GSE63314
Stably-paused genes revealed through inhibition of transcription initiation by the TFIIH inhibitor Triptolide
Transcription by RNA Polymerase II (Pol II) in metazoan is regulated in several steps, including  preinitiation complex (PIC) formation, initiation, Pol II escape, productive elongation, cotranscriptional  RNA-processing and termination. Genome-wide studies have demonstrated that  the phenomenon of promoter-bound Pol II pausing is widespread, especially for genes involved  in developmental and stimulus-responsive pathways. However, a mechanistic understanding of  the paused Pol II states at promoters is limited. For example, at a global level, it's unclear to  what extent the engaged paused Pol II is stably tethered to the promoter or undergoes rapid  cycles of initiation and termination. Here we used the small molecule Triptolide (TPL), an  XPB/TFIIH inhibitor, to block transcriptional initiation followed by measuring Pol II occupancy  by ChIP-seq. This inhibition of initiation enables us to investigate different states of paused Pol  II. Specifically, our global analysis reveals that most genes with paused Pol II, as defined by  pausing index, show significant clearance of Pol II during the period of TPL treatment. Our  study further identifies a group of genes with unexpectedly stably-paused Pol II, with unchanged  Pol II occupancy even after one hour of inhibition of initiation. This group of genes constitutes a  small portion of all paused genes defined by the conventional criterion of pausing index. These  findings could pave the way for evaluating the contribution of different elongation/pausing  factors on different states of Pol II pausing in developmental and other stimulus-responsive  pathways. Overall design: ChIP-Seq of total/Ser5P Pol II in HCT116 cells treated with DMSO or TPL in serum starvation/activation or normal conditions. Nascent RNA-seq in HCT116 cells treated with DMSO or TPL in starved condition.