Fig 2: SARS-CoV-2 disrupts chromatin architecture to antagonize the transcriptional activation of interferon response genes.a. Boxplots showing the expression deregulation of key interferon response (IFN) and pro-inflammatory genes after SARS-CoV-2 infection or IFN-beta treatment (1000u, 6-hr), as shown by RNA-Seq changes. For boxplots, the centre lines represent medians; box limits indicate the 25th and 75th percentiles; and whiskers extend 1.5 times the interquartile range (IQR) from the 25th and 75th percentiles. P-value: Mann-whitney U test.b,c. Heatmaps of select IFN or pro-inflammatory genes showing their fold changes in Pol2 ChIP-Seq or RNA-seq after SARS-CoV-2 infection or IFN-beta treatment (1000u, 6-hr).d,e. Snapshots of Hi-C contact matrices and calibrated ChIP-Seq tracks for indicated factors at two key loci coding for virus RNA sensors: DDX58 (coding for RIG-I) and IFIH1 (coding for MDA5). Left: Mock; right: SARS-CoV-2. Blue arrows point to reduced dot-shaped loops. The intra-TAD interactions were weakened throughout these two TADs. Red asterisks show reduced H3K27ac peaks by virus infection. Green arrows show H3K4me3 peaks that are not changed for these genes.f. A scatter plot showing a poor correlation between the C score only (from Hi-C contact, x-axis) and the true transcriptional changes of IFN genes by SARS-CoV-2 (y-axis, RPB1 ChIP-Seq). A liner regression fitted line and its 95% confidence interval are also shown.g. Similar to panel f, this is a scatter plot showing a poor correlation between the A score only (from enhancer H3K27ac activity, x-axis) and the true transcriptional changes of IFN genes by SARS-CoV-2 (y-axis, RPB1 ChIP-Seq).h. (Left) Design of gRNAs/CRISPRi that target the weakened enhancers seen in SARS-CoV-2 infected cells (the red asterisks); this IFIT locus is also shown in Fig.4b with more information there. (Right) RT-qPCR results showing that CRISPRi inhibition of the enhancers reduced IFIT gene expression in response to poly(I:C), a synthetic viral mimicry. gRNA1 and gRNA2 are two gRNAs targeting the same enhancer (see Extended Data Table 2).i. Similar to panel h, this is the RT-qPCR result showing that CRISPRi inhibition of the enhancer in DDX58 locus reduced its response to poly(I:C). The DDX58 enhancer location for the CRISPRi is shown in panel d. Data in panels h,i show Mean +/- SD from three biological replicates (n=3); p values: two tailed student’s T-test (*, p<0.05; **, p<0.01; ***, p<0.001)

Fig 3: Degradation of promoter-bound Pol II is signaled via GSK3.a Left panel: Representative western blots after cellular fractionation of nontreated (NT) GFP-RPB1 knock in (KI) cells or cells treated with the indicated kinase inhibitors (i) 30 min before UV irradiation. Fractionation was performed in non-irradiated cells (0 J) or 1 h after irradiation with 4 J/m2. BRG1 was used as loading control. NTD = RPB1 N-terminal domain, P-Ser = phospho-serine. Right panel: Quantification of the western blot signals of n = 2 independent experiments2, mean ± SD. b Left panel: Representative western blots after cellular fractionation of GFP-RPB1 knock in (KI) 1 h after irradiation with 4 J/m2 without (NT) or with GSK3 inhibitor treatment (GSK3i, CHIR-99021, 10 µM 30 min before UV). SSRP1 and BRG1 were used as loading controls. NTD = RPB1 N-terminal domain, P-Ser= phospho-serine. Right panel: Quantification of the indicated western blot signals, mean ± SD, n = 3 for ‘P-Ser2’ and ‘P-Ser5’, n = 4 for ‘NTD’. c Left panel: Fluorescence Recovery after photo-bleaching (FRAP) of GFP-RPB1 after UV irradiation with or without pretreatment for 30 min with GSK3 inhibitor (CHIR-99021, 10 µM). GFP-RPB1 was bleached and fluorescence intensity was measured every 0.4 sec for 4 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 analysed by FRAP. n = 19 cells for ‘0 J’ and ‘4 J 1–2 h’, n = 23 cells for ‘GSK3i 0 J’, n = 29 cells for ‘GSK3i 4 J 1–2 h’. *P = 0.05, **P = 0.01, ****P = 0.0001; analyzed by ordinary one-way ANOVA using Dunnett’s multiple comparisons test versus the respective 0 J condition.

