A damaged genome’s transcriptional landscape through multilayered expression profiling around in situ-mapped DNA double-strand breaks

Abstract

Of the many types of DNA damage, DNA double-strand breaks (DSBs) are probably the most deleterious. Mounting evidence points to an intricate relationship between DSBs and transcription. A cell system in which the impact on transcription can be investigated at precisely mapped genomic DSBs is essential to study this relationship. Here in a human cell line, we map genome-wide and at high resolution the DSBs induced by a restriction enzyme, and we characterize their impact on gene expression by four independent approaches by monitoring steady-state RNA levels, rates of RNA synthesis, transcription initiation and RNA polymerase II elongation. We consistently observe transcriptional repression in proximity to DSBs. Downregulation of transcription depends on ATM kinase activity and on the distance from the DSB. Our study couples for the first time, to the best of our knowledge, high-resolution mapping of DSBs with multilayered transcriptomics to dissect the events shaping gene expression after DSB induction at multiple endogenous sites.

Activation of Oncogenic Super-Enhancers Is Coupled with DNA Repair by RAD51

“…Together these results are consistent with previous studies showing enrichment of transcription-mediated DSBs (TM-DSBs) around promoters, enhancers Iannelli et al, 2017;Madabhushi et al, 2015;Bunch et al, 2015;Wei et al, 2016) and the recently reported loop anchors (insulators) (Canela et al, 2017(Canela et al, , 2019Gothe et al, 2019;Yan et al, 2017).…”

“…BLISS directly detects DSBs at a resolution of a single nucleotide and has sensitivity that allows identifying not only artificially induced but also naturally occurring physiological DSBs . BLISS was shown to be an accurate and sensitive quantitative method, in particular because it quantifies DSBs through unique molecular identifiers Iannelli et al, 2017). Yan et al (2017) were able to assess the genome-wide off-target activity of two CRISPR-associated RNA-guided endonucleases, Cas9 and Cpf1, demonstrating that Cpf1 has higher specificity than Cas9.…”

Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures

“…Further analysis revealed that out of the 1,211 predicted AsiSI sites in the human genome, 174 showed a signal significantly higher than background (Figures S1B and 1B). This is in relatively good agreement with our previous estimates of the number of induced DSBs inferred from γH2AX accumulation and with BLISS data in DIvA cells (Aymard et al., 2014, Iannelli et al., 2017). However, we could detect clear differences between γH2AX and BLESS signals, confirming that γH2AX does not strictly reflect DSB induction rate (Figure S1A, compare left and right panel; Figure S1C).…”

RNA at DNA Double‐Strand Breaks: The Challenge of Dealing with DNA:RNA Hybrids

“…The study of DNA repair in transcribed chromatin has received particular attention and it is well documented that DSBs have an inhibitory effect on transcription at nearby promoters. The process of DSB‐induced inhibition of transcription by RNA polymerase II (RNAPII) requires ATM and/or DNA‐PKc [ 15–17 ] as well as the chromatin remodeler BRG1. [ 18 ] This transcriptional arrest is characterized by reduced levels of elongating RNAPII along the gene body as indicated by loss of phosphorylation of the RNAPII carboxy terminal domain at Serine 2 and Serine 5 (CTD Ser2/5P).…”

“…Their RNAPII dependency has been further supported by the observation of a DSB‐dependent increase of RNAPII occupancy in a 4 kb window around break sites and a corresponding increase of transcripts mapping to sequences around the break. [ 16,25 ] Moreover, dilncRNAs have been detected in native RNA pull‐down experiments after the induction of a sequence specific DSB. [ 25 ] Together, these results strongly imply that dilncRNAs are transcribed by RNAPII and are produced de novo after a DSB event.…”