Abstract
Cohesin is a multiprotein ring complex that regulates 3D genome organization, sister chromatid cohesion, gene expression, and DNA repair. Cohesin is known to be ubiquitinated, although the mechanism, regulation, and effects of cohesin ubiquitination remain poorly defined. We previously used gene editing to introduce a dual epitope tag into the endogenous allele of each of 11 known components of cohesin in human HCT116 cells. Here we report that mass spectrometry analysis of dual-affinity purifications identified the USP13 deubiquitinase as a novel cohesin-interacting protein. Subsequent immunoprecipitation/Western blots confirmed the endogenous interaction in HCT116, 293T, HeLa, and RPE-hTERT cells; demonstrated that the interaction occurs specifically in the soluble nuclear fraction (not in the chromatin); requires the ubiquitin-binding domains (UBA1/2) of USP13; and occurs preferentially during DNA replication. Reciprocal dual-affinity purification of endogenous USP13 followed by mass spectrometry demonstrated that cohesin is its primary interactor in the nucleus. Ectopic expression and CRISPR knockout of USP13 showed that USP13 is paradoxically required for both deubiquitination and ubiquitination of cohesin subunits in human cells. USP13 was dispensable for sister chromatid cohesion in HCT116 and HeLa cells, whereas it was required for the dissociation of cohesin from chromatin as cells transit through mitosis. Together these results identify USP13 as a new cohesin-interacting protein that regulates the ubiquitination of cohesin and its cell cycle regulated interaction with chromatin.
Highlights
The cohesin complex is regulated by posttranslational modification
Ubiquitin-Specific Protease 13 (USP13) was identified by mass spectrometry in 5 of 11 independent dual-affinity purifications, using dual epitope– tagged endogenous SMC1A, SMC3, STAG2, WAPL, and
To further confirm the interaction between USP13 and cohesin, endogenous cohesin was immunoprecipitated with SMC3 antibodies from whole cell lysates prepared from genetically unmodified HCT116 cells and Western blot performed with USP13 antibodies
Summary
The cohesin complex is regulated by posttranslational modification. Acetylation of the SMC3 subunit is its most wellstudied modification and is required for establishment of sister chromatid cohesion [9, 10]. To further confirm the interaction between USP13 and cohesin, endogenous cohesin was immunoprecipitated with SMC3 antibodies from whole cell lysates prepared from genetically unmodified HCT116 cells and Western blot performed with USP13 antibodies (and STAG2 antibodies as a positive control).
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