FeaSion decodes the regulatory landscape and functional diversity of RNA polymerase II CTD phosphorylation

RNA polymerase II’s (RNAPII) C-terminal domain (CTD) contains five phosphorylation sites (pY1, pS2, pT4, pS5, and pS7). However, their regulatome and immediate functions remain elusive. Using the FeaSion (Feature-Screening-Function) strategy, we mapped RNAPII phosphorylation site-specific interactors and genomic occupancy, revealing links to preferential gene length, exon number, and transcription factor binding. CRISPR-FACS screens identified different candidate regulators modulating individual phosphorylation sites. Rapid replacement showed site-specific mutations influence different transcriptional processes, histone modifications (H3K36me3 and H2A.Zac), and preferentially affect developmental and signaling genes. Moreover, we demonstrate kinases CLK1/4 and YES1 directly regulate RNAPII transcription—via site-specific CTD phosphorylation—to control developmental, metabolic, and signal transduction programs. Our findings reveal an expanded regulatory network involving >100 kinase and phosphatases that potentially orchestrate CTD phosphorylation beyond their canonical functions, establishing a multilayered phospho-regulatory network with broad implications for gene expression control in development and disease.