Published: June 08, 2026, 05:43 UTC
A single DNA letter rewrites sex: How one base pair can flip the entire program of mammalian development
In the roughly three billion letters of the mammalian genome, a single one can be the difference between male and female. That is the startling conclusion of a study published April 9 in Nature Communications by a team at Bar-Ilan University in Israel, who showed that inserting one extra thymine nucleotide into a stretch of non-coding DNA is enough to trigger complete sex reversal in genetically female mice.
The finding does not rewrite the fundamentals of sex determination. The Y chromosome, and the SRY gene it carries, remain the primary switch. But the study reveals just how fragile that switch can be. It shows that a single nucleotide change in a regulatory element located half a million base pairs away from the sex-determining gene is capable of overriding the entire chromosomal program.
The hidden switch
The gene at the center of the story is Sox9, one of the most important genes in male sexual development. In a typical XY embryo, SRY protein produced from the Y chromosome activates Sox9 expression in the developing gonads, which then drives the bipotential tissue to become testes rather than ovaries. Without Sox9 activation, the default pathway is female.
Sox9 itself is controlled by a network of regulatory elements, including a 557-base-pair stretch of DNA called Enh13, located 565,000 base pairs upstream. Earlier work had shown that deleting Enh13 altogether causes XY mice to develop as females, confirming it is essential for testis formation. What remained unknown was whether subtler changes, the kind that occur naturally in human populations, could have similarly dramatic effects.
To test this, the Bar-Ilan team, led by PhD student Elisheva Abberbock and senior author Nitzan Gonen, used CRISPR to introduce two precise mutations into the Enh13 sequence of mouse embryos: a single base-pair insertion (adding one thymine) and a three-base-pair deletion, both within a binding site for the SOX9 protein itself.
What happened next surprised them.
From ovary to testis
Genetically female mice (XX) carrying either mutation developed as males. They grew male external genitalia, internal reproductive structures including seminal vesicles and vas deferens, and testes complete with organized seminiferous tubules and Sertoli cells. Their gonads produced SOX9, expressed no FOXL2 (a marker of female granulosa cells), and at the molecular level their adult gonadal transcriptomes were indistinguishable from those of XY males.
The reversal was not instantaneous. At embryonic day 13.5, the mutant gonads showed an ovotestis pattern, with both male and female tissue present in variable proportions between individuals and even between the left and right gonads of the same embryo. The one-base insertion produced more aggressive masculinization than the three-base deletion. But by birth, FOXL2 expression had retreated almost entirely, and by adulthood the ovarian program had been completely silenced.
The XX males were infertile. Without a Y chromosome, their testes were small and lacked sperm, as expected. But the body plan was male.
A regulatory rethink
The mechanism turned out to be subtler than simply strengthening the Sox9 signal. The researchers had expected the mutations to make the enhancer more responsive to SOX9 protein. Instead, they found the opposite: the mutations actually reduced SOX9 binding affinity to Enh13. What changed was how the enhancer responded to other transcription factors.
In normal female development, Enh13 is actively repressed by a set of pro-female factors, including RUNX1, GATA4, and NR5A1. These factors bind to the enhancer and keep it silent, preventing Sox9 activation. The one-base insertion, however, creates a new GATA4 binding motif (GATTG) adjacent to the existing RUNX1 site, shifting the balance from repression to cooperative activation. The three-base deletion works differently, altering the helical spacing between transcription factor binding sites so that RUNX1 and GATA4 now interact in a way that drives Sox9 expression rather than suppressing it.
In other words, Enh13 functions as a binary switch that integrates both activating and repressive signals. The mutations do not break the switch. They flip its default position.
This dual-function model is a new way of thinking about how sex determination works at the genomic level. Enh13 is not simply a testis-specific enhancer. It is a regulatory element that must be actively repressed in females and actively activated in males, and the same binding sites are involved in both processes.
From mice to humans
The study has direct implications for human medicine. More than 50 percent of cases of Differences of Sex Development, or DSD — conditions in which chromosomal sex does not match physical development — remain genetically unexplained. Most clinical genetic testing focuses on protein-coding genes. But this study shows that mutations in non-coding DNA, the vast majority of the genome that regulatory testing typically ignores, can be just as powerful.
Three cases of human XX male development associated with Enh13 duplications have already been described in the literature. The Bar-Ilan study now provides a mechanistic explanation: the enhancer is so sensitive to dosage and sequence that even a single extra copy or a single extra base can override the female program.
The Enh13 sequence itself is evolutionarily conserved. The spacing between key transcription factor binding sites is maintained across at least 11 mammalian species, suggesting the mechanism is broadly relevant to mammalian biology.
Caveats
The study was conducted entirely in mice on a C57BL/6J genetic background. While the Enh13 region is conserved, its human orthologue, eSR-A, has not been functionally tested in the same way. The mechanistic model, particularly the proposed RUNX1-GATA4 cooperativity shift, was inferred from luciferase reporter assays in cell culture and needs structural validation through techniques such as cryo-electron microscopy or chromatin immunoprecipitation sequencing.
The XX mutant males were infertile, which is expected given the absence of Y-chromosome genes required for spermatogenesis, but it limits the ability to assess behavioral or reproductive aspects of the masculinization. The variable ovotestis phenotype also suggests that even in an isogenic mouse strain, stochastic factors influence how the mutation plays out in individual embryos.
Reference: Abberbock E, Ridnik M, Stévant I, et al. “A single-nucleotide enhancer mutation overrides chromosomal sex to drive XX male development.” Nature Communications, 9 April 2026. DOI: 10.1038/s41467-026-71328-9