An international team of researchers says they have identified a key enhancer of Sox9, a gene critical for male sex development, and demonstrated that deleting that noncoding DNA results in male-to-female sex reversal in mice. The study (“Sex Reversal following Deletion of a Single Distal Enhancer of Sox9”) published in Science, deepens understanding of the normal process of sex determination in mammals and could also have important implications for patients with differences in sex development (DSDs), in which reproductive organs don't develop as expected, according to the scientists.

“Cell fate decisions require appropriate regulation of key genes. Sox9, a direct target of SRY, is pivotal in mammalian sex determination. In vivo high-throughput chromatin accessibility techniques, transgenic assays, and genome editing revealed several novel gonadal regulatory elements in the 2-megabase gene desert upstream of Sox9. Although others are redundant, Enh13, a 557–base pair element located 565 kilobases 5′, is essential to initiate mouse testis development; its deletion giving XY females with Sox9 transcript levels equivalent to XX gonads. Our data are consistent with the time-sensitive activity of SRY and indicate a strict order of enhancer usage. Enh13 is conserved and embedded within a 32.5-kilobase region whose deletion in patients is associated with XY sex reversal, suggesting it is also critical in humans,” write the investigators.

The research involved a collaboration between the laboratories of the late Danielle Maatouk, Ph.D., assistant professor of obstetrics and gynecology at Northwestern University Feinberg School of Medicine and corresponding author Robin Lovell-Badge, Ph.D., of the Francis Crick Institute in London. Dr. Maatouk’s work focused on sex determination, specifically on exploring how noncoding elements regulate gene expression and impact this process. 

The Sox9 gene is crucial for male differentiation and the proper formation of testes. If Sox9 is mutated or incorrectly expressed, an individual who is chromosomally male (XY) can develop ovaries instead of testes. Previously, it was known that some patients with DSDs have changes in their genome near the Sox9 gene that alter its expression and lead to sex reversal. But it was unclear exactly why.

In the current study, the scientists identified an enhancer that is necessary to regulate expression of the Sox9 gene. When the scientists deleted the enhancer in mouse models, they discovered that Sox9 expression was decreased enough to cause complete sex reversal. Mouse embryos that were chromosomally male (XY) developed as phenotypically normal females, with ovaries that were indistinguishable from those of XX females.

Alexandra Garcia-Moreno and Isabella Salamone, both fifth-year doctoral students in Feinberg's Driskill Graduate Program in Life Sciences, and Christopher Futtner, a research associate, were also co-authors. This is the first time scientists have identified an enhancer of Sox9 that, when lost, causes sex reversal, according to Salamone, who adds that the findings could help improve the genetic diagnosis of patients with DSDs in the future. Currently, only about 20% of such patients receive a genetic diagnosis. 

“We believe that many undiagnosed patients have mutations in regulatory regions—such as the one that we identified near Sox9—and regulatory regions are usually not investigated by genetic testing,” says Salamone, who is now completing her degree in the laboratory of Elizabeth McNally, M.D., Ph.D., director of the Center for Genetic Medicine. “Often genes important for sex determination are also crucial for other developmental processes, and a mutation in one gene or its regulatory region can impact a patient's health in many ways. As we begin to understand the genetic underpinnings of these disorders, we can improve our care of these patients.”

Going forward, the team is investigating other enhancers involved in the regulation of Sox9 and other sex-determining genes and hopes to also understand how Sox9 expression is repressed in females, leading to the development of ovaries. 

“The datasets we've produced can be used as a road map to regulatory regions of other genes important for gonad development,” notes Futtner.








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