Paf1C regulates RNA polymerase II progression by modulating elongation rate

Abstract

Elongation factor Paf1C regulates several stages of the RNA polymerase II (Pol II) transcription cycle, although it is unclear how it modulates Pol II distribution and progression in mammalian cells. We found that conditional ablation of Paf1 resulted in the accumulation of unphosphorylated and Ser5 phosphorylated Pol II around promoter-proximal regions and within the first 20 to 30 kb of gene bodies, respectively. Paf1 ablation did not impact the recruitment of other key elongation factors, namely, Spt5, Spt6, and the FACT complex, suggesting that Paf1 function may be mechanistically distinguishable from each of these factors. Moreover, loss of Paf1 triggered an increase in TSS-proximal nucleosome occupancy, which could impose a considerable barrier to Pol II elongation past TSS-proximal regions. Remarkably, accumulation of Ser5P in the first 20 to 30 kb coincided with reductions in histone H2B ubiquitylation within this region. Furthermore, we show that nascent RNA species accumulate within this window, suggesting a mechanism whereby Paf1 loss leads to aberrant, prematurely terminated transcripts and diminution of full-length transcripts. Importantly, we found that loss of Paf1 results in Pol II elongation rate defects with significant rate compression. Our findings suggest that Paf1C is critical for modulating Pol II elongation rates by functioning beyond the pause-release step as an “accelerator” over specific early gene body regions.

A CSB-PAF1C axis restores processive transcription elongation after DNA damage repair

“…Unirradiated cells depleted for PAF1 by auxin-mediated degradation showed normal nascent transcription in the first 50 kb and moderately reduced nascent transcription toward the end of long genes ( Figure 7B). This is in line with a role of PAF1 as elongation factor (Hou et al, 2019), but also shows that under our experimental conditions there is no dramatic impact of PAF1 depletion on general transcription (Supplemental Figure 5G). Following UV irradiation, PAF1-depleted cells showed a strong and progressive loss of reads into gene bodies at 3 hrs after UV irradiation.…”

“…During transcription regulation, PAF1C is thought to travel with RNAPII to facilitate efficient elongation through chromatin (Hou et al, 2019;Kim et al, 2010). Indeed, while we could detect PAF1 and RNAPII binding to TTS sites in unirradiated cells, this was substantially reduced at 8 hrs after UV irradiation.…”

“…The PAF1 complex strongly associates with RNAPII and CSB after UV irradiation PAF1C is an evolutionary conserved pausing and elongation complex that consists of 5 subunits (Figure 2A) (Chen et al, 2015b;Chen et al, 2017;Hou et al, 2019;Yu et al, 2015). Considering that CSB interacts robustly with RNAPII only in the presence of transcription-blocking DNA damage, it is possible that we indirectly detect PAF1C through the UV-induced association of CSB with the RNAPII complex.…”

“…One intriguing possibility is that transcription recovery after repair involves the activation of paused RNAPII from promoter-proximal regions. In particular because PAF1C regulates RNAPII pause release during transcription and subsequently stimulates processive elongation throughout genes (Chen et al, 2015b;Chen et al, 2017;Hou et al, 2019;Vos et al, 2018a;Yu et al, 2015).…”

“…The regulation of RNAPII pause release in response to environmental cues involves positive elongation factors, such as p-TEFb and the PAF1 complex (PAF1C) (Chen et al, 2018;Vos et al, 2018b;Yu et al, 2015). Both PAF1C and DSIF also act beyond pause release by stimulating the acceleration of RNAPII in promoter-proximal regions to ensure processive transcription elongation throughout genes (Fitz et al, 2018;Hou et al, 2019;Kim et al, 2010;Wada et al, 1998).…”