A Sox2 Enhancer Cluster Regulates Region-Specific Neural Fates from Mouse Embryonic Stem Cells

Abstract

Embryonic development depends on spatially and temporally orchestrated gene regulatory networks. Expressed in neural stem and progenitor cells (NSPCs), the transcription factor sex-determining region Y box 2 (Sox2) is critical for embryogenesis and stem cell maintenance in neural development. Whereas Sox2 is regulated by a distal cluster of enhancers in embryonic stem cells (ESCs), enhancers closer to the gene have been implicated in Sox2 transcriptional regulation in the neural lineage. Using functional genomics data, and deletion analysis we show that a downstream enhancer cluster regulates Sox2 transcription in NSPCs derived from mouse ESCs. By generating allelic mutants using CRISPR-Cas9 mediated deletions, we show that this proximal enhancer cluster, termed Sox2 regulatory regions 2-18 (SRR2-18), is a cis regulator of Sox2 transcription during neural differentiation. Transcriptome analyses demonstrate that loss of even one copy of SRR2-18 disrupts the region-specific identity of NSPCs. Biallelic deletion of this Sox2 neural enhancer cluster causes reduced SOX2 protein, less frequent interaction with transcriptional machinery, and leads to perturbed chromatin accessibility genome-wide further affecting the expression of neurodevelopmental and anterior-posterior regionalization genes. Furthermore, homozygous NSPC deletants exhibit self-renewal defects and impaired differentiation into cell types found in the brain. Altogether, our data define a cis-regulatory enhancer cluster controlling Sox2 transcription in NSPCs and highlight the sensitivity of neural differentiation processes to decreased Sox2 transcription, which influences their differentiation into posterior neural fates, specifically the caudal neural tube.