Fig 1: Cohesin Contributes to Transcriptional Repression at DNA Double-Strand Breaks(A) Cartoon of reporter construct (Tang et al., 2013) in which induction of the mCherry-tagged FokI endonuclease results in double-strand break (DSB) induction in a region upstream of a doxycycline-inducible reporter gene. Ongoing transcription of the reporter gene can be visualized by the presence of a YFP-MS2 fusion protein that binds stem-loop structures in the nascent transcript.(B, D, and H) Quantification of ongoing transcription in U2OS reporter cells (263 IFII) treated with small interfering RNA (siRNA) targeting SA2, PDS5B, BRG1, or ARID2 (B), SMC3, Rad21, SA2, or BRG1 (D), or SA2, SA1, or PDS5A (H). NTC, non-targeting control. After addition of doxycycline to induce transcription (Tx), transcriptional repression was monitored in cells with or without induction of the FokI endonuclease (DSB) by quantification of YFP-positive cells. Cells treated with 10 µM ATM inhibitor are indicated (ATMi). 150 cells were analyzed per condition per repeat. Data are presented as the mean ± SD; n = 4 (B), n = 3 (D and H). *p < 0.05, **p < 0.01 using Student’s t test.(C, E, and G) Western blot analysis of whole-cell extracts prepared from cells treated with siRNA targeting SA2, PDS5B, BRG1, or ARID2 (C), SMC3 or Rad21 (E), or SA2, SA1, or PDS5A (G). NTC, non-targeting control. a-Tubulin was used as a loading control.(F) Representative images of U2OS reporter cells analyzed in (B). Arrow indicates location of FokI-induced DSB (mCherry) and/or YFP-MS2 transcript.(I) Representative images of cells assayed for transcriptional activity by monitoring EU incorporation after DNA damage induced by laser microirradiation and treatment with the indicated siRNA.(J) Quantification of EU signal (new mRNA synthesis) across the path of laser micro-irradiation in cells treated as in (I). Data are presented as mean ± SEM. A minimum of 30 cells were analyzed per repeat (n = 3–5 biological repeats).See also Figure S1.