DROSHA, DICER and Damage-Induced long ncRNA control BMI1-dependent transcriptional repression at DNA double-strand break. Esposito&Capozzo et al.
Description
At DNA Double strand break sites, local chromatin modifications and damage-induced transcriptional silencing in cis (DISC) occur concomitantly with RNA PolII dependent de novo transcription of damage-induced long non-coding RNAs (dilncRNAs). We hypothesized that the two events could be reconciled in a unique mechanism. We discovered that the two RNAi machinery factors DICER, DROSHA, together with dilncRNAs and the shorter products DDRNAs, sustain the recruitment of the Polycomb protein BMI1 at the sites of break, fostering local H2AK119Ub deposition and DISC. We studied the process mainly in the DIvA cellular system, where endogenous annotated DSBs are enzymatically induced. We analyzed transcriptional repression of 6 DSB-bearing genes via RT-qPCR and investigated BMI1 recruitment at damage sites via ChIP, PLA or DI-PLA in cells depleted for DICER and DROSHA or where dilncRNAs-DDRNAs axis was impaired via antisense oligonucleotides; dissected BMI1-DRSOHA, BMI1-dilncRNAs and BMI1-DDRNAs interaction at the site of break; generated transcriptomic data in DIvA cells depleted for DICER/DROSHA.
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Steps to reproduce
The data were produced using U2OS-MS2-YFP, DIvA (U2OS AsISI) and HeLa-GFP cell lines. Standard protocols for RNA-seq, RT-qPCR, immunofluorescence, laser micorirradiation, PLA, DI-PLA, ChIP, RIP, Co-IP were used; and adapted strand-specific gene specific RT-qPCR protocol was used for dilncRNAs detection, and the miScript RT-qPCR protocol was used for DDRNAs detection. Images for immunofluorescence and live imaging were acquired with a Zeiss confocal microscope.
Institutions
- Istituto di Genetica Molecolare Consiglio Nazionale delle Ricerche