Fig. 3: CDK12 is recruited on DNA damaged sites proximal to transcribed genes. | Nature Communications

Fig. 3: CDK12 is recruited on DNA damaged sites proximal to transcribed genes.

From: CDK12 controls transcription at damaged genes and prevents MYC-induced transcription-replication conflicts

Fig. 3

a, b Analysis of proximity of CDK12 and γH2AX foci by PLA in U2OS cells upon CDK12 silencing (shCDK12) or mock (shNT, non-targeting). a Representative pictures of mock silenced cells (shNT). shNT: mock n = 284 cells; 30’ n = 317 cells; 4 h n = 301 cells. shCDK12: mock n = 283 cells; 30’ n = 303 cells; 4 h n = 310 cells. b Box plot of PLA signals (nuclear dots) at different times post-irradiation (10 Gy). The box extends from the 25th to 75th percentiles, the error bar indicates minimum and maximum. P values calculated by Mann-Whitney test. c Kinetics of recruitment of mCherry-CDK12 to laser-damaged sites in U2OS cells treated or not with 100 µM DRB or 1 µM triptolide (TPL). Left, representative pictures; the red boxes highlight the irradiated areas. Right, the time-series plot shows the average and the standard deviation. DMSO: n = 5 cells; DRB: n = 13 cells; TPL: n = 12 cells. ****: P-value < 0.0001 (Two way anova with sidak’s multiple comparisons test). d Colocalization of mCherry-CDK12 and γH2AX foci at SceI-induced DSBs in U2OS-TRE-I-SceI-19 cells treated or not with 100 µM DRB or 1 µM triptolide (TPL). Left, pictures of mCherry-CDK12 and γH2AX signals. Right, box plots of two independent experiments. MOCK n = 43 cells, n = 60 cells; DRB n = 52 cells, n = 61 cells; TPL n = 50 cells, n = 57 cells. e, f IF analysis of locus specific double strand breaks induced by CRISPR/Cas9 editing at select loci. The error bar is the standard deviation. e Representative images. f Bar plot reporting the average colocalization of CDK12 and γH2AX foci in DRB treated cells (average of two experiments). DMSO POLR1B −1kb: n = 28 cells, n = 31 cells; DRB POLR1B −1kb: n = 37 cells, n = 26 cells; DMSO POLR1B −2kb: n = 23 cells, n = 32 cells; DRB POLR1B: −2kb n = 29 cells, n = 30 cells; DMSO IFRD2: −1kb n = 27 cells, n = 29 cells; DRB IFRD2 −1kb: n = 28 cells, n = 25 cells; DMSO IFRD2 −2kb: n = 23 cells, n = 25 cells; DRB IFRD2 −2kb: n = 12 cells, n = 27 cells; DMSO MCM2 −1kb: n = 24 cells, n = 35 cells; DRB MCM2 −1kb: n = 21 cells, n = 28 cells; DMSO MCM2 −2kb: n = 23 cells, n = 27 cells; DRB MCM2 −2kb: n = 18 cells, n = 30 cells; DMSO DISTAL#1: n = 19 cells, n = 30 cells; DRB DISTAL#1: n = 23 cells, n = 24 cells. The error bar is the standard deviation. Source data are provided as a Source Data file.

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