Kinetics of RNA polymerase 2 recruitment and H3K36me3 deposition after stimulation of murine T-cells. Mus musculus

Activation of immune cells results in extremely rapid functional changes, yet it remains enigmatic how this is accomplished. By combining time-courses of RNA-polymerase-II (RNAPII) Chip-Seq, 4sU-Seq, RNA-Seq and ribosome profiling during Th1 activation we provide a genome-wide view on temporal dynamics for ~10.000 genes. Gene responses vary in time and magnitude and allow the identification of unknown immediate early (IEG) and posttranscriptionally regulated genes. However, >90% are regulated transcriptionally with coupled changes in translation. Surprisingly, not poised, but rather re-positioned RNAPII correlates with transcriptional changes and coincides with fluctuations in cotranscriptional splicing, which lags behind at first. Unspliced transcripts remain here at the chromatin, but are already poly-adenylated. Later splicing readjusts, correlating with progressive Ser-2 phosphorylation of the CTD domain of RNAPII and activation of the elongation-complex pTEFb. Thus, rapid re-positioning of RNAPII and regulating the phosphorylation state of its CTD dictates almost all events during immune responses, including post-transcriptional processes. Overall design: ChIP-Seq of total RNA-Pol2 and H3K36me3 after 30min and 2h of T-cell stimulation compared to non-restimulated (NR) T-cells