Profiling RNA Polymerase II Phosphorylation Genome-Wide in Fission Yeast.
The RNA polymerase II carboxyl-terminal domain (CTD) consists of tandem repeats of consensus sequence Tyr 1 -Ser 2 -Pro 3 -Thr 4 -Ser 5 -Pro 6 -Ser 7 . Dynamic posttranslational modifications of the CTD generate a CTD code crucial for the cotranscriptional recruitment of factors that control transcription, chromatin modification, and RNA processing. Analysis of CTD phosphorylation by ChIP (Chromatin ImmunoPrecipitation) coupled with high-throughput DNA sequencing (ChIP-seq) is a powerful tool to investigate the changes in CTD phosphorylation during the transcription cycle. In this chapter, we describe a ChIP-seq protocol to profile the different CTD phospho-marks in fission yeast. Using this protocol, we have found that Tyr1P, Ser2P, and Thr4P signals are highest at gene 3' ends, whereas Ser5P is enriched across the gene bodies.
Kecman T, Heo DH, Vasiljeva L