Extended Data Fig. 5: Co-transcriptional DNA:RNA hybrids accumulation in DIS3-deficient cells impairs optimal RAD21 localization.
a. Model suggesting impaired cohesin scanning in the presence of DNA:RNA hybrids in Dis3C/C cells. While DIS3-proficient cells resolve DNA:RNA hybrids using DIS3 activity, DIS3-deficient cells accumulate these structures (and associated proteins) which impair cohesin scanning (and/or decreases the processing speed of the cohesin complex) and ultimately perturb loop extrusion. Transcription inhibitors decrease the amount of co-transcriptional DNA:RNA hybrids and potentially restore cohesin positioning, even in the absence of DIS3. RAD21 ChIP–sequencing in activated B cells using transcription inhibitors. Rosacre/+ Dis3C/+ and Rosacre/+ Dis3C/C cells were treated with IL4 for 24 h and with transcription (TC) inhibitors or DMSO as control for 4 h before fixation (one experiment). b. Number and percentage of RAD21 peaks decreased in the absence of DIS3 and rescued after transcription inhibitors treatments. All peaks with a fold increase >1 after TC inhibitors treatment were considered. c. Genome-wide analysis of RAD21 binding with or without transcription inhibitors in Dis3C/C. Here we focused on RAD21 peaks which were decreased by 2-fold compare to the control Dis3C/+ without treatment. In these conditions 1,012 peaks show efficient rescue. The box and whiskers plots represent these values, the bottom and top whiskers represent the minimum and maximum scores, respectively, outside the interquartile range. The box plots show the lower quartile, median and upper quartile. This experiment was performed one time, two-tailed Wilcoxon rank-sum test. d to i. Multiple examples of RAD21 restoration after transcription inhibitor treatment. IGV tracks show the defect of RAD21 positioning in the absence of DIS3 (Dis3C/+ versus Dis3C/C, grey tracks) while transcription inhibitors restore RAD21 localization even in the absence of DIS3 (blue tracks). Black arrows indicate the RAD21 peaks which are strongly affected by DIS3-deficiency and rescued after transcription inhibitors treatments.
