RESUMEN
Alternative splicing (AS) is a key process underlying the expansion of proteomic diversity and the regulation of gene expression. Here, we identify an evolutionarily conserved embryonic stem cell (ESC)-specific AS event that changes the DNA-binding preference of the forkhead family transcription factor FOXP1. We show that the ESC-specific isoform of FOXP1 stimulates the expression of transcription factor genes required for pluripotency, including OCT4, NANOG, NR5A2, and GDF3, while concomitantly repressing genes required for ESC differentiation. This isoform also promotes the maintenance of ESC pluripotency and contributes to efficient reprogramming of somatic cells into induced pluripotent stem cells. These results reveal a pivotal role for an AS event in the regulation of pluripotency through the control of critical ESC-specific transcriptional programs.
Asunto(s)
Empalme Alternativo , Reprogramación Celular , Células Madre Embrionarias/metabolismo , Factores de Transcripción Forkhead/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células Madre Pluripotentes/metabolismo , Proteínas Represoras/metabolismo , Animales , ADN/metabolismo , Células Madre Embrionarias/citología , Genes Homeobox , Humanos , Ratones , Células Madre Pluripotentes/citología , Isoformas de Proteínas/metabolismoRESUMEN
Previous investigations of the core gene regulatory circuitry that controls the pluripotency of embryonic stem (ES) cells have largely focused on the roles of transcription, chromatin and non-coding RNA regulators. Alternative splicing represents a widely acting mode of gene regulation, yet its role in regulating ES-cell pluripotency and differentiation is poorly understood. Here we identify the muscleblind-like RNA binding proteins, MBNL1 and MBNL2, as conserved and direct negative regulators of a large program of cassette exon alternative splicing events that are differentially regulated between ES cells and other cell types. Knockdown of MBNL proteins in differentiated cells causes switching to an ES-cell-like alternative splicing pattern for approximately half of these events, whereas overexpression of MBNL proteins in ES cells promotes differentiated-cell-like alternative splicing patterns. Among the MBNL-regulated events is an ES-cell-specific alternative splicing switch in the forkhead family transcription factor FOXP1 that controls pluripotency. Consistent with a central and negative regulatory role for MBNL proteins in pluripotency, their knockdown significantly enhances the expression of key pluripotency genes and the formation of induced pluripotent stem cells during somatic cell reprogramming.
Asunto(s)
Empalme Alternativo , Reprogramación Celular , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteínas de Unión al ARN/metabolismo , Empalme Alternativo/genética , Secuencias de Aminoácidos , Animales , Diferenciación Celular/genética , Línea Celular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Factores de Transcripción Forkhead/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Cinética , Ratones , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas Represoras/metabolismoRESUMEN
How species with similar repertoires of protein-coding genes differ so markedly at the phenotypic level is poorly understood. By comparing organ transcriptomes from vertebrate species spanning ~350 million years of evolution, we observed significant differences in alternative splicing complexity between vertebrate lineages, with the highest complexity in primates. Within 6 million years, the splicing profiles of physiologically equivalent organs diverged such that they are more strongly related to the identity of a species than they are to organ type. Most vertebrate species-specific splicing patterns are cis-directed. However, a subset of pronounced splicing changes are predicted to remodel protein interactions involving trans-acting regulators. These events likely further contributed to the diversification of splicing and other transcriptomic changes that underlie phenotypic differences among vertebrate species.