Fig 4: UV-induced proteasomal degradation of promoter-bound Pol II.a, b Left panel: Fluorescence Recovery after photo-bleaching (FRAP) of GFP-RPB1 after UV irradiation with or without pre-treatment for 30 min with (a) proteasome inhibitor (Mg132, 10 µM) or (b) VCP inhibitor (VCPi) (NMS-873, 5 µM). 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 analysed by FRAP. Cells from two independent experiments were analyzed. 0 J/m2 n = 42, 4 J/m2 0–1 h n = 24, 4 J/m2 1–2 h n = 30, MG132 0 J/m2 n = 22, MG132 4 J/m2 0–1 h n = 16, MG132 4 J/m2 1–2 h n = 29, VCPi 0 J/m2 n = 14, VCPi 4 J/m2 0–1 h n = 16, VCPi 4 J/m2 1–2 h n = 28. (c) Top panel: Representative western blots after cellular fractionation of GFP-RPB1 knock in (KI) cells after the indicated treatments. SSRP1 and BRG1 were used as loading controls. WCL = whole cell lysate, NTD = RPB1 N-terminal domain, P-Ser= phospho-serine. Bottom panel: Quantification of the indicated western blot signals, NTD pellet fraction n = 3, Ser2 pellet fraction and NTD supernatant n = 2, P-Ser5 n = 4, mean ± SE. d Left panel: Representative images of GFP-RPB1 KI cells that were fixed and stained for P-Ser5 after UV irradiation without or with pre-treatment for 30 min with proteasome inhibitor (Mg132, 10 µM) or VCP inhibitor (VCPi) (NMS-873, 5 µM). Right panel: GFP-RPB1 and P-Ser5 fluorescence intensities (FI) were normalized were normalized to 0 J/m2 levels, which were set to 1. RFI Relative FI. n > 200 cells, error bars represent SD. *P = 0.05, ****P = 0.0001, ns nonsignificant; analyzed by ordinary one-way ANOVA using Dunnett’s multiple comparisons test versus the respective 0 J condition (a–d).

Fig 5: Tethering RUVBL2 to an active gene promoter enhances Pol II cluster formation.a Schematic showing the system used for simultaneous imaging of tethered proteins and recruited proteins, as published previously60. b Representative images of endogenous RUVBL1 and RUVBL2. Scale bar, 5 µm. c The relative IF signals of the target proteins at the LacO loci under mock (RUVBL1 n = 24, RUVBL2 n = 30) and Dox treatments (RUVBL1 n = 18, RUVBL2 n = 30). Box plots indicate median (middle line), 25th and 75th percentile (box) and 5th and 95th percentile (whiskers), as well as outliers (single black points), differences between groups were analyzed by two-tailed Student’s t tests (****p < 0.0001). d The mean and total intensity of LacR-BFP fusion signals at LacO loci per cell were plotted in bar graphs. The cotransfected cells (n = 30) in different fields from 3 transfection replicates were used for quantification. Differences between groups were analyzed by two-tailed Student’s t tests. “ns” indicates no significant difference. The error bars indicate mean + /- SDs. e Representative images of endogenous Pol II before and after transcriptional activation. Scale bars, 5 µm. f The relative IF signals of Pol II and LacR-BFP fusions at the LacO loci were plotted as boxplot. Cells from a minimum of 10 different fields (BFP n = 11, RUVBL1 n = 28, RUVBL2 n = 29 in mock state, and BFP n = 13, RUVBL1 n = 24, RUVBL2 n = 16 in Dox activation state) were used for quantification. Differences between groups were analyzed with two-tailed Student’s t tests (ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). g Nuclear Pol II (RPB1) density was quantified from 3 biological replicates. Two-tailed Student’s t tests were performed to determine the significance of differences between groups, and the error bars indicate the mean + /- SD. h IF assay was performed to examine endogenous RUVBL2. Cells (BFP n = 22, CTD n = 19) from a minimum of 10 different fields were used for clock scanning and quantification, and the error bars indicate the mean + /- SDs. The clock scan densities were analyzed with two-tailed Student’s t-tests (****p < 0.0001). Scale bars, 5 µm.