A gene desert required for regulatory control of pleiotropic Shox2 expression and embryonic survival [C-HiC]
Gene deserts spanning more than 500kb of non-protein coding genomic sequence are considered evolutionarily ancient and stable and are enriched in the vicinity of developmental regulator genes (Ovcharenko 2005). These extensive genomic regions typically harbor numerous conserved elements with predicted gene regulatory potential pointing to critical tissue-specific functions during development. Nevertheless, the biological necessity and underlying funtional enhancer landscapes of most gene deserts near developmental transcription factors (TFs) remain unknown, and it is unclear how precise pleiotropic expression patterns emerge from gene desert sequence. Here, we investigated the cis-regulatory architecture and function of a gene desert flanking the mouse Shox2 transcriptional regulator which itself is essential for embryonic limb, craniofacial, and cardiac pacemaker development. By combining epigenomic enhancer prediction, transgenic reporter validation and region-specific chromatin capture (C-HiC), we define the embryonic in vivo enhancer landscape and chromatin topology of the Shox2 gene desert. Targeted and context-specific genomic deletions uncover the gene desert not only as a regulator of embryonic survival through enhancer-mediated control of cardiac Shox2 expression, but also link distinct subsets of tissue-specific gene desert enhancers to the regulation of craniofacial patterning and proximal limb development. Our results hence identify the Shox2 gene desert as a fundamental genomic unit indispensable for pleiotropic patterning, robust organ morphogenesis and embryonic development progression by serving as a dynamic hub for tissue-specific developmental enhancers. Overall design: We used region capture Hi-C (C-HiC) in mouse embryos (mus musculus) with a probe set covering a 3,5Mb genomic interval containing the Shox2 gene. We performed CHi-C from embryonic forelimbs, mandibular processes, and hearts of wildtype embryos at E11.5 to define the tissue-specific 3D chromatin structure at the Shox2 locus.