Chromatin spatial analysis by METALoci unveils sex-determining 3D regulatory hubs

Mammalian sex is determined by opposing networks of ovarian and testicular genes that are well characterized; however, its epigenetic regulation is still largely unknown. Here we explore the 3D chromatin landscape of sex determination in vivo by profiling fluorescence-activated cell-sorted embryonic mouse gonadal populations in both sexes before and after sex determination. Through conventional Hi-C analyses, we show that chromatin structures, particularly topologically associating domains, remain largely unchanged during sex determination, suggesting a preformed configuration. We further integrate Hi-C data with ChIP-seq experiments using METALoci, a spatial autocorrelation analysis that identifies three-dimensional (3D) regulatory hubs across the genome. We uncover a prominent rewiring of chromatin interactions during sex determination, affecting the 3D regulatory hubs of hundreds of genes that display time-specific and sex-specific expression. By combining predictive approaches and validations in transgenic mice, we identify a 3D regulatory hub for the protesticular gene Fgf9. The deletion of this gonad-specific hub allows mutant mice to survive through development, overcoming lung lethality associated with Fgf9 loss of function while exhibiting male-to-female sex reversal. Through the reconstruction of gene regulatory networks, we identify a function for Meis genes, which act redundantly to specify sexual identity during ovarian and testicular development. Our results underscore the dynamic role of the 3D genome during sex determination, highlighting the potential of epigenomic approaches to uncover regulators of developmental processes.