A gene desert required for regulatory control of pleiotropicShox2expression and embryonic survival

Abstract

ABSTRACTThe Shox2 homeodomain transcriptional regulator is known for its critical functions during mouse embryogenesis, enabling accurate development of limbs, craniofacial structures, neural populations and the cardiac conduction system. At the genomic level, theShox2gene is flanked by an extensive gene desert, a continuous non-coding genomic region spanning over 500 kilobases that contains a multitude of evolutionarily conserved elements with predictedcis-regulatory activities. However, the transcriptional enhancer potential of the vast majority of these elements in combination with the biological necessity of the gene desert have not yet been explored. Using transgenic reporter assays in mouse embryos to validate an extensive set of stringent epigenomic enhancer predictions, we identify several novel gene desert enhancers with distinct tissue-specific activities inShox2expressing tissues. 4C-seq chromatin conformation capture further uncovers a repertoire of gene desert enhancers with overlapping activities in the proximal limb, in a compartment essential forShox2-mediated stylopod formation. Leveraging CRISPR/Cas9 to delete the gene desert region contained in theShox2topologically associated domain (TAD), we demonstrate that this complexcis-regulatory platform is essential for embryonic survival and required for control of region-specificShox2expression in multiple developing tissues. While transcription ofShox2in the embryonic limb is only moderately affected by gene desert loss,Shox2expression in craniofacial and cardiac domains is nearly abolished. In particular,Shox2transcripts in the sinus venosus (SV) encompassing the sinoatrial node (SAN) were depleted in embryos lacking the gene desert, likely accounting for the embryonic lethality due toShox2-dependency of the SAN pacemaker. Finally, we discover a 1.5kb SV enhancer within the deleted gene desert region, which may act as a genomic module controlling the development of the cardiac conduction system. In summary, our results identify a gene desert indispensable for pleiotropic patterning and highlight the importance of these extensive regulatory landscapes for embryonic development and viability.