Ubiquitylation of Pol II controls early stages of the transcription cycle [dxChIP-seq]
Control of RNA Polymerase II (Pol II) through ubiquitylation is essential for the DNA-damage response. Here we reveal a distinct ubiquitylation pathway in human cells that targets excessive and defective Pol II molecules at the initial stages of the transcription cycle. This pathway, mediated by ARMC5CUL3 ubiquitin ligase, drives homeostatic Pol II turnover, and is further enhanced when early stages of transcription - initiation and pausing - are perturbed. Upon ARMC5 loss, Pol II accumulates in the free pool and in the promoter-proximal zone, but is not permitted into elongation. We identify Integrator subunit 8 (INTS8) as a gatekeeper preventing the release of excess Pol II molecules into gene bodies. Combined loss of ARMC5 and INTS8 has strong detrimental effects on cell growth, with ARMC5 loss exacerbating transcriptional defects seen in INTS8-depleted cells. These findings uncover that ARMC5CUL3 and INTS8 form a collaborative checkpoint, monitoring the quantity and quality of transcription complexes before they are licensed into elongation. Overall design: We aimed to study how ARMC5 loss affects Pol II occupancy on chromatin and how is a partial resistance to XBP inhibition by triptolide achieved in ARMC5 KO cells in HEK293 Trex flp In background. We treated WT and ARMC5 KO cells with triptolide at 300 nM for 2 h. We then performed double cross-linking, first we incubated the washed cells with disuccinimidyl glutarate (DSG) for 15 minutes, followed by washing and incubation with formaldehyde. Immunoprecipitation was performed against total Pol II (D8L4Y Antibody) and Serine 5 phosphorylated Pol II (3E8 Antibody).