RESUMEN
Copy number variations (CNVs) often include noncoding sequences and putative enhancers, but how these rearrangements induce disease is poorly understood. Here we investigate CNVs involving the regulatory landscape of IHH (encoding Indian hedgehog), which cause multiple, highly localized phenotypes including craniosynostosis and synpolydactyly. We show through transgenic reporter and genome-editing studies in mice that Ihh is regulated by a constellation of at least nine enhancers with individual tissue specificities in the digit anlagen, growth plates, skull sutures and fingertips. Consecutive deletions, resulting in growth defects of the skull and long bones, showed that these enhancers function in an additive manner. Duplications, in contrast, caused not only dose-dependent upregulation but also misexpression of Ihh, leading to abnormal phalanges, fusion of sutures and syndactyly. Thus, precise spatiotemporal control of developmental gene expression is achieved by complex multipartite enhancer ensembles. Alterations in the composition of such clusters can result in gene misexpression and disease.
Asunto(s)
Enfermedades del Desarrollo Óseo/genética , Elementos de Facilitación Genéticos/genética , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Hedgehog/fisiología , Osteogénesis/genética , Animales , Secuencia de Bases , Variaciones en el Número de Copia de ADN , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Deformidades Congénitas del Pie/genética , Eliminación de Gen , Dosificación de Gen , Duplicación de Gen , Técnicas de Inactivación de Genes , Genes Reporteros , Proteínas Hedgehog/deficiencia , Proteínas Hedgehog/genética , Ratones , Ratones Endogámicos C57BL , Polidactilia/genética , Secuencias Reguladoras de Ácidos Nucleicos , Análisis de Secuencia de ADN , Cráneo/anomalías , Transcripción GenéticaRESUMEN
Structural variations (SVs) contribute to the variability of our genome and are often associated with disease. Their study in model systems was hampered until now by labor-intensive genetic targeting procedures and multiple mouse crossing steps. Here we present the use of CRISPR/Cas for the fast (10 weeks) and efficient generation of SVs in mice. We specifically produced deletions, inversions, and also duplications at six different genomic loci ranging from 1.1 kb to 1.6 Mb with efficiencies up to 42%. After PCR-based selection, clones were successfully used to create mice via aggregation. To test the practicability of the method, we reproduced a human 500 kb disease-associated deletion and were able to recapitulate the human phenotype in mice. Furthermore, we evaluated the regulatory potential of a large genomic interval by deleting a 1.5 Mb fragment. The method presented permits rapid in vivo modeling of genomic rearrangements.
