RESUMEN
The cohesin complex spatially organizes interphase chromatin by bringing distal genomic loci into close physical proximity, looping out the intervening DNA. Mutation of cohesin complex subunits is observed in cancer and developmental disorders, but the mechanisms through which these mutations may contribute to disease remain poorly understood. Here, we investigate a recurrent missense mutation to the hinge domain of the cohesin subunit SMC1A, observed in acute myeloid leukemia. Engineering this mutation into murine embryonic stem cells caused widespread changes in gene expression, including dysregulation of the pluripotency gene expression program. This mutation reduced cohesin levels at promoters and enhancers, decreased DNA loops and interactions across short genomic distances, and weakened insulation at CTCF-mediated DNA loops. These findings provide insight into how altered cohesin function contributes to disease and identify a requirement for the cohesin hinge domain in three-dimensional chromatin structure.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Regulación Neoplásica de la Expresión Génica , Mutación , Neoplasias/genética , Dominios y Motivos de Interacción de Proteínas , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos , Perfilación de la Expresión Génica , Histonas , Ratones , Neoplasias/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , CohesinasRESUMEN
Cohesin is a ring-shaped protein complex that controls dynamic chromosome structure. Cohesin activity is important for a variety of biological processes, including formation of DNA loops that regulate gene expression. The precise mechanisms by which cohesin shapes local chromosome structure and gene expression are not fully understood. Recurrent mutations in cohesin complex members have been reported in various cancers, though it is not clear whether many cohesin sequence variants have phenotypes and contribute to disease. Here, we utilized CRISPR/Cas9 genome editing to introduce a variety of cohesin sequence variants into murine embryonic stem cells and investigate their molecular and cellular consequences. Some of the cohesin variants tested caused changes to transcription, including altered expression of gene encoding lineage-specifying developmental regulators. Altered gene expression was also observed at insulated neighborhoods, where cohesin-mediated DNA loops constrain potential interactions between genes and enhancers. Furthermore, some cohesin variants altered the proliferation rate and differentiation potential of murine embryonic stem cells. This study provides a functional comparison of cohesin variants found in cancer within an isogenic system, revealing the relative roles of various cohesin perturbations on gene expression and maintenance of cellular identity.
Asunto(s)
Proteínas de Ciclo Celular/genética , Diferenciación Celular , Proteínas Cromosómicas no Histona/genética , Regulación Neoplásica de la Expresión Génica , Mutación , Neoplasias/genética , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Proteínas Cromosómicas no Histona/metabolismo , Elementos de Facilitación Genéticos , Masculino , Ratones , Ratones Endogámicos C57BL , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Neoplasias/metabolismo , CohesinasRESUMEN
Chromosome structure is a key regulator of gene expression. CTCF and cohesin play critical roles in structuring chromosomes by mediating physical interactions between distant genomic sites. The resulting DNA loops often contain genes and their cis-regulatory elements. Despite the importance of DNA loops in maintaining proper transcriptional regulation and cell identity, there is limited understanding of the molecular mechanisms that regulate their dynamics and function. We report a previously unrecognized role for WIZ (widely interspaced zinc finger-containing protein) in DNA loop architecture and regulation of gene expression. WIZ forms a complex with cohesin and CTCF that occupies enhancers, promoters, insulators, and anchors of DNA loops. Aberrant WIZ function alters cohesin occupancy and increases the number of DNA loop structures in the genome. WIZ is required for proper gene expression and transcriptional insulation. Our results uncover an unexpected role for WIZ in DNA loop architecture, transcriptional control, and maintenance of cell identity.
Asunto(s)
Factor de Unión a CCCTC/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Sitios de Unión/genética , Cromatina , Inmunoprecipitación de Cromatina/métodos , Cromosomas/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias , Expresión Génica/genética , Regulación de la Expresión Génica/genética , Genoma/genética , Humanos , Masculino , Ratones , Regiones Promotoras Genéticas/genética , CohesinasRESUMEN
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.