Genome binding/occupancy profiling by high throughput sequencing Other
Summary
The coordinated transcription of genes involves the regulated release of RNA polymerase II (RNAPII) from promoter-proximal sites into active elongation. DNA lesions in transcribed strands block elongation and induce a strong transcriptional arrest. The transcription-coupled repair (TCR) pathway efficiently removes transcription-blocking DNA lesions, but this is not sufficient to resume transcription. Through proteomics screens, we find that the TCR-specific CSB protein loads the evolutionary conserved PAF1 complex (PAF1C) onto RNAPII in promoter-proximal regions in response to DNA damage. PAF1C is dispensable for TCR-mediated repair, but is essential for recovery of RNA synthesis after UV irradiation, suggesting an uncoupling between DNA repair and transcription recovery. Moreover, we find that PAF1C promotes RNAPII pause release in promoter-proximal regions and subsequently acts as a processivity factor that stimulates transcription elongation throughout genes. Our findings expose the molecular basis for a non-canonical PAF1C-dependent pathway that restores transcription throughout the human genome after genotoxic stress.
Overall design
BrU-seq and ChIP-seq in human cell lines either untreated (mock) or 3h, 8h or 26h after UV-C treatment with varying dose (as indicated per sample). ChIP-seq was performed targeting RPB1 or PAF1 in either WT or CSB knockout U2OS cells or in U2OS cells in which PAF1 was depleted using the auxin-inducible degron system. BrU-seq was also performed on U2OS cells with and without auxin-induced depletion of PAF1.
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