USP13 interacts with cohesin and regulates its ubiquitination in human cells

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.

Genetic variation as a long-distance modulator of RAD21 expression in humans

“…For example, both USP37 and USP32 can reduce resistance to cisplatin-targeting therapies in cancer 30 , suggesting the involvement of USP32 in drug resistance. Further evidence to this role of USP32 and cohesin has been found in a screen of deubiquitinating enzyme interactions, where USP32 had a high-confidence Protein-Protein interaction with SMC1A 26 (which has been found to be mono-ubiquitinated in mitosis 29 ). Additionally, USP32 mutations have been found in human leukemia cells, which frequently carry recurrent cohesin deficits, including mutations in SMC1 31 and RAD21 32 .…”

“…Ubiquitylation is a reversible process, removed by deubiquitylating enzymes, of which one class is the ubiquitin-specific proteases (USPs). Ubiquitination of the cohesin complex helps to regulate chromosome segregation and cohesion during mitotic progression through control of replication fork integrity and the cellular response to replication stress 28 , 29 . While previous findings have pointed to Ubiquitin-specific proteases USP13 and USP37, the mechanisms and effects of cohesin ubiquitination remain largely undefined 29 .…”

“…Ubiquitination of the cohesin complex helps to regulate chromosome segregation and cohesion during mitotic progression through control of replication fork integrity and the cellular response to replication stress 28 , 29 . While previous findings have pointed to Ubiquitin-specific proteases USP13 and USP37, the mechanisms and effects of cohesin ubiquitination remain largely undefined 29 . Of note, in mitosis RAD21 is poly-ubiquitinated (targeting RAD21 for degradation), but the role of USPs in this hasn’t been fully elucidated.…”

Genetic Variation as a Long-Distance Modulator of RAD21 Expression in Humans

“…For example, both USP37 and USP32 can reduce resistance to cisplatintargeting therapies in cancer 30 , suggesting the involvement of USP32 in drug resistance. Further evidence to this role of USP32 and cohesin has been found in a screen of deubiquitinating enzyme interactions, where USP32 had a high-confidence Protein-Protein interaction with SMC1A 26 (which has been found to be mono-ubiquitinated in mitosis 29 ). Additionally, USP32 mutations have been found in human leukemia cells, which frequently carry recurrent cohesin deficits, including mutations in SMC1 31 and RAD21 32 .…”

Role of USP13 in physiology and diseases

“…USP13 deficiency led to total loss of ubiquitin of Cohesin subunits rather than increased ubiquitination. Besides, the ubiquitination of Cohesin is essential for its disassociation from chromatin ( He et al, 2020 ). Therefore, USP13 is required for Cohesin ubiquitination and its release from chromatin during mitosis.…”