Gonadal supporting cells acquire sex-specific regulatory elements during mammalian sex determination

Purpose: In this study we employed unbiased, genome-wide techniques to identify regulatory elements during murine sex determination. Methods: We performed ATAC-seq on 60K FACS-purified XX and XY gonadal cells before and after sex determination to map nucleosome depleted regions (NDRs) indicative of regulatory elements. To determine whether these are active enhancers, we performed ChIP-seq for H3K27ac, a histone modification that marks active enhancers in both sexes and time points. Transient transgenics was performed on select enhancers to determine whether they are functional in gonads during the sex determination stage. Results: We have produced a genome wide map of potential regulatory elements and active enhancers during the process of murine sex determination. Furthermore, we validated the power of our dataset by identifying a novel enhancer downstream of Bmp2, a female-specific gene. Conclusions: This work supplies a powerful resource for identifying chromatin regulatory elements active during mammalian sex determination. ATAC-seq was performed in duplicate 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 supporting cells from E10.5 and E13.5 mouse embryonic gonads. Each replicate contained pooled cells from multiple embryos.