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

Domain: neuroscience
Published: 2026-04-09
Editorial outcome: major_revision

Abstract
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.

Introduction
In eutherian mammals, the sex of the individuals is established through a succession of events that eventually provide them with functional reproductive organs. First, genetic sex is defined at fertilization by the provision of an X or a Y chromosome by the sperm to an X-bearing oocyte. Later, during gestation, the undifferentiated bipotential gonad adopts a testicular or ovarian fate upon a process called gonadal sex determination1–3. Once differentiated, the gonads produce sex hormones necessary for the development of internal and external genitalia, secondary sexual characteristics, and then control puberty and fertility of the individual4.

Gonadal sex determination is mediated by two antagonistic genetic programs involving the activation of pro-male or pro-female factors, but also the silencing of the alternative sex genes. Testis development is initiated by the activity of the Sry gene, located on the Y chromosome, which activates its downstream autosomal target gene, Sox95–8. SOX9, the master regulator of Sertoli cell differentiation and testis development, drives the transcription of downstream target genes critical for testis formation5,8–10. Indeed, human individuals and mice with mutations in SRY or SOX9 are born as XY females8,11–15. On the contrary, forced expression of SOX9 in both human individuals and mice drives testis differentiation of XX gonads16–19. Ovary development relies on the activity of several pro-female factors, including the WT1(-KTS) isoform20, WNT4/RSPO1/β-CATENIN21–25, FOXL226–28 and RUNX129. Knockouts (KOs) of individual pro-female factors result in only partial XX sex reversal, while double KOs of either Runx1, Rspo1 or Wnt4 together with Foxl2 amplify the XX female-to-male sex reversal phenotype29–32, suggesting a redundant function of the pro-female factors during ovarian development. It is proposed that repression of Sox9 expression is required for proper ovary development, yet the molecular mechanisms remain largely unknown33.

We have previously identified Enhancer 13 (Enh13) as the critical regulator of the Sox9 gene in the testis34. Enh13 is a 557 bp-long enhancer, located 565 kb upstream of the Sox9 gene. Deletion of Enh13 in both mice and humans leads to XY male-to-female sex reversal34,35. Upon Enh13 deletion, Sox9 expression level drops by 80%, leading to ovarian development despite the presence of a Y chromosome, the Sry gene, and two intact Sox9 copies34. Furthermore, we have recently shown that mutating together the SOX9 and SRY transcription factor binding sites (TFBS) within Enh13 is enough to induce XY female to development in vivo36. While the role of Enh13 in activating testis development is now well established, our results did not suggest it may also have a function during ovarian development34,36. However, several XX patients with Differences of Sex Development (DSD) (46, XX DSD) were identified carrying a~3.7-5kb duplication that includes the human Enh13 (also called eSR-A)37,38, suggesting that having an additional Enh13 copy may lead to SOX9 expression and testis development in the absence of SRY.

Here, we show that XX mice carrying a 3bp deletion or a 1bp insertion within the SOX9 binding site (BS) of Enh13 present with female-to-male sex reversal. Adult XX mice appear externally and internally as males, but with small testes devoid of sperm due to the lack of the Y chromosome6,39. At embryonic stages, XX homozygous gonads appear as ovotestis to different extents across individuals and depending on the mutation, with the 1bp insertion appearing more like a testis compared to the 3bp deletion. Transcriptomic analysis of E12.5 gonads shows that indeed XX homozygous gonads express both the testicular and ovarian genes, while XY homozygous gonads are indistinguishable from wild type (WT) XY gonads. TFBS analysis suggests that Enh13 contains binding sites for pro-female factors and that the SOX9 BS overlaps with a RUNX1 BS. Indeed, reporter assays indicate that Enh13 can be repressed by RUNX1 and other pro-female factors. Delving into the mechanism exerted by the 3bp deletion and 1bp insertion XX mutants, we show that the mutations do not abolish SOX9 binding but rather prevent repression by the bipotential factors NR5A1, GATA4, and RUNX1 to allow sufficient activating capacity to induce Sox9 expression above the minimal threshold present in the female gonad. Once expressed, SOX9 further enhances its own expression and drives testis development in XX individuals. These findings implicate Enh13 as a regulatory element essential for both testicular and ovarian development, placing it at the critical junction of sexual differentiation pathways. This also highlights that even small changes within regulatory elements can induce dramatic phenotypic outcomes.

