GSE26658
Total RNA Pol2 ChIP-seq in mES cells treated with flavopiridol (Cdk9 inhibitor) after DSIF and NELF knockdown
Recent studies have found that promoter-proximal pausing occurs at most Pol2-regulated genes.  DSIF and NELF function as negative elongation factors and promote Pol2 pausing.  P-TEFb, whose enzymatic activity lies in the kinase Cdk9, positively regulates the transition into productive elongation by phosphorylating subunits in DSIF, NELF and Pol2.  To gain insights into the interplay between these factors in regulating transcriptional pause release, we tested if knockdown of either pausing factor could bypass P-TEFb function.  We find that P-TEFb function is still required for transcriptional elongation after DSIF or NELF knockdown. Overall design: mES cells were infected with a shRNA hairpin to knockdown Spt5, Spt4, NelfE or control.  The resulting cells were then treated with flavopiridol (1uM for 60 minutes).  DNA was enriched by chromatin immunoprecipitation (ChIP) and analyzed by Solexa sequencing. A sample of whole cell extract was sequenced and used as the background to determine enrichment.  ChIP was performed using an antibody against total RNA Pol2 (Rpb1 N-terminus, Santa Cruz sc-899).