Identification and Validation of SOXB1 Bound Developmental Enhancers

Abstract

Enhancers are regions of non-coding DNA bound by transcription factors that influence gene expression, and are essential for the precise regulation of embryonic development. Many enhancers have been identified through reporter assays and bioinformatic techniques, but these are unable to show the functional contribution of the enhancer to endogenous expression. The SOXB1 proteins, expressed within the neural progenitor and spermatogonial stem cell populations are important TFs, the absence of which leads to developmental defects in both humans and mice. NES is an intermediate filament protein, and is thought to be regulated via a SOXB1 bound enhancer, commonly used in transgenic mice models to direct expression to NPCs. Through CRISPR mediated deletion of the Nes enhancer we show it is active from 9.5dpc in the CNS, and is responsible for up to 70% of endogenous Nes expression. Further, we identify possible trans activity of the enhancer, a new field of mammalian enhancer research. SOX3 ChIP-Seq identified a region upstream of the Wnt-receptor gene, Fzd3 that appeared to be an enhancer. Subsequent deletion via CRISPR confirmed enhancer activity in the CNS. Combined with LacZ reporter mouse models we show the enhancer directs expression specifically to the floor plate, an important region for axon guidance for which FZD3 is required. SOX3s role within the postnatal testes is largely unknown. Through ChIP-Seq we identified putative enhancers bound by SOX3, and present evidence that it is important in the regulation of the complex chromatin reorganisation that occurs during spermatogenesis. Together, this data presents new insights into the role of SOXB1s in development as well as highlighting the importance of functional validation of putative enhancers which can be achieved through CRISPR.