Extended Data Fig. 7: MNNG/H2O2-induced CycT1 PARylation inhibits P-TEFb’s transcriptional elongation activity.
a, A procedure for isolating newly synthesized, 4-thiouridine (4sU)-labeled RNA for subsequent analysis by real-time RT-PCR. b, Transcription from the cellular FOS and JUN gene promoters in untreated or MNNG-treated cells was investigated by 4sU-labeling followed by qRT-PCR. The mRNA levels in untreated cells were artificially set to 1.0. The error bars indicate mean ± s.d. with n = 3 biologically independent samples; statistical analysis was performed using two-tailed unpaired t-tests; ***P < 0.001. c, Transcription from the promoters of HIV-1, FOS and JUN in untreated or H2O2-treated cells was analyzed as in b. The error bars indicate mean ± s.d. with n = 3 biologically independent samples; statistical analysis was performed using two-tailed unpaired t-tests; ***P < 0.001; **P = 0.0085. d, A diagram of double strand break (DSB) induction in U2OS-265 cells. ER-mCherry-LacI-FokI-DD is stabilized and directed to LacO repeats to introduce DSBs by the addition of Shield-1 and 4-OH. Transcription of the reporter gene is induced with Doxycycline after DSB induction. e, Transcription from the reporter and GAPDH gene promoters in untreated or DSB-induced cells was measured by qRT-PCR (mean ± s.d., n = 3 biologically independent samples) and the ratios of “Reporter gene vs. GAPDH” are shown. The signals were normalized to non-DSB conditions, which were set to 1.0. f, A diagram illustrating the experimental design involving the incubation with MNNG for the indicated periods of time with the last 20 min of the incubation performed also in the presence of 5-EU to label the newly synthesized. g, HeLa cells were untreated or treated with MNNG for the indicated time periods. Newly synthesized RNA was labeled with 5-EU following the scheme in d and then detected using Alexa fluor 488. Scale bar = 50 μM. Right: Quantification of the 5-EU fluorescence intensity per cell. Red lines indicate the mean 5-EU intensity in each group. n represents the number of cells examined in a representative assay out of 3 independent experiments: MNNG 0 min (n = 181), MNNG 20 min (n = 181), MNNG 40 min (n = 180), MNNG 60 min (n = 183); statistical analysis was performed using two-tailed unpaired t-tests; ****P < 0.0001. h, HeLa cells expressing GFP-CycT1 were treated with MNNG for the indicated time periods and examined by fluorescence microscopy. Scale bar = 10 μM. Bottom: Quantification of the enrichment of GFP-CycT1, which is calculated as the ratio of mean fluorescence intensity per nuclear condensate versus the mean fluorescence intensity of the entire cell nucleus. The box plots show the minimum, first quartile, median, third quartile and maximum with n represents the number of cells examined in a representative assay out of 3 independent experiments: MNNG 0 min (n = 71), MNNG 20 min (n = 84), MNNG 40 min (n = 67), MNNG 60 min (n = 65); statistical analysis was performed using two-tailed unpaired t-tests; ****P < 0.0001. i, Volcano plot showing differential gene expression based on genome-wide sequencing of 4sU-labeled RNA in DMSO- or MNNG-treated cells. The negative log10 p-values test the significance of enrichment (y axis) and are plotted against the average log2 fold changes in expression (x axis). Genes not differentially expressed are displayed as black dots. Colored dots are genes showing at least 2-fold changes (red indicates increase and green decrease) in expression with p-values less than 0.01. p-values were determined using Fisher’s exact test. j, Scatterplot of log10 fold changes in the expression of genes in MNNG- versus i-CDK9-treated cells. The r value denotes the Pearson correlation coefficient; and the p value (< 2.2e-16) displays statistical significance of the correlation as calculated by two-tailed test. k, & l, HeLa cells were depleted of CycT1 and reconstituted with WT or Mut2 CycT1. Transcription from the JUN (k) and FOS (l) gene promoters in untreated or MNNG-treated cells was investigated by 4sU-labeling followed by qRT-PCR analysis. The mRNA levels in untreated cells were artificially set to 1.0. The error bars indicate mean ± s.d. with n = 3 biologically independent samples; statistical analysis was performed using two-tailed unpaired t-tests; ***P < 0.001.
