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

Platinum-based compounds and ultraviolet (UV) irradiation produce bulky DNA lesions that stall RNA polymerase II (RNAPII), activating transcription-coupled nucleotide excision repair (TC-NER), RNAPII degradation, and global transcriptional shutdown. However, the consequences of RNAPII bypassing such lesions remain unclear. We identified the acetyltransferase p300 as a key regulator of TC-NER-dependent RNAPII removal from damaged chromatin via a USP7-dependent mechanism. Loss of p300 permits RNAPII to bypass transcription-blocking lesions, sustaining transcription and full-length mRNA production despite DNA damage. This leads to continued translation, endoplasmic reticulum (ER) stress, and activation of the unfolded protein response (UPR), compromising cell viability. Notably, this stress response resensitizes tumors resistant to platinum-based chemotherapy. Our findings reveal a vulnerability in tumor cells that evade transcriptional shutdown and define a synthetic lethal interaction between p300 inhibition and platinum-induced DNA damage, offering a targeted strategy to overcome chemoresistance. Overall design: Chromatin immunoprecipitation DNA sequencing (ChIP-seq) for RNA Polymerase II (RPB1) was performed in WT or P300 knockout (?p300) HeLa cells at 0 or 2 hours post-UV irradiation (20 J/m²). For RPB1ser2 ChIP-seq experiments using DRB (5,6-Dichloro-1-ß-d-ribofuranosylbenzimidazole), WT or ?p300 HeLa cells were treated with siRNA of CSB or a control pool from Dharmacon. 24 after the transfection, cells were treated with 100 µM DRB for 3 hours. Following this treatment, the cells were split into two groups: one group was exposed to UV irradiation (20 J/m²), while the other group (control) was not irradiated. Five minutes after UV exposure, DRB was removed from both groups to allow transcription to restart. Samples were collected immediately after DRB washout (0 minutes) and at 45 minutes post-UV irradiation for ChIP-seq analysis. For RPB1 ChIP-seq experiments using A485, WT HeLa cells were treated with either DMSO or 1 µM A485 1 hour before UV treatment (20 J/m²). For some groups, 1 µM of the USP7 inhibitor FT671 was added 1 hour before UV irradiation. Cells were harvested 2 hours post-UV irradiation. For the siRNA experiments, cells were transfected with a pool of siRNAs from Dharmacon using Polyplus reagent 24 hours before treatment with either DMSO or 1 µM A485. One hour post-treatment, cells were divided into two groups: one group was exposed to UV radiation (20 J/m²) and the other (control) was not exposed. Cells were harvested 2 hours post-UV radiation. Chromatin immunoprecipitation DNA sequencing (ChIP-seq) for RNA Polymerase II (RPB1) was performed in WT or P300 knockout (?p300) HeLa cells at 0 or 2 hours post-UV irradiation (20 J/m²), with or without 1 h of treatment with Tripolide 50 nM. Cells were harvested 2 hours post-UV irradiation.