Continuous transcription initiation guarantees robust repair of all transcribed genes and regulatory regions

Abstract

Inhibition of transcription caused by DNA damage-impaired RNA polymerase II (Pol II) elongation conceals a local increase in de novo transcription, slowly progressing from Transcription Start Sites (TSSs) to gene ends. Although associated with accelerated repair of Pol II-encountered lesions and limited mutagenesis, it is still unclear how this mechanism is maintained during genotoxic stress-recovery. Here we uncover a widespread gain in chromatin accessibility and preservation of the active H3K27ac mark after UV-irradiation. The concomitant increase in Pol II escape from promoter-proximal pause (PPP) sites of most active genes, PROMPTs and enhancer RNAs favors unrestrained initiation, as evidenced by the synthesis of nascent RNAs including start RNAs. Accordingly, drug-inhibition of PPPrelease replenishes levels of pre-initiating Pol II at TSSs after UV. Our data show that such continuous engagement of Pol II molecules ensures maximal transcription-driven repair throughout expressed genes and regulatory loci. Importantly, revealing this unanticipated regulatory layer of UV-response provides physiological relevant traction to the emerging concept that Pol II initiation rate is determined by pause-release dynamics.

Genomic Characterization of Cisplatin Response Uncovers Priming of Cisplatin-Induced Genes in a Resistant Cell Line

“…Here, we measured alteration in chromatin accessibility three hours after cisplatin treatment, and did not find a significant shift in accessibility in either the sensitive or resistant cells. This is in contrast to a recent report that UV-treatment increases chromatin accessibility in skin fibroblasts [ 35 ]. In our hands, the transcriptional response to cisplatin is also more attenuated than the response to UV even in the same cell type (data not shown).…”

Exons and introns exhibit transcriptional strand asymmetry of dinucleotide distribution, damage formation and DNA repair

“…Transcription-coupled repair (in XP-C cells) also shows slightly higher repair in exons; however, the difference is not statistically significant (Figure 5C and D ). Since transcription coupled repair is initiated by actively elongating RNA Pol II, and global genome repair is highly influenced by chromatin accessibility, we analyzed elongating RNA Pol II occupancy and ATAC-seq accessibility profiles from VH10 skin fibroblasts over introns and exons (data obtained from ( 41 , 42 ), Figure 5E – J ). Like transcription-coupled repair, RNA pol II showed higher, but not statistically significant, different levels on exons.…”

“…BigWig files of ChIP-seq data of elongating RNA polymerase II (RNAPII) and ATAC-seq data in un-irradiated human VH10 cells (Fibroblast cells immortalized with hTERT) were obtained from GEO accession numbers GSE83763 and GSE125181 ( 41 , 42 ), respectively. Introns and exons coordinates of hg19 were obtained as described above where first and last intron/exon were removed in order to avoid biases.…”

aniFOUND: analysing the associated proteome and genomic landscape of the repaired nascent non-replicative chromatin

“…Duplicated alignments were eliminated using samtools markdup -r. Replicates were downsampled to the minimum sample read depth per biological condition and merged in order to create a consensus dataset. Genomic annotations of hg19 RefSeq transcription start sites (TSSs) and enhancers were retrieved by UCSC table browser ( 34 ) and FANTOM5 project ( 35 ), while active and inactive characterization of TSSs, enhancers, and asPROMPTs was applied using previously published RNA Pol II-ser2P, H3K27ac and H3K27me3 ChIP-seq data from VH10 hTERT-immortalized human skin fibroblasts ( 22 , 29 ) and CAGE-seq data ( 35 ) from dermal and skin fibroblasts, as described in Liakos et al. ( 22 ).…”

“…There are two sub-pathways of NER, the transcription coupled NER (TC-NER), which is triggered by an actively transcribing RNA polymerase II (Pol II) and removes DNA lesions from the transcribed strand of genes and gene regulatory regions ( 21 , 22 ) and the global genomic NER (GG-NER) that removes lesions from the entire genome. The two sub-pathways mainly differ at the step of damage recognition.…”