Brd4 has no regular binding site, we cloned the three BLIMP1 promoter regions (-2045→-3207, -845→-2087 and +333→-883 bp from TSS) into a pGL4 – vectors, respectively, and measured luciferase activity in human 293 cells. As shown in Fig 4C, we demonstrated that transfection with Brd4 siRNA reduced the promoter activity of BLIMP1 in regions of -845→-2087 bp (Blimp1-S2) and +333→-883 bp (Blimp1-S3), suggesting that Brd4 is a critical factor in activating BLIMP1 promoter activity. To address whether Brd4 directly targets the BLIMP1 promoter, ChIP-qPCR assays were performed with primers located in the promoter region of BLIMP1 using anti-Brd4 antibody in B220+ B cells from spleen of FL/FL mice. We first used JASPAR DATABASE to predict both Brd4 binding site (-853 ~-838 and -487 ~ -476) in BLIMP1 promoter (Fig. 4D). Then we demonstrated that Brd4 was significantly enriched in the BLIMP1 promoter region between -853 and -838 (Fig. 4E) in B220+ B cells from spleen of FL/FL mice, providingin vivo evidence that Brd4 directly binds and activates the endogenous BLIMP1 promoter.