Extended Data Fig. 6: G2/M short-arrest and release protocol confirms that cohesive-cohesin alone does not form focalized peaks without extrusive-cohesin.
From: Extensive mutual influences of SMC complexes shape 3D genome folding
a, Schematic showing the G2/M short-arrest and release protocol to obtain condensin-deficient mitotic chromosomes loaded with cohesive-cohesin alone. b, Metaphase spread analysis using the G2/M short-arrest and release protocol showing extensive sister-chromatid cohesion in the WAPL replete mitotic cells with Sororin(9A) mutant. In comparison, cells without Sororin(9A) mutant lost the cohesion between sister chromatids. Two independent experiments were performed. Scale bar: 10 μm. c, Quantification of (b), showing the percentage of “closed arm” during mitosis for indicated conditions. Data are presented as mean values (n = 2 independent experiments for both conditions). d, Venn-diagrams showing very few RAD21 peak calls in condensin-deficient mitotic cells with cohesive- but not extrusive-cohesin (G2/M short-arrest and release protocol). e, Venn-diagrams showing very few Sororin(9A) peak calls in condensin-deficient mitotic cells with cohesive- but not extrusive-cohesin (G2/M short-arrest and release protocol). There are 13 peaks that do not fall into the three listed categories. f, CPM normalized browser tracks showing loss of flag-tagged Sororin(9A) (cohesive-cohesin) peaks in the condensin-deficient mitotic chromosomes without extrusive-cohesin. Samples obtained through original or short-arrest and released protocols were shown. Tracks for independent biological replicates were shown. g, CPM normalized density heatmap plots of the flag-tagged Sororin(9A), showing that cohesive-cohesin peaks were almost completely lost in the absence of extrusive-cohesin. Samples generated by both original or short-arrest and release protocols were shown. h, Bar graphs showing the dramatic loss of flag-tagged Sororin(9A) ChIP-seq signals in the absence of extrusive-cohesin. Peaks colocalized to CTCF, CRE or both were shown separately. Data are presented as mean values +/- SEM (n = 5, 2 and 3 biological replicates for extrusive-cohesin replete, extrusive-cohesin deficient (long-arrest/release) and extrusive-cohesin deficient (short-arrest/release) samples respectively). P values were calculated using two-sided student’s t-test.
