Supplementary Figure 4: BRD4 knockdown and reconstitution system.
From: BRD4 assists elongation of both coding and enhancer RNAs by interacting with acetylated histones
a, Diagram of YFP-BRD4, YFP-BRD4-mBD, and their short forms. These BRD4 constructs contain nucleotide substitutions around the target site of BRD4 shRNA (marked by an asterisk) so that they are resistant to BRD4 shRNA while keeping the amino-acid coding unchanged. b, Transient transcription reporter assays using HIV-LTR-Luc and minP-Luc (minimal promoter Luc) luciferase reporters. HIV-LTR-Luc is sensitive to P-TEFb activity, while minP-Luc is not. Of note, low levels of P-TEFb-independent luciferase activities were observed both with BRD4 and BRD4short on minP-Luc, which were comparable with those observed with BRD4short on HIV-LTR-Luc. c, Work flow to establish cells stably expressing YFP-BRD4 or YFP-BRD4-mBD (or their short forms) in place of endogenous BRD4. NIH3T3 cells were sequentially infected with shRNA retrovirus and YFP-BRD4 expressing retrovirus as shown. The transduced cells were selected with G418 and puromycin. d, Left panel: immunoblot with anti-BRD4 antibody. The positions of YFP-tagged BRD4 proteins and endogenous BRD4 are shown. Right panel: immunoblot with anti-BRD4 antibody for endogenous BRD4, and with anti-YFP (GFP) antibody for YFP-BRD4short proteins. e, Microarray gene expression analysis of the recovery of BRD4-dependent genes after BRD4 shRNA-mediated knockdown. Paired differences in the recovery ratios (RRs) of 410 BRD4 target genes are binned and plotted as in Fig.3g. In the right, BRD4 target genes are classified by gene ontology. In parenthesis shown are the numbers of genes in each group and P values of paired t test on the RR compared between the YFP-BRD4 and YFP-BRD4-mBD reconstituted cells, all of which are statistically significant. Bars represent means ± s.e.m. of the RRs.
