RESUMEN
Long non-coding RNAs (lncRNAs) are an emerging class of transcripts that can modulate gene expression; however, their mechanisms of action remain poorly understood. Here, we experimentally determine the secondary structure of Braveheart (Bvht) using chemical probing methods and show that this â¼590 nt transcript has a modular fold. Using CRISPR/Cas9-mediated editing of mouse embryonic stem cells, we find that deletion of 11 nt in a 5' asymmetric G-rich internal loop (AGIL) of Bvht (bvhtdAGIL) dramatically impairs cardiomyocyte differentiation. We demonstrate a specific interaction between AGIL and cellular nucleic acid binding protein (CNBP/ZNF9), a zinc-finger protein known to bind single-stranded G-rich sequences. We further show that CNBP deletion partially rescues the bvhtdAGIL mutant phenotype by restoring differentiation capacity. Together, our work shows that Bvht functions with CNBP through a well-defined RNA motif to regulate cardiovascular lineage commitment, opening the door for exploring broader roles of RNA structure in development and disease.
Asunto(s)
Células Madre Embrionarias de Ratones/metabolismo , Miocitos Cardíacos/metabolismo , ARN Largo no Codificante/genética , Proteínas de Unión al ARN/genética , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Composición de Base , Secuencia de Bases , Sitios de Unión , Proteína 9 Asociada a CRISPR , Diferenciación Celular , Linaje de la Célula/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Endonucleasas/genética , Endonucleasas/metabolismo , Eliminación de Gen , Edición Génica , Regulación de la Expresión Génica , Humanos , Ratones , Células Madre Embrionarias de Ratones/citología , Miocitos Cardíacos/citología , Conformación de Ácido Nucleico , Fenotipo , Unión Proteica , ARN Largo no Codificante/química , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/metabolismo , Transducción de SeñalRESUMEN
The classic model of eukaryotic gene expression requires direct spatial contact between a distal enhancer and a proximal promoter. Recent Chromosome Conformation Capture (3C) studies show that enhancers and promoters are embedded in a complex network of looping interactions. Here we use a polymer model of chromatin fiber to investigate whether, and to what extent, looping interactions between elements in the vicinity of an enhancer-promoter pair can influence their contact frequency. Our equilibrium polymer simulations show that a chromatin loop, formed by elements flanking either an enhancer or a promoter, suppresses enhancer-promoter interactions, working as an insulator. A loop formed by elements located in the region between an enhancer and a promoter, on the contrary, facilitates their interactions. We find that different mechanisms underlie insulation and facilitation; insulation occurs due to steric exclusion by the loop, and is a global effect, while facilitation occurs due to an effective shortening of the enhancer-promoter genomic distance, and is a local effect. Consistently, we find that these effects manifest quite differently for in silico 3C and microscopy. Our results show that looping interactions that do not directly involve an enhancer-promoter pair can nevertheless significantly modulate their interactions. This phenomenon is analogous to allosteric regulation in proteins, where a conformational change triggered by binding of a regulatory molecule to one site affects the state of another site.