Journal: Nature Communications

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

doi: 10.1038/s41467-021-21520-w

Figure Lengend Snippet: 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/m 2 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/m 2 ( n = 1) and 9 J/m 2 ( 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. . 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/m 2 . 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/m 2 , 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/m 2 ( 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/m 2 . j Representative images of U2OS WT or CSB-KO stained for Ub-H2B at 0 or 8 h after 9 J/m 2 . Scale bar indicates 10 µm. Boxplots of the quantification of these images are presented in Supplementary Fig. . 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/m 2 UV. Total reads per plot were normalized to total Ub-H2B levels quantified by microscopy as in j and Supplementary Fig. . Data are averages of two replicates per condition.

Article Snippet: Cells were further permeabilized by incubation with 0.5% TritonX100 in PBS for 5 min. Then nuclei were consecutively blocked with 100 mM Glyine in PBS for 10 min, washed extensively with PBS and blocked with 0.5% BSA and 0.05% tween20 in PBS (WB-buffer) for 10 min. Ub-H2B was visualized by labeling the cells for 2 h with rabbit anti-ub-H2B (K120) (Cell signaling (mAb#5546, D11); 1:200 in WB-buffer).

Techniques: ChIP-sequencing, Irradiation, Binding Assay, Staining, Microscopy

Journal: Nature Communications

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

doi: 10.1038/s41467-021-21520-w

Figure Lengend Snippet: a Outline of the BrU-seq approach to measure nascent transcription across the genome. b Metaplots of nascent transcription in genes of >100 kb, between 50 and 100 kb, or between 25 and 50 kb in one replicate of either TIR1 cells (upper panels) or PAF1-AID cells (lower panels) that were either mock-treated, or UV-irradiated (7 J/m 2 ) and analyzed at the indicated timepoints (3, 8, or 24 h). The relative distribution of nascent transcript read density (in reads per thousand base-pairs per million reads) was normalized to the absolute nascent transcript intensities measured in parallel to the BrU-seq experiments using the same cells and timepoints (see Fig. ). A replicate experiment is shown in Supplementary Fig. . c Heatmaps of BrU-seq data from the first replicate of unirradiated (mock) or UV-irradiated (3 or 24 h after 7 J/m 2 ) TIR1 control or PAF1-AID cells. Data was mapped and processed as for ChIP-seq and data is presented for the top 3000 genes with PAF1 binding at the TSS followed by ranking according to gene length. d UCSC genome browser track showing the nascent transcript read density across the ZFR gene in unirradiated and UV-irradiated TIR1 and PAF1-AID cells. Also shown are the PAF1, RNAPII, and Ub-H2B read densities for the same gene for comparison.

Article Snippet: Cells were further permeabilized by incubation with 0.5% TritonX100 in PBS for 5 min. Then nuclei were consecutively blocked with 100 mM Glyine in PBS for 10 min, washed extensively with PBS and blocked with 0.5% BSA and 0.05% tween20 in PBS (WB-buffer) for 10 min. Ub-H2B was visualized by labeling the cells for 2 h with rabbit anti-ub-H2B (K120) (Cell signaling (mAb#5546, D11); 1:200 in WB-buffer).

Techniques: Irradiation, ChIP-sequencing, Binding Assay