Results
Small mutations in the SOX9 BS of Enh13 lead to XX male development in adulthood

In our previous study aiming to identify the functional elements of Enh13, we have generated mice carrying mutations in several TFBS, including NR5A1, SOX9 and SRY36. Using CRISPR-Cas9, we obtained two different mouse strains with mutations within the SOX9 BS, one with a 20bp deletion that completely abolished the SOX9 BS (Supplementary Fig. 1A, termed Enh13SOX9*−20bp) and one with a 3bp deletion that removed 3 nucleotides at the center of the SOX9 BS (Fig. 1A, termed Enh13SOX9*−3bp). When looking at the sequence of the Enh13SOX9*−3bp, we noticed that we recreated a quasi-identical SOX9 BS (CACAATGG, Fig. 1A, B), so we did not consider using this strain in our previous study. Heterozygous and homozygous XX and XY Enh13SOX9*−20bp mice presented normal phenotypes, i.e., XX females with ovaries, and XY males with testes, demonstrating that the SOX9 BS alone is not responsible for the Enh13 activity (Supplementary Fig. 1B-C), but instead acts redundantly with the SRY BS to control the Sox9 gene expression36. While XX and XY heterozygous and XY homozygous mice carrying the Enh13SOX9*−3bp also presented normal phenotypes (Supplementary Fig. 2A, B), XX mice homozygous for the 3bp deletion were born sex reversed highlighted by male external genitalia and small testes (Fig. 1C). Immunostaining of adult gonads with markers of Sertoli cells of the testis (SOX9), granulosa cells of the ovary (FOXL2) and gonocytes (DDX4) revealed that XX Enh13SOX9*−3bp-/- homozygous gonads show no FOXL2 expression and instead contain seminiferous tubules, marked by SOX9 expression. These seminiferous tubules lack DDX4-positive gonocytes at 6-week-old mice (Fig. 1D), a phenotype known to occur in XX males as they are not able to support spermatogenesis in the absence of Y chromosome genes6,39.

To better characterise the phenotype, we explored the expression levels of Sox9, Sox8, a downstream target gene of SOX9 in Sertoli cells14, and Foxl2 in adult gonads. XX WT and heterozygous gonads exhibited low expression of Sox9 and Sox8 while having high Foxl2 expression (Fig. 1E). XY WT gonads had high expression of Sox9 and Sox8 and no detectable Foxl2 expression. XX homozygous gonads for the 3bp deletion were similar to XY WT gonads, with high expression levels of Sox9 and Sox8 and no detectable Foxl2 expression, confirming a female-to-male sex reversal in adulthood (Fig. 1E).

Early E10.5 gonads of XY and XX both express low levels of (cytoplasmic) SOX9, after which Sox9 is highly upregulated in the testes and decreased in ovaries40,41. In XY gonads, SRY induces Sox9 expression by binding to Enh13, and subsequently, SOX9 controls its own expression by binding to both the SOX9 and SRY BSs of Enh1336. Since SOX9 protein can bind to both SRY and SOX9 TFBS, we tested whether the elevated Sox9 expression observed in the XX Enh13SOX9*−3bp-/- is due to SOX9 binding to both the mutated SOX9 BS as well as the SRY BS. To that aim, we generated mice carrying the same 3bp deletion in the SOX9 BS along with a deletion removing the entire SRY BS (Supplementary Fig. 3A, termed Enh13SOX9*−3bpSRY*−12bp). Heterozygous and homozygous XY mice developed as males, while XX heterozygous mice presented as female mice (Supplementary Fig. 3B, C). Homozygous XX mice presented as males (Supplementary Fig. 3B, C), demonstrating that removal of the SRY BS, to which SOX9 can bind, does not affect the female-to-male sex reversal phenotype, suggesting that the mutated SOX9 BS is still functional and able to induce Sox9 expression. These results denote that, in this sensitized context, the loss of the SRY BS does not prevent testis development in XY. The 3bp deletion alone allows Sox9 expression to rise above the critical level required to trigger testicular differentiation in XX and XY gonads. Moreover, the female-to-male sex reversal is solely due to the 3bp deletion and does not involve the SRY motif, to which SOX9 can bind.

We next wanted to examine whether other insertion/deletions (INDELs) within the SOX9 BS of Enh13 will cause a similar phenotype. To that aim, we generated a mouse strain carrying 1bp insertion in the SOX9 BS (Fig. 1F, termed Enh13SOX9*+1bp). Heterozygous XX mice presented as females while heterozygous and homozygous XY mice presented as males (Supplementary Fig. 3D). Similarly to what we observed with the 3bp deletion, XX homozygous mice carrying only a 1bp insertion in Enh13 were born as XX males with small testes (Fig. 1G). Immunostaining of adult gonads confirmed complete sex reversal and the presence of seminiferous tubules lacking gonocytes (Supplementary Fig. 3E). Altogether, these findings underscore the extraordinary sensitivity of the Sox9 regulatory network to subtle enhancer perturbations, showing that specific mutations are capable of inducing Sox9 expression above a critical threshold required to initiate testicular development in an XX context.

