Journal: Nature Communications

Article Title: DNA damage-induced transcription stress triggers the genome-wide degradation of promoter-bound Pol II

doi: 10.1038/s41467-022-31329-w

Figure Lengend Snippet: a Pol II promoter occupancy on the RPLP1 and RPL3 genes in GFP-RPB1 KI cells after the indicated treatments determined by GFP ChIP followed by q-PCR. Wild-type cells without GFP-RPB1 were analysed as negative control (WT Ctrl). VCPi= VCP inhibitor (NMS-873, 5 µM 30 min before UV irradiation), Mg132=proteasome inhibitor (10 µM, 30 min before UV irradiation). Average Pol II occupancy around the transcription start site of n = 3 independent experiments is shown, error bars indicate SE. All conditions were normalized to promoter occupancy in nonperturbed conditions (NT -UV), which was set to 1. * P ≤ 0.05, ** P ≤ 0.01, ns nonsignificant; analyzed by ordinary one-way ANOVA using Dunnett’s multiple comparisons test. b Isolation of ubiquitylated proteins using tandem ubiquitin-binding entities (TUBEs) from the supernatant of GFP-RPB1 KI cells after cellular fractionation. Cellular fractionation was optimized to separate hyper-phosphorylated, elongating Pol IIo in the pellet from hypo-phosphorylated, free and promoter-bound Pol IIa in the supernatant. Isolated ubiquitylated proteins were stained for promoter-paused Pol II (P-Ser5) and ubiquitin (FK2). FK2 staining confirms equal purification of ubiquitylated proteins. Brg1 staining was used as loading control. Note that ubiquitylated, P-Ser5 Pol II (pull-down) migrates higher compared to P-Ser5 Pol II in the input due to increased molecular weight. P-Ser = phospho-serine. The experiment has been performed two times with similar results. c , d Pol II occupancy around the transcription start site was analysed by Pol II ChIP-seq before and 1 h after 4 J/m 2 UV, and with or without VCP inhibition before UV irradiation. Pol II signal at representative individual genes ( c ), and heatmaps of Pol II on all refseq genes before and 1 h after 4 J/m 2 UV ( d ). Values were normalized to spike in controls. Heat maps are colour-scaled according to read densities form low (red) to high (blue) in units of RPM.

Article Snippet: Transcription levels were measured by pulse labeling with the nucleotide analogue 5′ethynyl uridine (EU) (Jena Bioscience).

Techniques: Negative Control, Irradiation, Isolation, Ubiquitin Proteomics, Binding Assay, Cell Fractionation, Staining, Purification, Control, Molecular Weight, ChIP-sequencing, Inhibition

Journal: Nature Communications

Article Title: DNA damage-induced transcription stress triggers the genome-wide degradation of promoter-bound Pol II

doi: 10.1038/s41467-022-31329-w

Figure Lengend Snippet: a Top panel: representative images of GFP-RPB1 KI cells pulse-labelled with EU for the last 30 min before fixation to visualize transcription rates by click-chemistry-based azide-594 coupling (top row), or stained with an anti-P-Ser5 RPB1 antibody (bottom row) in non-perturbed conditions (0 J) or after irradiation with 4 J/m 2 of UV at the indicated time points. Bottom panel: EU and P-Ser5 fluorescence intensities (RFI) were normalized to 0 J/m 2 levels, which were set to 1. n = 242, 329, 224, 291, 221, 301, 282, 222, 294, 202, 212, 201 cells (left to right) measured in 2 independent experiments. b Western blots after cellular fractionation of GFP-RPB1 knock in (KI) cells after the indicated time points after irradiation with 4 J/m 2 of UV. Cellular fractionation was optimized to separate hyper-phosphorylated, elongating Pol IIo in the pellet from hypo-phosphorylated, free and promoter-bound Pol IIa in the supernatant. SSRP1 and BRG1 were used as loading controls. WCL = whole cell lysate, NTD = RPB1 N-terminal domain, P-Ser= phospho-serine. Experiment has been performed two times with similar results. c GFP-RPB1 KI cells transfected with the indicated siRNAs were pulse-labelled with EU for the last 30 min before fixation to visualize nascent transcription rates in non-perturbed conditions (0 J) or after irradiation with 8 J/m 2 of UV at the indicated time points. EU relative fluorescence intensities (RFI) were normalized to 0 J/m 2 levels, which were set to 1. n = 430, 381, 372, 186, 163, 174 cells (left to right) measured in 2 independent experiments. d Left and middle panel: GFP-RPB1 FRAP measurements in unperturbed cells (0 J/m 2 ) or after irradiation with 4 J/m 2 . Time points indicate how long cells were left to recover before beginning FRAP measurements. GFP-RPB1 was bleached and fluorescence intensity was measured every 0.4 sec for 3 min, background-corrected and normalized to prebleach fluorescence intensity (FI), which was set to 100. RFI relative FI. Right panel: Mean ±SD of pre-bleach GFP-RPB1 FI as a measure for Pol II protein levels in nuclei analyzed by FRAP. n = 16 cells measured in 2 independent experiments. *** P ≤ 0.001,**** P ≤ 0.0001; ns nonsignificant, analyzed by ordinary one-way ANOVA using Dunnett’s multiple comparisons tests.

