A single-nucleotide enhancer mutation overrides chromosomal sex to drive XX male development

Mammalian sex determination is governed by two mutually antagonistic genetic programs that must be precisely balanced. Activation of Sox9 initiates testis development, while its repression is essential for ovarian fate. The distal enhancer, Enh13, is essential for testicular Sox9 expression, with its deletion or inactivation resulting in complete XY sex reversal. Here, we show that subtle mutations within Enh13, including a single-nucleotide insertion, produce the reciprocal phenotype: complete XX female-to-male sex reversal. Pro-female factors can strongly repress Enh13, suggesting they mediate Sox9 silencing in ovaries. The small enhancer alterations facilitate inappropriate Sox9 upregulation in the absence of Sry, triggering the testicular transcriptome and repressing ovarian gene expression. Mechanistically, these mutations disrupt the repressive effect of RUNX1, NR5A1 and GATA4, thereby reprogramming enhancer activity. Our findings identify Enh13 as a central regulatory hub, integrating opposing sex-specific cues, hence acting as a binary switch for gonadal fate. A single base pair insertion in the Sox9 enhancer, Enh13, mediates XX female-to-male sex reversal. This subtle non-coding mutation reconfigures transcriptional regulation, enhances Sox9 expression, revealing Enh13 as a binary switch governing sexual fate.