GSE67483
Pol II chromatin immunoprecipitation in S. cerevisiae
In Saccharomyces cerevisiae short non-coding RNA (ncRNA) generated by RNA Polymerase II (Pol II) are terminated by the NRD complex consisting of Nrd1, Nab3 and Sen1. We now show that Pcf11, a component of the cleavage and polyadenylation complex (CPAC), is generally required for NRD-dependent transcription termination through the action of its CTD interacting domain (CID). Pcf11 localizes downstream of Nrd1 on NRD terminators, and its recruitment depends on Nrd1. Furthermore mutation of the Pcf11 CID results in Nrd1 retention on chromatin, delayed degradation of ncRNA and restricts Pol II CTD Ser2 phosphorylation and Sen1-Pol II interaction. Finally, the pcf11-13 and sen1-1 mutant phenotypes are very similar as both accumulate RNA:DNA hybrids and display Pol II pausing downstream of NRD terminators. We predict a mechanism whereby Nrd1 and Pcf11 exchange on chromatin facilitates Pol II pausing and CTD Ser2-P phosphorylation. This in turn promotes Sen1 activity that is required for NRD-dependent transcription termination in vivo. Overall design: ChIP-seq with antibody against pol II in wild type and Pcf11 mutants: Pcf11-2, Pcf11-9 and Pcf11-13 grown at 25C and 37C along with input samples