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

Abstract

The 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, the Shox2 gene 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 predicted cis-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 in Shox2 expressing 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 for Shox2-mediated stylopod formation. Leveraging CRISPR/Cas9 to delete the gene desert region contained in the Shox2 topologically associated domain (TAD), we demonstrate that this complex cis-regulatory platform is essential for embryonic survival and required for control of region-specific Shox2 expression in multiple developing tissues. While transcription of Shox2 in the embryonic limb is only moderately affected by gene desert loss, Shox2 expression in craniofacial and cardiac domains is nearly abolished. In particular, Shox2 transcripts 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 to Shox2-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.

Temporal constraints on enhancer usage shape the regulation of limb gene transcription

“…Prior studies have tested 13 of these enhancers in vivo , with five driving limb reporter activity at specific embryonic stages, validating our approach for enhancer identification ( Fig. 4B, Supplementary Table S4 ) (Abassah-Oppong et al, 2023; Osterwalder et al, 2018). These findings suggest that a significant number of early enhancers is required to initiate and maintain Shox2 transcription during the early stages of limb budding, while fewer are necessary to sustain expression at later stages.…”

“…The light gray box next to the Shox2 gene is the Rsrc1 gene. B. Schematic representation of the pattern displayed by enhancers previously validated through in vivo LacZ reporter assays (Abassah-Oppong et al, 2023; Osterwalder et al, 2018).…”

“…Enhancers bear the ability to be bound by transcription factors integrating the cellular environments and ultimately to translate it into an integrated transcriptional output (Spitz and Furlong, 2012). Within the same chromatin domains known as Topologically Associating Domains (TADs), genes can dynamically interact with multiple enhancers, referred to as enhancer repertoires, collectively shaping their expression patterns (Abassah-Oppong et al, 2023; Andrey and Mundlos, 2017; Furlong and Levine, 2018; Kvon et al, 2021; Robson et al, 2019; Stadhouders et al, 2019). While multiple studies have explored how distinct enhancers may act with distinct or overlapping spatial activities (Frankel et al, 2010; Kvon et al, 2014; Osterwalder et al, 2018; Perry et al, 2010; Petit et al, 2017; Robson et al, 2019; Werner et al, 2007), their temporal specificities remain less clear.…”

“…The regulatory landscape of Shox2 is embedded within a 1.1 Mb TAD that splits into a centromeric side featuring a 500 kb gene desert and a telomeric side, largely composed of the introns of the Rsrc1 gene. Numerous limb enhancers have been identified on both the telomeric and centromeric sides, collectively contributing to Shox2 limb expression (Abassah-Oppong et al, 2023; Osterwalder et al, 2018; Rosin et al, 2013; Ye et al, 2016). Although these enhancer regions have been shown to drive expression in the limb and other Shox2 -expressing tissues, the specific time windows of their activity have not been determined.…”

A single-cell transcriptome atlas of human early embryogenesis

“…In situ data on limb staged from E9.5 to E11.5 mouse were collected from literatures and the EMBRYS database [37][38][39][40][41][42][43][44][45][46] (Extended Data Fig. [10][11].…”