Restriction mechanism of peri-centromeric DNA loop expansion necessary to ensure accurate chromosome segregation [ChIP-seq]

Centromeres in budding yeast are surrounded by ~10-kb DNA loops generated by the cohesin complex. These peri-centromeric loops are shown to play a role in faithful chromosome segregation. It remains not fully understood how the dynamics of the peri-centromeric loops are controlled. Here we found that gene deletion of two cohesin regulators, Wpl1 and Eco1, resulted in larger peri-centromeric loops that connected the centromeres and genome regions up to ~ 300 kb distant in G2/M-arrested cells. Deletion of Wpl1 and Eco1 synergistically contributed to the loop size enlargement. ChIP-seq revealed that cohesin and its ATPase activator, Scc2, were colocalized at the anchor sites of the extended peri-centromeric loops, specifically in the double-deletion mutant Δwpl1 Δeco1. Consistently, acute depletion of Scc2 in the G2/M phase resulted in the disappearance of the extended loops. Given that Scc2 is bound prominently only at the centromeres in wild-type cells, our results suggest that Wpl1 and Eco1 jointly promote the dissociation of Scc2 from cohesin and inhibit excessive expansion of peri-centromeric DNA loop size. Notably, we found that nuclear division after benomyl block and release was significantly delayed in the Δwpl1 Δeco1 double-deletion mutant, and this delay was rescued by Scc2 depletion in the G2/M phase. This indicates that cohesin regulators cooperatively regulate the size of the peri-centromeric DNA loops to ensure faithful chromosome segregation. Chromatin immunoprecipitation-sequencing (ChIP-seq) targeting Scc1, Scc2 and Rpo21 in budding yeast S.cerevisiae in several conditions (wt, ∆eco1_∆wpl1, ∆wpl1, Gal-Ub-Eco1, Pds5-AID, etc.)