Cohesin can remain associated with chromosomes during DNA replication

Abstract

SUMMARYTo ensure disjunction to opposite poles during anaphase, sister chromatids must be held together following DNA replication. This is mediated by cohesin, which is thought to entrap sister DNAs inside a tripartite ring composed of its Smc and kleisin (Scc1) subunits. How such structures are created during S phase is poorly understood, in particular whether they are derived from complexes that had entrapped DNAs prior to replication. To address this, we used selective photobleaching to determine whether cohesin associated with chromatin in G1 persists in situ after replication. We developed a non-fluorescent HaloTag ligand to discriminate the fluorescence recovery signal from labeling of newly synthesized Halo-tagged Scc1 protein (pulse-chase or pcFRAP). In cells where cohesin turnover is inactivated by deletion of WAPL, Scc1 can remain associated with chromatin throughout S phase. These findings suggest that cohesion might be generated by cohesin that is already bound to un-replicated DNA.

ESCO1/2's roles in chromosome structure and interphase chromatin organization

“…Remarkably, in esco1 esco2 wapl mutants, the cohesin staining on interphase chromatin also revealed a different structure, similar, at a glance, to the one reported by Tedeschi et al (2013) in G0-arrested MEFs or haploid human cells deleted for WAPL . Notably, this phenotype is not observed in equivalent single wapl mutants in interphase DT40 cells or in U2OS cells mutated for WAPL (Rhodes et al 2017). We suspect that these differences are intrinsic to the chromatin organization features and cohesin dynamics in proliferating versus G0-arrested cell lines and may also be influenced by the cohesin subunit detected and/or differences in the expression levels of ESCO1/2 in different systems or cell cycle phases (Tedeschi et al 2013;Haarhuis et al 2017).…”

“…S5B) as initially described in post-mitotic MEFs. Recently, U2OS cells deficient in WAPL were also reported to lack a clear vermicelli phenotype (Rhodes et al 2017). These differences are likely caused at least in part by the fact that DT40 and U2OS cells are dividing cells, and cohesin spends less time on chromatin before its removal by separase.…”

“…Different pools of cohesin binding have been described, with cohesin bound in G1 being more loosely associated with chromatin, whereas the acetylated cohesin was bound longer to chromatin and was resistant to removal by WAPL (Ladurner et al 2016;Rhodes et al 2017). We performed chromatin fractionation experiments using synchronously growing cells treated with Auxin for 6 h and enriched in interphase (see Fig.…”

Determining cellular CTCF and cohesin abundances to constrain 3D genome models

“…Indeed, it is currently the preferred choice for live-cell single molecule imaging. Combined with the development of Cas9-mediated genome-editing (Ran et al, 2013), endogenous Halotagging of proteins has thus become the gold standard (Chong et al, 2018;Hansen et al, 2017;Komatsubara et al, 2019;Rhodes et al, 2017aRhodes et al, , 2017bStevens et al, 2017;Teves et al, 2016aTeves et al, , 2018Youmans et al, 2018), because it avoids the now well-established limitations and potential artifacts associated with protein overexpression (Hansen et al, 2017;Shao et al, 2018;Teves et al, 2016a). Now that we have determined the absolute abundance of CTCF in U2OS cells and cross-validated it using FCS-calibrated confocal imaging ( Figure 1A-D, F),…”

CTCF and cohesin regulate chromatin loop stability with distinct dynamics

“…Finally, it came to our attention that during extended FRAP experiments (in the multi hour range), incomplete washout of Halo- or SNAP-dye can lead to artifactual FRAP recovery (Rhodes et al, 2017). This is most likely through dye binding to new protein produced after the bleach pulse.…”