Unique roles of cohesin-SA2 in proliferation and gene regulation: impact on embryonic development

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 14-02-2020Cohesin is a multiprotein complex composed of four subunits: SMC1, SMC3, Rad21 and SA. Within its ring-shape, two DNA fibers can be held together. Cohesin is best known for its role in cohesion by stably entrapping sister chromatids. In addition, cohesin can also form loops by bringing together distal regions from the same chromatid. This property establishes cohesin as a major organizer of interphase chromatin, which is essential for gene regulation among other processes. In somatic vertebrate cells, the SA subunit can be either SA1 or SA2, thus giving rise to two coexisting cohesin variants: cohesin-SA1 and cohesin-SA2. Despite their similarity, both proteins are functionally non-redundant, best exemplified by the lethality of SA1-null embryos. STAG2, the gene encoding SA2, is one of the most frequently mutated genes across multiple cancer types, and recently germline mutations have also been identified in developmental syndromes known as cohesinopathies. In this Thesis, we generated a Stag2 conditional knock out (cKO) mouse model to shed light onto the functional specificities of cohesin variants and address the consequences of SA2 loss both in vitro, in murine embryonic fibroblasts (MEFs), and in vivo, for embryonic development. We found that SA2 is dispensable in vitro, but its depletion leads to slower proliferation and reduced fidelity of chromosome segregation. We also show that the genome wide distribution of cohesin-SA2 in MEFs is not identical to that of cohesin-SA1. While both complexes can be found at sites also bound by the insulator protein CTCF, a fraction of cohesin-SA2 is present at enhancers independently of CTCF. These sites cannot be occupied by cohesin-SA1 even when SA2 is absent, which likely alters gene expression. We demonstrate that the association of cohesin-SA2 with chromatin is more dynamic than that of cohesin-SA1, a feature that likely explain the distinct distribution of the two variants and the different transcriptional signatures in MEFs lacking SA1 or SA2. Despite being dispensable for cell viability in vitro, cohesin-SA2 is critical for embryonic development. SA2-null embryos die by mid-gestation, displaying a systemic developmental delay along with specific heart defects that result from a general reduction in proliferation and impaired morphogenesis resulting from altered transcription. We uncovered that a particular pool of cardiac progenitors requires SA2 to efficiently deploy into the elongating heart tube and contribute to its differentiation. These results support a unique role for cohesin-SA2 during embryogenesis that cannot be assumed by cohesin-SA1, and suggest a causal relationship between STAG2 mutations and cardiac anomalies in cohesinopathy patients.La cohesina es un complejo multiproteico en forma de anillo compuesto por SMC1, SMC3, Rad21 y SA, que abraza la fibra de ADN. Además de su papel en el apareamiento de cromátidas hermanas o cohesión, puede formar lazos de cromatina al abrazar regiones distales de una misma cromátida. Esta propiedad establece a la cohesina como un importante organizador de la cromatina interfásica, esencial para la regulación génica entre otros procesos. En células somáticas de organismos vertebrados, la subunidad SA puede ser SA1 o SA2, dando lugar a dos variantes que coexisten en todas las células: cohesina-SA1 y cohesina-SA2. A pesar de su similitud, los dos complejos no son redundantes, siendo la mejor prueba de ello la letalidad de los embriones murinos carentes de SA1. STAG2, el gen que codifica SA2, es uno de los más mutados en cáncer, y recientemente se han identificado mutaciones en la línea germinal en pacientes de síndromes de desarrollo conocidos como cohesinopatías. En esta Tesis hemos generado un modelo de ratón knock out condicional de Stag2 para esclarecer la especificidad funcional de las variantes de cohesina y estudiar las consecuencias de eliminar SA2 tanto in vitro, en fibroblastos embrionarios de ratón, como in vivo, durante el desarrollo embrionario. Observamos que SA2 es dispensable in vitro, pero su depleción conlleva una proliferación más lenta y una menor fidelidad en segregación cromosómica. También mostramos que la distribución genómica de las dos variantes no es idéntica. Mientras ambas ocupan sitios donde también se une CTCF, una fracción de los complejos cohesina-SA2 se encuentra en enhancers independientemente de CTCF. Estos sitios no pueden ser ocupados por cohesina-SA1, incluso en ausencia de cohesina-SA2, lo que altera la expresión génica. Demostramos que la asociación de cohesina-SA2 a cromatina es más dinámica que la de cohesina-SA1, lo que probablemente determina su distribución genómica y contribuye a explicar que los cambios transcripcionales en células carentes de SA1 o SA2 sean muy diferentes. A pesar de ser dispensable para la viabilidad celular in vitro, la cohesina-SA2 es crítica para el desarrollo embrionario. En su ausencia, los embriones mueren a mitad del período de gestación y presentan un retraso de crecimiento sistémico y defectos específicos en el corazón. Estos defectos se deben a una menor proliferación y a alteraciones transcripcionales que afectan a la migración de una población de progenitores cardiacos hacia el tubo cardiaco durante su elongación. Así pues, hemos identificado un papel único de la cohesina-SA2 en la morfogénesis del corazón en el desarrollo embrionario que sugiere una relación causal entre mutaciones en STAG2 y las anomalías cardiacas de los pacientes de cohesinopatías

