Bromodomain dependence of BRD4-dependent gene expression in mouse fibroblasts. Mus musculus

To study the role of the bromodomain (BD) in BRD4-dependent gene expression, we compared the function of wild type BRD4 and a mutant BRD4-mBD, which carries a point mutation in each of the two BDs. We first knocked down endogenous BRD4 by shRNA (shBRD4) and then stably reconstituted the cells with shBRD4-resistant YFP-BRD4 (wild type or mBD mutant). Following BRD4 reconstitution, the degree of recovery in gene expression was analyzed by Affymetrix Mouse Exon 1.0 ST array. Wild-type BRD4 restored gene expression significantly and far more efficiently than the mBD mutant. Overall design: NIH3T3 cells were sequentially infected with shRNA retrovirus (pSRneo-shCont or pSRneo-shBRD4) and YFP-BRD4 expressing retrovirus (pMSCVpuro-YFP, pMSCVpuro-YFP-BRD4, pMSCVpuro-YFP-BRD4-mBD). shBRD4 contains an oligonucleotide encoding an shRNA sequence against mouse BRD4 and shCont contains a scrambled oligonucleotide. The transduced cells were selected with G418 and puromycin. Two independent sets of cell lines (line B set and line D set) were produced from different laboratory stocks of NIH3T3 cells. Each set contained: YFP_shCONT, YFP_shBRD4, BRD4WT_shBRD4, and RD4mBD_shBRD4. The Affymetrix Mouse Exon 1.0 ST platform was used. No technical replicate was performed.