Embryonic gonads develop as ovotestis upon small mutations in the SOX9 BS of Enh13

We then sought to explore whether the sex reversal observed in XX adult gonads occurs during early gonadal development. We hence collected E13.5 gonads from XX and XY WT as well as XX Enh13SOX9*−3bp-/- embryos. XX gonads of Enh13SOX9*−3bp−/− embryos presented variable phenotypes of ovotestis. At E13.5, WT testes are organised into testis cords, containing Sertoli cells and germ cells, along with an interstitial compartment where Leydig cells reside, and present with a coelomic vessel. WT ovaries, on the other hand, are devoid of clear structural organisation at that stage and germ cells along with somatic cells are spread across the ovarian area. While some XX homozygous gonads appeared mostly as testes with coelomic vessel and testis cords, others had a coelomic vessel without clear testis cords and some presented with a testicular gonadal portion alongside an ovarian portion (Fig. 2A). Variability in ovotestis phenotypes were also evident between the right and left gonads of the same embryo wherein one the anterior portion was ovarian and the posterior was testicular, whereas the other gonad presented an anterior and posterior ovarian portions and a centralised testicular portion (Fig. 2A, first and third mutant gonads are the left and right gonads of the same embryo). It is important to note that all embryos are on the same C57BL6/J genetic background, and hence the variable phenotype does not stem from differences in genetic background as reported in other cases42. Notably, XX heterozygous gonads were indistinguishable from XX WT gonads (Supplementary Fig. 4A) while XY heterozygous and homozygous gonads were indistinguishable from XY WT gonads (Supplementary Fig. 4B).

To better explore this ovotestis phenotype, we performed immunostaining on E13.5 embryonic gonads using SOX9, FOXL2 and TRA98 (marks gonocytes). XX Enh13SOX9*−3bp-/- gonads presented with ovotestis with an anterior ovarian portion alongside a testicular posterior portion, or an ovotestis where the testicular tissue is centrally located, flanked by ovarian domains at both poles (Fig. 2B). FOXL2 and SOX9 positive cells were mutually exclusive (Fig. 2B, larger magnification). Immunostaining of XX heterozygous gonads (Supplementary Fig. 4C) or XY heterozygous and homozygous gonads (Supplementary Fig. 4D) demonstrated they are similar to XX WT or XY WT, respectively.

To confirm the ovotestis phenotype, we measured the expression levels of Sox9, Sox8 and Foxl2 by qPCR at E13.5. Overall, Sox9 levels in XX homozygous gonads are comparable to WT XY gonads, though sample variability likely reflected differences in the extent of ovotestis phenotype (Fig. 2C). Heterozygous XX gonads had similar expression levels to XX WT controls (Fig. 2C). Sox8 and Foxl2 expression in homozygous XX gonads presented an intermediate level of expression compared to the XX WT and XY WT gonads, consistent with a mixed gonadal identity.

Whereas XX individuals from the three mutant mouse lines: Enh13SOX9*−3bp-/-, Enh13SOX9*+1bp-/- and Enh13SOX9*−3bpSRY*−12bp-/- showed complete XX female-to-male sex reversal in adulthood, we wanted to explore whether they all present with an ovotestis phenotype at embryonic stages. To that aim, we examined E13.5 gonads from the Enh13SOX9*+1bp-/- and Enh13SOX9*−3bpSRY*−12bp-/- by both bright field (Supplementary Fig. 4E-F) and immunostaining (Fig. 2D, Supplementary Fig. 4G). XX homozygous mutant gonads from the Enh13SOX9*+1bp-/- (Fig. 2D) and Enh13SOX9*−3bpSRY*−12bp-/- (Supplementary Fig. 4G) strains also presented an ovotestis phenotype, with a more testis-like appearance compared to the Enh13SOX9*−3bp-/- strain. Gonads displayed SOX9 expression along with a very limited FOXL2-positive region, typically restricted to one or two poles of the gonad (Fig. 2D, Supplementary Fig. 4G).

Since E13.5 mutant gonads presented as ovotestes to different extents, and at 6 weeks, these gonads were testis expressing SOX9, but no FOXL2, this prompted us to explore mutant gonads also at two intermediate time points: E15.5 and P1 (Supplementary Fig. 5). Bright field analysis confirmed that E15.5 and P1 gonads from both mutants (Enh13SOX9*+1bp-/- and Enh13SOX9*−3bp-/-) presented as a testis (Supplementary Fig. 5A, D). Immunostaining at E15.5 showed that FOXL2 is still expressed in either just the anterior pole or both poles, much more so in the Enh13SOX9*−3bp mutants rather than the Enh13SOX9*+1bp mutants (Supplementary Fig. 5B, C). Analysis of mutant gonads at P1 presented mostly SOX9-positive cells with very few FOXL2-positive cells, located mostly in the cortex, more in the Enh13SOX9*−3bp mutants rather than the Enh13SOX9*+1bp mutants (Supplementary Fig. 5E, F). Altogether, these analyses indicate that the Enh13SOX9*+1bp mutant appears to have a stronger ability for sex reversal than the Enh13SOX9*−3bp mutant, as more FOXL2-positive cells persist postnatally at P1 in the Enh13SOX9*−3bp mutant.

