Understanding the Epigenetics of Sex Determination

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A team including Northwestern Medicine scientists has identified a key enhancer of Sox9 — a gene critical for male sex development — and demonstrated that deleting the enhancer results in male-to-female sex reversal in mice.

The study, published in Science, deepens understanding of the normal process of sex determination in mammals. The findings could also have important implications for patients with differences in sex development (DSDs), in which reproductive organs don’t develop as expected.

The Science study was a collaboration between the laboratories of the late Danielle Maatouk, PhD, assistant professor of Obstetrics and Gynecology, and corresponding author Robin Lovell-Badge, PhD, of the Francis Crick Institute in London. Alexandra García-Moreno and Isabella Salamone, both fifth-year doctoral students in Feinberg’s Driskill Graduate Program in Life Sciences (DGP), and Christopher Futtner, a research associate, were also co-authors.

Maatouk’s research focused on sex determination, the process during which embryos develop either testes or ovaries. Her laboratory was specifically focused on exploring how non-coding elements — parts of DNA that don’t encode for proteins — regulate gene expression and impact this process.

The late Danielle Maatouk, PhD, an assistant professor of Obstetrics and Gynecology, was co-senior author of the study published in Science.

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 (a short region of DNA that can increase gene transcription) 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.

This is the first time scientists have identified an enhancer of Sox9 that, when lost, causes sex reversal, according to Salamone.

The findings could help improve the genetic diagnosis of patients with DSDs in the future; currently, only about 20 percent 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,” said Salamone, who is now completing her degree in the laboratory of Elizabeth McNally, MD, PhD, director of the Center for Genetic Medicine and a member of the Simpson Querrey Center for Epigenetics. “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 data sets we’ve produced can be used as a road map to regulatory regions of other genes important for gonad development,” Futtner said. “This is the first paper that is being published, with two more to follow in the next few months that represent the work we did in Danielle’s lab.”

Maatouk passed away in November 2016. Maatouk and her husband, Futtner, joined Northwestern in 2013, after she completed a post-doctoral fellowship at Duke University. Maatouk received her PhD in molecular genetics from the University of Florida.

“Danielle was an incredibly gifted scientist and a great mentor to her graduate students,” Salamone said. “I learned so much in her lab, and I’m so happy that we continued and finished this work that Danielle started.”

The study was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001107), the UK Medical Research Council (FC001107), and the Wellcome (FC001107); and by the UK Medical Research Council (U117512772). The study was also supported by the Agence Nationale pour la Recherche (ANR blanc TestisDev) and Northwestern University Feinberg School of Medicine.