Abstract
Faithful transcription of DNA is constantly threatened by different endogenous and environmental genotoxic effects. Transcription coupled repair (TCR) has been described to stop transcription and quickly remove DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked transcription. This repair mechanism has been well characterized in the past using individual target genes. Moreover, numerous efforts investigated the fate of blocked RNA polymerase II (Pol II) during DNA repair mechanisms and suggested that stopped Pol II complexes can either backtrack, be removed and degraded or bypass the lesions to allow TCR. We investigated the effect of a non-lethal dose of UVB on global DNA-bound Pol II distribution in human cells. We found that the used UVB dose did not induce Pol II degradation however surprisingly at about 93% of the promoters of all expressed genes Pol II occupancy was seriously reduced 2–4 hours following UVB irradiation. The presence of Pol II at these cleared promoters was restored 5–6 hours after irradiation, indicating that the negative regulation is very dynamic. We also identified a small set of genes (including several p53 regulated genes), where the UVB-induced Pol II clearing did not operate. Interestingly, at promoters, where Pol II promoter clearance occurs, TFIIH, but not TBP, follows the behavior of Pol II, suggesting that at these genes upon UVB treatment TFIIH is sequestered for DNA repair by the TCR machinery. In agreement, in cells where the TCR factor, the Cockayne Syndrome B protein, was depleted UVB did not induce Pol II and TFIIH clearance at promoters. Thus, our study reveals a UVB induced negative regulatory mechanism that targets Pol II transcription initiation on the large majority of transcribed gene promoters, and a small subset of genes, where Pol II escapes this negative regulation.
Highlights
Proper cell homeostasis and function requires expression of the DNA encoded information
Numerous efforts investigated the fate of blocked polymerase II (Pol II) during DNA repair mechanisms and suggested that stopped Pol II complexes can either backtrack, be removed or bypass the lesions to allow repair
We carried out a genome-wide analysis of Pol II behavior upon a DNA damaging stress, UVB, which is relevant from the public health standpoint
Summary
Proper cell homeostasis and function requires expression of the DNA encoded information. Transcription of thousands of coding and non-coding RNAs by the RNA polymerase II (Pol II) is a regulated multistep process that can be divided into five stages: pre-initiation, initiation, promoter clearance, elongation and termination. The ‘‘canonical’’ Pol II occupancy ChIP-seq profile on an average expressed gene displays Pol II molecules engaged in the major phases of transcription [1,2,3,4,5,6,7] and can be divided in three major regions: i) the sharp and usually high peak centered about +50 bp downstream of the transcription start site (TSS), representing Pol II molecules that have entered the pre-initiation complex (PIC) during transcription initiation/clearance and stopped at promoter proximal pausing position. Analyses of short transcribed RNA molecules showed that these arrested polymerases are predominantly in a transcriptionally engaged state [4,8,9]; ii) the background-like low signals in the gene body (GB), representing quickly elongating Pol II molecules; and iii) the broad signal downstream from the 39 end of the annotated genes (EAGs) representing Pol IIs that have finished transcribing the premRNA and are slowly transcribing and approaching the termination site often 4–6 kb away from the 39end of the gene [10,11,12,13] (see below)
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