Additional file 4 of Contribution of variant subunits and associated factors to genome-wide distribution and dynamics of cohesin

Additional file 4: Movie S2. STAG2-GFP iFRAP

Additional file 5 of Contribution of variant subunits and associated factors to genome-wide distribution and dynamics of cohesin

Additional file 5: Movie S3. RAD21-GFP iFRAP

Distinct roles of cohesin-SA1 and cohesin-SA2 in 3D chromosome organization.

Two variant cohesin complexes containing SMC1, SMC3, RAD21 and either SA1 (also known as STAG1) or SA2 (also known as STAG2) are present in all cell types. We report here their genomic distribution and specific contributions to genome organization in human cells. Although both variants are found at CCCTC-binding factor (CTCF) sites, a distinct population of the SA2-containing cohesin complexes (hereafter referred to as cohesin-SA2) localize to enhancers lacking CTCF, are linked to tissue-specific transcription and cannot be replaced by the SA1-containing cohesin complex (cohesin-SA1) when SA2 is absent, a condition that has been observed in several tumors. Downregulation of each of these variants has different consequences for gene expression and genome architecture. Our results suggest that cohesin-SA1 preferentially contributes to the stabilization of topologically associating domain boundaries together with CTCF, whereas cohesin-SA2 promotes cell-type-specific contacts between enhancers and promoters independently of CTCF. Loss of cohesin-SA2 rewires local chromatin contacts and alters gene expression. These findings provide insights into how cohesin mediates chromosome folding and establish a novel framework to address the consequences of mutations in cohesin genes in cancer.We thank Y. Cuartero and J. Quilez (4D Genome-CRG) for technical help with the Hi-C experiments, D. Rico (Newcastle University), F.X. Real (CNIO) and M. Manzanares (CNIC) for comments on the manuscript, T. Hirano (RIKEN) and H. Yu (UT Southwestern) for reagents, and M. Quintela (CNIO) for MCF10A cells. This work has been supported by the Spanish Ministry of Economy and Competitiveness and FEDER funds (grant no. BFU2013-48481-R (A.L.), BFU2016-79841-R (A.L.) and BFU2013-47736-P (M.A.M.-R.), fellowship no. BES-2014-069166 (M.D.K.), and Centro de Excelencia Severo Ochoa grant no. SEV-2015-0510 (to CNIO) and SEV-2012-0208 (to CRG), the European Research Council (FP7/2010-2015, ERC grant agreement 609989; M.A.M.-R.), the EU Horizon 2020 Research and Innovation Program (agreement 676556; M.A.M.-R.), the CERCA Programme-Generalitat de Catalunya (M.A.M.-R.) and the La Caixa Foundation (PhD fellowship to A.K.).S