Transcriptomic analysis of E12.5 embryonic gonads demonstrates an ovotestis pattern

To gain insight into the molecular and transcriptomic consequences of small mutations within Enh13, and to determine whether different INDEL alleles elicit distinct gene expression profiles, we performed RNA-seq on E12.5 embryonic gonads. We chose the E12.5 stage as it is known that ovarian development is lagged ~1 day behind compared to testis differentiation that commences at E11.520,43. Therefore, analysing E12.5 gonads seemed to be an optimal stage to capture the early transcriptomic changes that eventually lead to ovotestis, followed by testis development. Pairs of E12.5 gonads from individual embryos were harvested and bulk RNA-seq was performed. We harvested 3 pairs of gonads from XX and XY WT and heterozygous embryos, while we collected 5 gonads from XY and XX homozygous embryos for the 3 different mutant mouse strains (Enh13SOX9*−3bp, Enh13SOX9*+1bp and Enh13SOX9*−3bpSRY*−12bp) as we assumed there may be a higher degree of variability there. Principal component analysis (PCA) demonstrated that XX WT and heterozygous gonads mostly clustered together (Fig. 3A). XY WT, heterozygous and homozygous gonads also clustered together, suggesting that the small mutations do not hamper testis development in the context of XY. PC1, which accounts for 27.7% of the variability, clearly separated the testis (right side) and the ovary (left side) (Fig. 3A). The XX homozygous gonads were positioned between the testis and the ovary, and a large degree of variability was evident between the three different mutant mouse strains and even between the samples of each strain. Spearman pairwise sample correlation analysis supported these findings and separated the XX homozygous samples from both the XX ovaries and XY testis (Supplementary Fig. 6A).

We next performed differential expression analysis on all samples to identify early genes driving the gonadal sex change (Fig. 3B, Supplementary Data 1). 1,803 genes were found to be differentially expressed and could be classified into 6 groups (a to f) based on their expression profile. Groups a-c exhibited genes with increased expression in the ovary, while groups e-f constitute genes with increased expression in the testis. Consistent with the ovotestis phenotype, the XX homozygous gonads presented a transcriptomic profile that is different from the testicular and ovarian ones, with expression of both testicular and ovarian genes together, albeit at lower levels (Fig. 3B).

We then examined the expression profile of specific genes associated with various cell types of the gonads. Analysis of bipotential genes, Gata4 and Zfpm2 (Fog2), which are expressed in supporting cell precursors, and later maintained in both Sertoli and pre-granulosa cells, revealed no major differences in expression between WT, heterozygous, and homozygous gonads from the Enh13SOX9*−3bp, Enh13SOX9*+1bp and Enh13SOX9*−3bpSRY*−12bp mutants (Supplementary Fig. 7A). Nr5a1 expression in the XX homozygous mutants was more similar to the male expression levels, whereas Wt1 expression in the homozygous mutants resembles more that of the WT XX gonads (Supplementary Fig. 7A). Markers expressed in pre-granulosa cells as Foxl2, Fst, Bmp2, Wnt4, Runx1, Rspo1, Lhx9 and Emx2 were highly expressed in XX WT and XX heterozygous gonads, but displayed a low-to-intermediate expression in the XX homozygous mutant gonads of the 3 mouse strains (Fig. 3C and Supplementary Fig. 7B). A mirrored expression pattern was observed for Sertoli cell markers, including Sox9, Sox8, Fgf9, Dhh, Amh, Gdnf and Aard. These genes were highly expressed in XY WT, heterozygous, and homozygous gonads, absent in XX WT and heterozygous gonads, but displayed intermediate to high expression in all XX homozygous mutant gonads (Fig. 3D and Supplementary Fig. 7C). Notably, Sox9 and Fgf9 showed the highest expression levels, whereas the remaining markers exhibited intermediate expression, likely reflecting their later activation during testis development, as E12.5 precedes their peak expression44–46. Examination of markers of germ cells and interstitial/stromal cells did not exhibit any dramatic change between all samples (Supplementary Fig. 7D, E).

Altogether, this transcriptomic analysis, at an early stage of the sex reversal process, reveals that transcriptomes of heterozygous XY and XX gonads, as well as homozygous XY gonads, are indistinguishable from their respective WT counterparts. In contrast, XX homozygous gonads from all three mutant strains exhibit a mixed ovarian and testicular transcriptome signature at E12.5.

The SOX9 BS mutations do not abolish the ability of SOX9 to bind and activate Enh13

We next wanted to explore the molecular mechanisms leading to these small mutations, in an XX environment, to enable activation of Sox9 expression, driving testis development. As the mutations lie within the SOX9 binding site, and since it still appears to be a putative SOX9 BS, even in the presence of the mutations (Fig. 1B), we hypothesised that these mutations might enhance SOX9 binding and promote activation, given its low-level cytoplasmic expression in XX gonads before sex determination40,47. To test this, we performed Protein Binding Microarray (PBM)48 and electro mobility shift assays (EMSA) with in vitro–translated SOX9 on synthetic Enh13 sequences (WT, –3bp, +1bp). Both assays confirmed SOX9 binding, albeit with lower affinity, with even further reduced affinity in the 3bp deletion mutant (Supplementary Fig. 8A–C). Luciferase assays in HEK293T cells supported this, showing that there is still enhancer activity in the mutants (Supplementary Fig. 8D). These findings rule out that the mutation has created a more potent SOX9 binding site that would lead to higher Sox9 transcriptional activation as the underlying mechanism.

