Bypass of blocking lesions by RNAPII reveals a novel stress induced by DNA damage [RNA-seq]

Ultraviolet (UV) irradiation or platinum-based drugs generate bulky DNA lesions that impede transcription elongation by RNA Polymerase II (RNAPII). This unscheduled transcription elongation block triggers a stress response, which includes initiating transcription-coupled DNA repair, removing and degrading the halted RNAPII, and a global transcriptional shutdown. However, the molecular and cellular consequences of RNAPII successfully bypassing these blocking lesions have remained unknown. Here, we have identified the acetyltransferase p300 as a regulator of the stress response when RNAPII stalls at blocking lesions. Indeed, p300 interacts with the halted RNAPII and controls its removal and degradation in a USP7-dependent mechanism. Notably, in p300-deficient cells, transcription persists despite DNA damage, as RNAPII bypasses blocking lesions, leading to mutant mRNAs and splicing variants accumulation. This persistent transcription activity is particularly detrimental post DNA damage as it results in toxic translation activity and higher DNA damage levels. These observations provide the first evidence that bypass of blocking lesions by RNAPII inhibits DNA repair as well as the cellular stress response. Our findings offer essential insights into the mechanisms of resistance to DNA damage and suggest strategies to enhance the efficacy of platinum-based drugs, such as cisplatin, in anti-tumor therapies. Overall design: To characterize the impact of p300 deficiency on transcription control post DNA damage, we determined the number of genes affected in WT and p300-deficient cells using RNAseq from Chromatin or polysome-enriched fractions RNAseq of Polysomes-enriched fractions (Poly), 5-EU labeled RNA purified from Polysome-enriched fractions (Poly_Purif), 5-EU labeled and purified RNA from DRB treated cells (DRB_5EU_Nonfrag) or total RNA samples (5EU) were collected and analyzed in HeLa WT and ?p300 (p300KO) cells at different time points after UV irradiation (20 J/m2). In some cases, the RNA was fragmented prior to the 5-EU purification step (DRB_5EU_Frag). RNAseq of 5-EU labeled RNA purified from Polysome-enriched fractions (Poly_Purif) were collected and analyzed in HeLa WT and ?p300 (p300KO) cells 2 hs after UV irradiation (20 J/m2) or treatment with actinomycin D (2 µg/ml)