4C-seq analysis of three-dimensional chromatin interactions at the mouse Shox2 locus during embryonic limb development

The SHOX2 transcription factor is required during mouse embryonic development for the formation of the proximal elements of limbs, the humerus and femur. The Shox2 gene is flanked by an extensive gene desert spanning over 500 kilobases (kb) that contains many evolutionarily conserved elements with predicted cis-regulatory activities. However, the transcriptional enhancer potential of the vast majority of these regions have not yet been assessed. Therefore, we have generated a map of the Shox2 regulatory landscape during limb development using chromosome conformation capture techniques (4C-Seq). We report that at least five enhancers, distributed over more than 500 kb interact with the Shox2 gene and control its expression in developing proximal limbs, as confirmed by transgenic mouse assays. Furthermore, by using two of the identified enhancer candidates as 4C-seq viewpoints, we also find evidence that three of these putative enhancers interact with each other as well as the Shox2 gene, perhaps forming a cooperative regulatory complex. We expect this study to provide insight into the regulation of the human SHOX gene, a closely-related homolog of Shox2, that is similarly flanked by a large gene desert. Notably, deletions within the gene desert downstream of human SHOX are implicated as a major cause of the limb deformities characteristic of Léri-Weill dyschondrosteosis. 4C-seq analysis was performed using a viewpoint adjacent to the Shox2 promoter to detect chromatin interactions in proximal limbs from wild-type E12.5 mouse embryos, using two independent replicates . A secondary 4C-seq analysis was performed with the same samples, using sequences of the putative enhancers LHB-A (LE2) and PDE4 (LE4) as viewpoints to confirm their interaction with Shox2.