Pro-female factors can repress Enh13 and hence Sox9 expression

As the 3bp deletion and 1bp insertion do not create a more potent SOX9 BS, this prompted us to explore which other TFBS are present within Enh13, and more so, around the SOX9 BS. We hence performed JASPAR TFBS analysis on the WT Enh13 and saw the presence of putative binding sites for bipotential factors (NR5A1, WT1, GATA4) and pro-male factors (SOX9, SRY, DMRT1) as previously reported34,36 (Fig. 4A). To our surprise, we could also identify many putative binding sites for pro-female TFs as the TCF/LEF (the WNT pathway), FOXL2, RUNX1 and WT132. Moreover, overlapping to the SOX9 BS, there was a RUNX1 BS (Fig. 4B). Although typically considered a pro-ovarian factor29, RUNX1 is expressed in both sexes at E11.5, before becoming enriched in pre-granulosa cells and downregulated in Sertoli cells29,45,46 (Fig. 4C). Given the overlap between the RUNX1 and SOX9 binding sites, we investigated whether the mutations affected RUNX1 binding. PBM and EMSA assays confirmed RUNX1 binding to Enh13, but revealed no difference in affinity between the WT and mutant sequences (Supplementary Fig. 9A, B).

Next, to explore the effect of pro-female factors on the ability to induce Sox9 expression via the WT Enh13, we employed Luciferase reporter assays. In the absence of any TF, Enh13 is not activated in HEK293T cells. Transfection with a combination of three critical bipotential factors: NR5A1, GATA4 and WT1 led to ~10-fold induction. The addition of FOXL2 and CHIR (agonist of the WNT pathway) did not lower enhancer activity. In contrast, addition of RUNX1 to the bipotential factors significantly lowered enhancer activity by two-fold, suggesting that the expression of RUNX1 in the ovary can repress Sox9 expression mediated via Enh13. Combination of RUNX1, FOXL2 and CHIR together did not lower enhancer activity more than the reduction observed with RUNX1 alone (Fig. 4D). We next wanted to simulate a state where the enhancer is highly activated so that we could assess the potential repressive effect of adding pro-female factors. We hence added SOX9 to the bipotential factors and observed 133-fold enhancer induction (Fig. 4E). This time, addition of FOXL2 and CHIR significantly lowered reporter activity to 68-fold induction. Addition of RUNX1 alone also lowered enhancer activity to even lower levels (49-fold). Combined addition of pro-female factors, FOXL2, CHIR and RUNX1 presented an additive effect and further lowered reported activity to 32-fold (Fig. 4E).

Altogether, these experiments suggest that Enh13 might be normally repressed by RUNX1 and other pro-female factors and imply that the repression of Sox9 expression, mandatory for proper ovary development, may be mediated via Enh13. It is possible that SOX9 and RUNX1 compete on partially overlapping binding sites in the testis and the ovaries, respectively.

The 3bp deletion and 1bp insertion mutations affect the cooperativity of TFs on Enh13 and enhance its activity

We next examined the mutation region more broadly and found it is highly conserved among mammals, both in nucleotide sequence and in the spacing between TFBS relative to the SOX9 BS (Fig. 5A). An NR5A1 binding site lies 24bp upstream, the RUNX1 site partially overlaps with the SOX9 BS, and a GATA4 binding site is located 6bp downstream (Fig. 5A). Notably, the RUNX1–SOX9 binding site module is fully conserved across all examined mammalian species, and the distance between this module and the adjacent NR5A1 site varies by no more than one nucleotide. The high degree of conservation in both sequence and spacing suggests that precise spatial organisation of these TFBS may be critical for cooperative binding and enhancer function, as it has been shown that even minor alterations in spacing can disrupt transcriptional synergy49. As SOX9 and RUNX1 binding affinities were not significantly altered (Supplementary Figs. 8, 9), we conducted a comprehensive JASPAR analysis of ~2300 vertebrate TFs, including all those expressed in the gonad at this stage. No major gain or loss of binding sites was found between WT and mutant Enh13 sequences to explain the phenotype (Supplementary Data 2; Supplementary Fig. 10A–C).

It has previously been shown that small mutations in regulatory elements could affect gene expression via alteration of the DNA helical positioning, hence modifying the 3D structure in a way that affects the cooperative binding of nearby TFBS50,51. To assess whether the mutations affect cooperative TF binding, we performed luciferase assays in HEK293T and COS7 cells using NR5A1, GATA4, RUNX1, and SOX9, the transcription factors with binding motifs immediately adjacent to the mutated region, conserved in both sequence and spacing, and co-expressed in the gonad prior to Sox9 activation. Transfection of HEK293T cells with RUNX1 or any other factor alone did not induce any reporter activity (Fig. 5B, Supplementary Fig. 11A). Co-transfection of NR5A1 and GATA4 led to a 61-fold induction of the WT Enh13, a comparable 72-fold induction for Enh13SOX9*−3bp (named here “–3bp”), and a markedly stronger 107-fold activation for Enh13SOX9*+1bp (named here “+1bp”). Notably, the addition of RUNX1 to NR5A1 and GATA4 reduced enhancer activity of the WT to 38-fold, but failed to repress the two mutant enhancers, which maintained elevated activity levels (70-fold for –3bp and 77-fold for +1bp). This suggests that the mutations impair a repressive input mediated by RUNX1 in the context of the WT enhancer. Importantly, this defect was recapitulated in COS7 cells, where the differences between the mutants and the WT were even more pronounced (Supplementary Fig. 11B). As expected, inclusion of SOX9 in the transfection cocktail strongly enhanced reporter activity, with even greater induction observed for the mutant enhancers compared to the WT (120-fold for WT, 173-fold for –3bp, and 182-fold for +1bp) (Fig. 5B).

