Figure 5. Cohesin contributes to transcription repression propagation but does not regulate the repression of the damaged gene.

Cells were treated with siRNAs to knock down SCC1 of cohesin. The DSB was induced by vfCRISPR in a live-cell transcription reporter (Figure 1A) stably integrated in U-2 OS cells (A–C) or endogenous ACTB in HEK293T cells (D).

(A) Heatmaps of TS and 53BP1 intensities were measured from live-cell imaging as in Figure 1 (siSCCI, n = 74 cells; siNeg, n = 98 cells). Cells with 53BP1 recruitment were selected.

(B) Percentages of repressed cells were measured for knockdown samples and control samples during 30 min tracking. Cells were considered repressed if the averaged TS intensities in the last 4 frames were lower than a threshold determined from siNeg control cells with 53BP1 recruitment. Error bar: SEM (n = 3 biological replicates).

(C) Transcription decay and response times were obtained by fitting the delayed decay model (Figure 1G) to the repressed TS intensities in (B) (siSCCI, n = 49 cells; siNeg, n = 73 cells). Error bar: quartiles. Unpaired Mann-Whitney test was performed.

(D) ChIP-qPCR experiments of RNAPII were performed in HEK293T cells when ACTB locus was damaged by vfCRISPR. Change of RNAPII occupancy (measured in RPM) upon light stimulation on ACTB locus and nearby genes were measured in siSCCI and siNeg samples. GAPDH on a different chromosome served as a control gene to indicate global transcriptome change upon UV exposure. FSCN1: 70 kb from DSB. PMS2: 487 kb from DSB. Error bar: SEM (n = 2 biological replicates).

(E) Working model: DSB induces rapid transcription repression, regulated by proteasome-mediated RNAPII removal on the damaged and surrounding genes.