Article Snippet: Transcription levels were measured by pulse labeling with the nucleotide analogue 5′ethynyl uridine (EU) (Jena Bioscience).

Techniques: Staining, Irradiation, Fluorescence, Western Blot, Cell Fractionation, Knock-In, Transfection

Journal: Nature Communications

Article Title: DNA damage-induced transcription stress triggers the genome-wide degradation of promoter-bound Pol II

doi: 10.1038/s41467-022-31329-w

Figure Lengend Snippet: a Fluorescence Recovery after photo-bleaching (FRAP) analysis of GFP-RPB1 in wild type (WT), TC-NER-deficient (CSB, XPA) cells, or in cells lacking the GG-NER damage recognizing protein XPC, in non-perturbed conditions (0 J/m 2 ), immediately after irradiation (4 J/m 2 0–1 h), 1 h (4 J/m 2 1–2 h) or 20 h (4 J/m 2 20–21 h) after irradiation. n > 16 cells from 2 independent experiments. b Average GFP-RPB1 fluorescence intensity (FI) of KI cells analyzed by FRAP in (A) before photo-bleaching, representing Pol II protein levels at the indicated treatment conditions. n = 13 cells for ‘XPC KO 4 J 0–1 h’, n = 15 cells for ‘XPA KO 4 J 20–21 h’, n = 31 cells for ‘XPC KO 0 J’, n = 32 cells for ‘WT 4 J 1–2 h’ and ‘CSB KO 0 J’, n = 16 cells for all other conditions. Cells were measured in 2 independent experiments. Error bars represent SD. c Left panel: Representative western blots after cellular fractionation of GFP-RPB1 in wilt type cells (WT) and cells with CRISPR/Cas9-mediated gene knockout of the indicated repair factors. Fractionation was performed in nonperturbed conditions (0 J) or 1.5 h and 20 h after irradiation with 4 J/m 2 (4 J), only the supernatant fraction containing cytoplasmic and nucleoplasmic proteins is shown. BRG1 was used as loading control. NTD = RPB1 N-terminal domain, P-Ser= phospho-serine. Right panel: Quantification of western blot band intensities of P-Ser5-modified Pol II. Mean ±SD of n = 6 western blot experiments for CSB and XPA KO, n = 7 independent western blots for XPC KO, and n = 8 independent western blots for WT. d Representative images (left) and quantification (right) of transcription rates in GFP-RPB1 KI cells after the indicated treatments as determined by EU pulse labelling for 30 min followed by click-chemistry based azide-594 coupling. Nuclear EU fluorescence intensity (FI) was normalized to 0 J levels, which was set to 1. n = 437, 397, 250, 333, 363, 428, 260, 380, 308, 354, 229, 240, 166, 196, 122, 121, 166, 166, 103, 89 cells (left to right) measured in 2 independent experiments, error bars represent SD. e , f Clonogenic survival of MRC5 wild-type GFP-RPB1 knock in (KI) cells transfected with the indicated siRNAs. Equal numbers of cells were seeded and colony forming ability was determined in triplicate 7 days after irradiation with the indicated UV doses ( e ), or 24 h exposure to the indicated dose of Illudin S. Relative colony number ± SE of 3 independent experiments is shown. g , h Clonogenic survival in either wildtype (WT) ( g ) or CSB knockout (KO) ( h ) cells upon GSK3 inhibition (GSK3i, CHIR-99021, 10 μM 30 min before UV for 24 h) upon irradiation with the indicated UV doses. Relative colony number ± SE of 3 independent experiments is shown. ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001; analyzed by ordinary one-way ANOVA using Dunnett’s multiple comparisons tests.

Article Snippet: Transcription levels were measured by pulse labeling with the nucleotide analogue 5′ethynyl uridine (EU) (Jena Bioscience).

Techniques: Fluorescence, Irradiation, Western Blot, Cell Fractionation, CRISPR, Gene Knockout, Fractionation, Control, Modification, Knock-In, Transfection, Knock-Out, Inhibition