Together, these results indicate that the mutations render the enhancer to be less susceptible to RUNX1/NR5A1/GATA4-mediated repression, thereby facilitating Sox9 activation. Moreover, the distinct responses of the two mutants to NR5A1 and GATA4 alone suggest that they may act through different molecular mechanisms to alter enhancer function. EMSA experiments with NR5A1 did not reveal any differences in binding between the WT and mutant probes. In contrast, EMSA with GATA4 identified a complex formed exclusively with the +1bp probe (Fig. 5C). This result is consistent with the creation of a de-novo GATA binding motif (GATTG) due to the T insertion in the +1bp mutant (Fig. 5D), a sequence known to support GATA factor binding52. GATA transcription factors have been reported to dimerize and enhance transcriptional output53, raising the possibility that the new motif facilitates cooperative GATA4 binding with the adjacent native site, thereby amplifying enhancer activity.

Lastly, seeing the different response between the –3bp and +1bp mutants, we wanted to assess the SOX9 levels in vivo, at E11.5 gonads. To that aim, we performed whole-mount immunostaining with SF1 and SOX9 on E11.5 gonads of WT XY and XX embryos as well as embryos of Enh13SOX9*+1bp-/- and Enh13SOX9*−3bp-/- (Supplementary Fig. 12). XX WT gonads present no SOX9 expression, while XY WT gonads exhibited SOX9 expression within the SF1-positive gonadal cells. While no SOX9 protein was evident in the –3bp mutant gonads, SOX9-positive cells were clearly present in the +1bp mutant gonads (Supplementary Fig. 12). This is consistent with the more testicular appearance of the +1bp mutants at E13.5 compared to the more ovotesticular phenotype of the –3bp mutants (Fig. 2B, D).

Overall, we demonstrate that Enh13 is used for both the activation and repression of Sox9 expression in the testis and ovary, respectively. We also show that the –3bp/+1bp mutations sufficiently modify the cooperative binding of TFs, to allow significantly higher activation of Sox9 even in the absence of SRY. This surpasses the activation threshold that must not be exceeded in XX gonads, leading to SOX9 auto-activation and testis development in XX gonads.

Methods
Animal Ethics Statement
All animals were maintained with appropriate husbandry according to Bar Ilan University ethics protocols 11-02-2020, 1-11-2020, and 2306-117-11. All mice were maintained under specific pathogen-free conditions. Animals were housed at a temperature of 20–24°C with a relative humidity of 35-65% under 12 hour light-12 hour dark cycle. All mice strains generated were maintained on a C57BL/6J genetic background. Primers used for genotyping are listed in Table S1. At least ten different animals of each genotype from all mouse strains were analysed.

Design and preparation of sgRNAs mRNA
Guide RNAs targeting each transcription factor binding site (TFBS) were designed using the IDT CRISPR/Cas9 design platform (https://eu.idtdna.com). Candidate guides were selected based on their position relative to the center of the TFBS, considering that Cas9 cleavage typically occurs approximately 3–4 bp upstream of the PAM sequence.
To prepare the crRNA–tracrRNA duplexes, crRNAs for all guides (sgRNA sequences are provided in Table S2) and tracrRNA (IDT, 224893246) were each resuspended to a concentration of 200 µM in Nuclease-Free Duplex Buffer (IDT, 11-01-03-01). Equal volumes of crRNA and tracrRNA were then combined at a 1:1 ratio to generate a final duplex concentration of 100 µM. Duplex formation was achieved by heating the mixture at 95°C for 5 min, followed by gradual cooling to room temperature. The annealed complexes were stored at −20°C for up to two weeks until use.
CRISPR/Cas9 ribonucleoprotein (RNP) complexes were assembled immediately before electroporation. The RNP mixture was prepared in Opti-MEM (Thermo Fisher Scientific, 31985-062) and consisted of 1.2 µM Cas9 Nuclease V3 (IDT, 1081059) and 6 µM sgRNA. In experiments where two RNP complexes were introduced simultaneously, the concentration of each sgRNA was adjusted to 3 µM. The assembled RNP mixture was incubated for 10 minutes at room temperature and subsequently kept on ice until electroporation of zygotes.

