Distinct and overlapping roles of STAG1 and STAG2 in cohesin localization and gene expression in embryonic stem cells

Abstract

Background: The three-dimensional organization of the genome in the nucleus plays an integral role in many biological processes, including gene expression. The genome is folded into DNA loops that bring together distal regulatory elements and genes. Cohesin, a ring-shaped protein complex, is a major player in the formation of DNA loops. Cohesin is composed of a core trimer and one of two variant STAG subunits, STAG1 or STAG2. It is not understood whether variant STAG proteins give rise to cohesin complexes with distinct functions. Recent studies have begun to characterize the roles of STAG1 and STAG2, with partially contradictory results. Results: Here, we generate stable single-knockout embryonic stem cell lines to investigate the individual contributions of STAG1 and STAG2 in regulating cohesin chromosomal localization and function. We report both overlapping roles for STAG1 and STAG2 in cohesin localization and somewhat distinct roles in gene expression. STAG1 and STAG2 occupy the same sites across the genome, yet do not exist together in a higher order complex. Despite their shared localization, STAG1 and STAG2 have both distinct and redundant effects on gene expression. Loss of both STAG1 and STAG2 causes widespread transcriptome dysregulation, altered cohesin DNA occupancy, and reduced cell proliferation. Conclusions: Together, this work reveals the requirement of at least one STAG protein for proper cohesin function. STAG1 and STAG2 have independent roles in cohesin localization and both overlapping and distinct roles in gene expression. The roles of STAG1 and STAG2 in mouse embryonic stem cells may be somewhat different than in other cell types, due to their relative expression levels. These results advance our understanding of the link between mammalian genome organization and gene expression during development and disease contexts.

A cohesive look at leukemogenesis: The cohesin complex and other driving mutations in AML

“…Concurrent knockout of both Stag1 and Stag2 is lethal, with mice rapidly developing bone marrow aplasia and pancytopenia, 44 a situation which phenocopies Smc3 loss. 31 However, many recent studies have investigated subunit-based differences in double-stranded DNA repair 51 as well as gene looping and transcriptional control in various cell types, including murine HSPCs, 44 ESCs 54 and AML cell lines. 55 In 2018, the first study to describe differential roles for STAG1 versus STAG2 in chromosome organization (in a variety of human cell types) identified differential interactions with CTCF sites, where both STAG1 and STAG2 are known to bind.…”

“…Interestingly, while deletion of either STAG1 / 2 alone leads to differential gene expression, deletion of both subunits enhances gene dysregulation. 54 …”

Functional impact of cancer-associated cohesin variants on gene expression and cellular identity

“…Murine embryonic stem cells (V6.5, male, derived from a C57BL/6(F) × 129/sv(M) cross) were grown under standard ESC culture conditions ( Arruda et al 2020 ; Justice et al 2020 ). mESC media contained DMEM KO (Thermo Fisher Scientific, 10829-018), 15% fetal bovine serum (VWR, 97068-085), homemade leukemia inducing factor (LIF), 100 U/ml penicillin, 100 µg/ml streptomycin (Thermo Fisher Scientific, 15140-122), 100 µM beta-mercaptoethanol (Thermo Fisher Scientific, 21-985-023), 1× Non-essential amino acids (Thermo Fisher Scientific, 11140-050), and 1× Glutamax (Thermo Fisher Scientific, 35050-061).…”

“…Genome-edited mESC lines were generated as previously described, with modifications ( Arruda et al 2020 ; Justice et al 2020 ). mESCs were transfected with a single-stranded donor oligonucleotide (ssODN) repair template, and plasmids encoding a synthetic guide RNA (sgRNA), Cas9 and a fluorescent gene (eGFP or mCherry) using Lipofectamine 2000 (Thermo Fisher, 11-668-027).…”

“…Cohesin subunits STAG2 and SMC1A acquire somatic mutations in diverse tumor types, including bladder cancer and myeloid neoplasia ( Kon et al 2013 ; Ley et al 2013; Kim et al 2016 ). The STAG2 gene is X-encoded and is a frequent target of inactivating mutations, which are only partially compensated for by its paralogue, STAG1 ( Hill et al 2016 ; Arruda et al 2020 ). Some cancers with inactivated STAG2 exhibit decreased cell viability and disrupted cell cycle, but conflicting data exist on whether these defects are attributable to cohesin’s role in mediating sister chromatid cohesion ( Solomon et al 2011 , 2013 ; Balbás-Martínez et al 2013 ; Lawrence et al 2014 ; Taylor et al 2014 ; Mullenders et al 2015 ; Kim et al 2016 ; Viny and Levine 2018 ; Benedict et al 2020 ).…”