Unmasking a hidden code during transcription: A practical strategy to reveal Thr4 phosphorylation in RNA polymerase II.

RNA Polymerase II (Pol II) is core to cell survival as the responsible enzyme for transcription of mRNA as well as various non-coding RNA species. The post-translational modification (PTM) of the C-terminal domain (CTD) of its largest subunit RPB1, principally through phosphorylation, enables docking of relevant proteins to govern the transcription cycle and RNA processing. The detection of phosphorylated Thr4 of the CTD heptad motif is exacerbated by epitope masking due to phosphorylations on nearby residues, thereby decreasing antibody recognition of the target epitope. Here, we introduced ChIP-phos-seq, wherein we conducted chromatin immunoprecipitation with phosphatase treatment followed by sequencing. This method addressed the overlooked phosphorylated Thr4 by removing masking phosphomarks with the targeted, specific phosphatase, Ssu72. In order to perform ChIP-phos-seq, Ssu72 was purified and characterized for specificity and activity through mass spectrometry and kinetics assays. The phosphatase treatment was optimized and used as a tool to target the masked phosphorylated Thr4 epitopes recognized in an antibody pulldown. Sequencing the DNA bound to Pol II then allows the CTD Thr4 phosphorylation to be profiled and analyzed for its presence on different gene types, lengths, and ontologies. With a clearer view of phosphorylated Thr4, a better understanding of how Thr4 phosphorylation contributes to transcription regulation and transcript processing is achievable.