GSE99320
Sex reversal following the deletion of a single far upstream enhancer of Sox9.
Purpose: In this study we employed unbiased, genome wide techniques to identify novel enhancers of Sox9 that may cause Disorders of Sex Development (DSDs) when disrupted in the mouse. Methods: We performed ATAC-seq on 60K FACS-purified gonadal cells before and after sex determination to map nucleosome depleted regions (NDRs) indicative of regulatory elements. We then selected 16 putative enhancers present in Sertoli cells. To determine whether these are active enhancers, we performed ChIP-seq for H3K27ac, a histone modification that marks active enhancers. Transient transgenics was performed on select enhancers to determine whether they drive Sertoli-specific expression in vivo. Finally, we selected a single active Sertoli-specific enhancer to delete with CRISPR. Results: We identified a single enhancer upstream of Sox9 that causes complete male-to-female sex reversal in mice when deleted. Conclusions: Our study is the first to identify a single enhacer supstream of Sox9 which drives Sertoli-specific expression in vivo and causes complete male-to-female sex reversal when deleted in the mouse. Furthermore, this enhancer overlaps a region in humans (XY SR) associated to DSDs. Overall design: ATAC-seq was performed in triplicate on FACS-sorted XX and XY gonadal supporting cells from E10.5 and E13.5 mouse embryonic gonads. ChIP-seq for H3K27ac was performed in duplicate on FACS-sorted  XX and XY gonadal suuported cells from E13.5 mouse embryonic gonads.