RESUMO
The investigation of cohesin binding sites throughout different mammalian genomes by ChIP-sequencing has been fundamental to discover how cohesin and CTCF collaborate to form chromatin loops and to gain insight in the intricate regulation of cohesin. Here we describe a detailed ChIP protocol that has been successfully used for different cohesin subunits and cohesin regulators in various cell lines.
Assuntos
Cromatina , Proteínas Cromossômicas não Histona , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Mamíferos/metabolismo , CoesinasRESUMO
The NIPBL/MAU2 heterodimer loads cohesin onto chromatin. Mutations in NIPBL account for most cases of the rare developmental disorder Cornelia de Lange syndrome (CdLS). Here we report a MAU2 variant causing CdLS, a deletion of seven amino acids that impairs the interaction between MAU2 and the NIPBL N terminus. Investigating this interaction, we discovered that MAU2 and the NIPBL N terminus are largely dispensable for normal cohesin and NIPBL function in cells with a NIPBL early truncating mutation. Despite a predicted fatal outcome of an out-of-frame single nucleotide duplication in NIPBL, engineered in two different cell lines, alternative translation initiation yields a form of NIPBL missing N-terminal residues. This form cannot interact with MAU2, but binds DNA and mediates cohesin loading. Altogether, our work reveals that cohesin loading can occur independently of functional NIPBL/MAU2 complexes and highlights a novel mechanism protective against out-of-frame mutations that is potentially relevant for other genetic conditions.