Figure 7.

The P-TEFb-mediated Ser2 phosphorylation is dispensable for pause release. (A) WB analysis of the effect of ΔCTR-Spt5 on HMBA-induced Ser2p and pause release. The cell lysates (top) and LSEN (bottom) from 293T cells with indicated WT- or ΔCTR-Spt5-f transfection and HMBA treatment were analyzed for the levels of indicated proteins. (B) WB analysis of Ser2p, Ser5p and indicated proteins in cell lysates of HeLa cells with co-infection of shSpt5 and shCdk9 as indicated. (C) qRT-PCR analysis for the mRNA levels of representative genes in cells with indicated shRNA(s) co-infection as in Figure 1F. All values were expressed as Mean ± SD of three replicates. **P < 0.01; P-values were assessed using two-tailed Student's t-test. (D) Effect of depletion of Cdk9 and Spt5 on the enrichment of Pol II on the promoter region of tested genes. The DNAs ChIPed from HeLa cells co-infected with indicated shRNA(s) were analyzed by qPCR as in Figure 1E. All values were expressed as Mean ± SD of three replicates. **P < 0.01; P-values were assessed using two-tailed Student's t-test. (E) WB analysis of the effect of knockdown AFF4 on Ser2p and pause release. LSEN and cell lysates from HeLa cells with shRNA(s) infection and HMBA treatment were analyzed for the levels of indicated proteins. (F) ChIP-Seq analysis of the effect of AFF4 knockdown on HMBA-induced pause release as in Figure 1D. Diagrams display the proportion of pause-released genes in HCT116 cells with or without AFF4 knockdown under HMBA treatment. The criterion of Pol II pausing was defined as TR (the relative ratio of promoter read density/gene body read density) > 4.0, P-value < 0.005.