Zygote harvesting and electroporation
For zygote collection, C57BL/6J female mice aged 4–7 weeks were hormonally stimulated by intraperitoneal injection of 5 IU PMSG (Pregnant Mare Serum Gonadotropin; ProSpec, hor-272). After 48–50 hours, females received a second intraperitoneal injection of 5 IU hCG (Human chorionic gonadotropin; Sigma Aldrich, CG10). Immediately following hCG administration, females were paired with adult C57BL/6J males at a 1:1 ratio. The following morning, females were examined for the presence of a vaginal plug (VP). Females with a confirmed VP were euthanized by cervical dislocation, and their oviducts were dissected to isolate fertilized zygotes.
The oviducts were removed and the ampulla was gently opened to release cumulus–oocyte complexes into M2 medium (Sigma Aldrich, M7167) supplemented with 300 µg/ml hyaluronidase (Sigma Aldrich, H4272) to disperse the surrounding cumulus cells.
Zygotes were collected using a mouth-controlled glass pipette and transferred into a dish containing 2 ml of fresh M2 medium. They were subsequently washed through several M2 droplets to ensure complete removal of residual cumulus cells. Cleaned zygotes were then transferred to KSOM culture medium (Mercury, MR-106-D) and incubated for 30 minutes in a flat-bed CO₂ incubator (5.3% CO₂, 5% O₂ at 37°C; Esco, Miri 2070047). Embryos exhibiting visible polar bodies and pronuclei were selected, rinsed in M2 droplets, followed by three washes in Opti-MEM, and finally transferred into the electroporation chamber containing the prepared RNP mixture.
Electroporation was carried out using a NEPA21 electroporator (NepaGene). A series of pulses was applied to transiently disrupt the zona pellucida and facilitate entry of the CRISPR/Cas9 ribonucleoprotein complexes into the zygotes. Depending on the experimental setup, either a large glass electrode plate (CUY505P5, 5mm gap) containing 50 µl of RNP solution and 20–150 embryos or a small glass electrode plate (CUY501P1, 1mm gap) containing 5 µl of RNP solution and 5–50 embryos was used.
For the large electrode plate, poring pulse parameters were set to 225V, 1ms pulse width, 50ms interval, and 4 pulses. Transfer pulses were applied at 20V with a 50ms pulse width, 50ms interval, and 5 pulses. For the small electrode plate, poring pulses were delivered at 40V with a pulse width of 3.5ms, a 50ms interval, and 4 pulses. Transfer pulses were set to 5V with a 50ms pulse width, 50ms interval, and 5 pulses. Electrical impedance was measured both before and after embryo loading according to the manufacturer’s instructions.
Following electroporation, embryos were washed sequentially in M2 and KSOM droplets and then cultured overnight in KSOM medium in a CO₂ incubator (5.3% CO₂, 5% O₂ at 37°C). Embryos that progressed to the 2-cell stage were subsequently transferred surgically into the oviducts of pseudopregnant CD1 recipient females.

gDNA isolation and genotyping genetically altered mice
Genomic DNA (gDNA) was extracted from tail biopsies of embryos or ear punch samples from adult mice. For adult tissue, ear punches were incubated for 15 min at 95°C in lysis buffer containing 10 mM NaOH and 0.1 mM EDTA (pH 8), followed by neutralization with 40 mM Tris-HCl (pH 5). Embryonic samples were processed using the PCRBIO Rapid Extract Lysis Kit (PCRBIO, PB15.11-S) according to the manufacturer’s instructions.
Founder mice carrying mutations in the SOX9 or combined SOX9 and SRY binding sites within Enh13 were initially screened by Sanger sequencing of a 616 bp PCR fragment spanning the entire Enh13 region (genotyping primers listed in Table S1). Identified founders were crossed with C57BL/6J mice to establish germline transmission. F1 offspring were verified by Sanger sequencing, after which mutation-specific PCR assays were designed for routine genotyping. Heterozygous F1 animals were crossed to establish stable mutant lines. PCR products were purified using either the Universal DNA Purification Kit (TIANGENE, TI-DP214-03) or the EPPiC Fast Kit (A&A Biotechnologies, 1021-500F) before sequencing. INDEL mutations in founders and F1 pups were analyzed using the online tool DECODR. For samples carrying sequential mutations within the same allele, TA cloning (Promega pGEM®-T, A3600) was performed to resolve the mutation structure. The purified 616 bp Enh13 PCR fragment was ligated into the pGEM®-T vector, transformed into competent E. coli (NEB, C3019), plated on LB agar containing 100 µg/mL ampicillin, and incubated overnight at 37°C. Individual colonies were isolated, plasmid DNA was purified using the NucleoSpin Plasmid EasyPure Kit (Macherey-Nagel, 740727), and inserts were sequenced and analyzed using BLAST.
For routine genotyping, the 616 bp Enh13 fragment was amplified and digested with BstXI FD (Thermo Scientific, FD1024). This enzyme cleaves the wild-type allele into fragments of 367 bp and 249 bp but does not cut alleles carrying mutations in the SOX9 binding site, which remain as a 616 bp fragment. Digested products were run on a 2% agarose gel.
All PCR reactions were performed using 2X Red PCR Master Mix (PCRBIO, PB10.23-10) according to the manufacturer’s instructions. Chromosomal sex of all mice and embryos was determined by PCR (primers listed in Table S1).

