Fig 2: The UV-induced repositioning of RNAPII into promoter-proximal regions requires PAF1C.a Schematic representation of the traveling ratio of PAF1, which is calculated by dividing the reads of the gene body (TSS to +1000 bp; blue) over the reads in the promoter-proximal region (-750bp to TSS; red). b Quantification of the traveling ratio (or shift) in PAF1 binding after UV. All replicates and UV doses (6 and 9 J/m2, total n = 4) were pooled, which revealed a set of 478 genes that shows a uniform UV-induced shift into promoter-proximal regions in all replicate experiments at 8 h after 6 and 9 J/m2 (All Shift) and a set of 2522 genes that show a UV-induced shift only in a subset of the conditions (Mixed Shift). c UCSC genome browser track showing the read density of PAF1 and RNAPII signal across the POLG2 gene (All Shift) and SLC40A1 gene (Mixed Shift) in unirradiated (mock) and UV-irradiated cells. d Averaged metaplots of PAF1 ChIP-seq around the TSS of the 478 All Shift genes (upper panel) or the 2522 Mixed Shift genes (lower panel) in unirradiated cells (mock, n = 3) and 8 h after UV with 6 J/m2 (n = 1) and 9 J/m2 (n = 3). e The ratio of the RNAPII traveling ratios for the 478 All Shift genes compared to the 2522 Mixed Shift genes at 8 h after UV with 6 J/m2 (n = 3) and 9 J/m2 (n = 2). f The ratio of the RNAPII traveling ratios for 3000 genes relative to the average traveling ratio in the unirradiated control (set to 1) shown for TIR1 (upper panel) or PAF1-AID clone 14 (lower panel). Shown are unirradiated cells (mock, n = 2), 8 h after 6 J/m2 (6J8h, n = 2), or 9 J/m2 (9J8h, n = 2). g Data as in f, but for 902 genes with consistent shift of RNAPII in all replicates at 6 J/m2 or 9 J/m2 compared to mock in TIR1 cells. The y-axes in panels e–g indicate percent of all genes. Indicated percentages and n indicate genes with a normalized traveling ratio above 1.

Fig 3: The RNAPII–PAF1C interaction is mediated by CSB.a Representative images of U2OS cells treated with DMSO or 10 µM LDC00067 (CDK9 inhibitor), followed by pulse-labeling with 5-ethynyl-uridine (5-EU). Scale bar indicates 10 µm. Boxplots represent the median, 5th and 95th percentile of all cells of two independent experiments. b Co-immunoprecipitation of GFP-RPB1 in the presence of DMSO or 10 µM LDC00067 for the indicated time. c Co-immunoprecipitation of GFP-PAF1 from U2OS cells (WT or CSB-KO). d Co-immunoprecipitation of endogenous RNAPII-S2 from U2OS cells (WT or indicated KO). e As in d, but now including U2OS CSB-KO cells reconstituted with GFP-CSB. b–e All co-immunoprecipitation figures are representative examples of at least two independent experiments.

Fig 4: The UV-induced repositioning of PAF1 and associated ubiquitylation of H2B requires CSB.a Representative heatmaps around TSS from single PAF1 ChIP-seq data of the top 3000 genes that bind PAF1 in CSB-KO cells at 0 and 8 h after 9 J/m2 UV. b UCSC genome browser track showing the read density of PAF1 signal across the PSMD3 gene. Tracks represent pooled reads of three wild-type mock and three wild-type 9J8h PAF1 ChIP-seq replicates (blue) and two CSB-KO mock and two CSB-KO 9J8h PAF1 ChIP-seq replicates (red). c The left panel shows averaged metaplots of PAF1 ChIP-seq of the top 3000 genes in CSB-KO unirradiated cells (mock, n = 2) or CSB-KO cells 8h after UV irradiation with 6 J/m2 (n = 1) and 9 J/m2 (n = 2). The right panel shows the UV-induced redistribution of PAF1 calculated by subtracting the mock from the +UV distribution profiles. d Quantification of UV-induced PAF1 traveling ratios (or shift) in the 478 all shift genes as defined in WT cells in Fig. 6a, b. Genes shifting in all CSB-KO replicates are in blue, genes not shifting in at least 1 of the replicates are indicated in red. e The UV-induced redistribution of PAF1 calculated by subtracting the mock from the +UV distribution profiles in WT and CSB-KO cells. f Representative heatmaps around the TSS from single ChIP-seq data on RNAPII of the top 3000 genes that bind PAF1. Heatmaps are show for CSB-KO cells at 0 and 8 h after 6 J/m2. g Averaged non-normalized metaplots around the TSS of RNAPII ChIP-seq of the top 3000 genes in unirradiated (mock, n = 2) or UV-irradiated (8 h after 6 J/m2, n = 2) CSB-KO cells showing differences in total RNAPII binding in different conditions. h As in g for unirradiated wild-type cells (mock, n = 3) or UV-irradiated wild-type cells, 8 h after 6 J/m2 (n = 3). i UCSC genome browser tracks showing the read density of ubiquitylated H2B (Ub-H2B) signal across the KANSL1 gene in wild-type and CSB-KO cells 0 or 8 h after 9 J/m2. j Representative images of U2OS WT or CSB-KO stained for Ub-H2B at 0 or 8 h after 9 J/m2. Scale bar indicates 10 µm. Boxplots of the quantification of these images are presented in Supplementary Fig. 6. k Averaged metaplots of Ub-H2B ChIP-seq of 820 genes of >100 kb in WT or CSB-KO cells at 0 or 8 h after 9 J/m2 UV. Total reads per plot were normalized to total Ub-H2B levels quantified by microscopy as in j and Supplementary Fig. 6. Data are averages of two replicates per condition.

Fig 5: PAF1C loss impairs transcription recovery after UV irradiation.a Experimental outline of the nascent transcription analyses. b Validation of the knockdown of XPA and PAF1 in U2OS cells by western blot analysis. c Validation of the knockdown of endogenous PAF1 in U2OS cells ectopically expressing siRNA-resistant PAF1 by western blot analysis. d Representative images of U2OS cells transfected with the indicated siRNAs after pulse-labeling with 5-ethynyl-uridine (5-EU). Cells with green outlines express GFP-tagged siRNA-resistant PAF1. Scale bar indicates 10 µm. e Quantification of nascent transcript levels (NTL) from d. Boxplots represent the median, 5th and 95th percentile of all cells of three independent experiments. f Validation of auxin-induced degradation of endogenous PAF1 in two independent U2OS PAF1-AID knockin clones. g Representative images of auxin-treated U2OS TIR1 control or two PAF1-AID clones after pulse-labeling with 5-ethynyl-uridine (5-EU). Scale bar indicates 10 µm. h Quantification of nascent transcript levels from g. Boxplots represent the median, 5th and 95th percentile of all cells of four independent experiments. b, c, f Representative figure of at least two replicates.