Time mating, gonad harvesting, preparation and imaging
Gonads were harvested from embryos and mice carrying the desired mutations. Embryos were collected following timed matings at embryonic day E12.5 or E13.5, with day 0.5 defined by the detection of a vaginal plug (VP). Bright-field images of gonads were acquired using a Nikon Eclipse Ts2R microscope with an exposure time of 10ms and processed using NIS-Elements D software.
For immunostaining of the gonads from E13.5 embryos and 6-week-old mice were dissected and fixed overnight at 4°C in 4% paraformaldehyde (Sigma Aldrich, P6148) prepared in phosphate-buffered saline (PBS). Samples were then washed three times in PBST (PBS containing 0.1% Triton; Sigma Aldrich, 9002-93-1) at room temperature, incubated overnight in 20% sucrose (Fisher BioReagents, BP220-1) at 4°C, embedded in OCT compound (Leica, 14020108926), and stored at −80°C until immunostaining.

Immunofluorescence staining
Immunofluorescence staining for E13.5, E15.5, P1, and 6-week samples was performed on 10 μm-thick sagittal cryostat sections (Leica, CM3050-S). Antigen retrieval was performed for embryonic samples with DAKO (Target retrieval solution, Agilent, S1699) at 65°C for 30 min. Samples were then blocked in PBST containing 10% donkey serum (Sigma Aldrich, D9663) for 1 hour at room temperature (RT) and incubated with primary antibodies (diluted in PBST containing 1% donkey serum) overnight at 4°C. Following three PBST washes, secondary antibodies were added for 1 hour at RT. Slides were then washed, dried, and mounted (Polysciences 18606-20). All immunofluorescence slides were also stained with 4’,6-diamidino-2-phenylindole (DAPI; Invitrogen, D1306) to visualize nuclear DNA.
For E11.5 gonads, whole-mount immunostaining was performed. Gonads were fixed in 4% paraformaldehyde for 20 minutes at RT. Blocking was performed in 10% donkey serum in 0.3% PBST for 3 hours at RT. Samples were then incubated with primary antibodies in a solution of 10% donkey serum in 0.3% PBST at 4°C overnight. After six washes with PBST, samples were incubated overnight with secondary antibodies and DAPI in a solution of 10% donkey serum in 0.3% PBST at 4°C. The next day, samples were washed and transferred onto a glass slide with mounting media (Polysciences 18606-20).
All primary and secondary antibodies, as well as dyes used are listed in Table S3. Images were obtained with a Leica Microsystems SP8 confocal microscope.

RNA isolation, cDNA preparation, and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
RNA was isolated from a pair of E13.5 embryos by dissecting out the gonad-mesonephros complexes. The gonad was separated from the mesonephros, and a pair of gonads was snap frozen and stored in −80°C until isolation. For 6-week-old gonads, RNA was isolated using Qiagen RNeasy Plus mini kit (Qiagen, 74134) and from E13.5 gonad pairs using Qiagen RNeasy Plus micro kit (Qiagen, 74034). RNA concentration was measured using a NanoDrop spectrophotometer. cDNA synthesis was performed using SuperScript™ III Reverse Transcriptase (Thermo Scientific, 18080085) with 1 µg of RNA from adult gonads or 200 ng from E13.5 gonads, following the manufacturer’s instructions.
qRT-PCR reactions were performed in duplicate using Power SYBR Green PCR Master Mix (Thermo Scientific, AB-4367659) with 140 nM forward and reverse primers (Table S4), and run on a QuantStudio 1 Real-Time PCR System (Thermo Scientific). Gene expression was analysed using the comparative CT (ΔΔCT) method and normalized to the housekeeping gene Hprt. Statistical analysis was performed using GraphPad Prism 9. Data were analysed by one-way ANOVA followed by Dunnett’s post-hoc test, with p<0.05 considered statistically significant. Dataset normality was evaluated using the Shapiro–Wilk test, and potential outliers were assessed using Grubbs’ test. The number of gonads analysed for each stage is indicated in the corresponding graphs.

RNA-seq and library prep
Gonad pairs of E12.5 mice were dissected, separated from the mesonephros and snap-frozen in liquid nitrogen. RNA was extracted using Qiagen RNeasy Plus micro kit (Qiagen, 74034). 140 ng of total RNA was taken from each sample for library preparation. mRNA was transcribed to cDNA and prepared into libraries using Poly(A) mRNA Magnetic Isolation Module (NEB, E7490S), along with NEBNext Ultra Express RNA library prep kit (NEB, E3330S) and cleaned using AMPure beads (Beckman Coulter, Bc-a63881) according to manufacturer's instructions. Unique indexes were added to each sample using ++NEBNext Multiplex Oligos for Illumina kit (NEB-E6440S) according to the manufacturer's instructions and cleaned using AMPure beads. cDNA and library concentrations were analysed using Qubit ds HS Assay Kit (Invitrogen, 2326054) and sample size distribution using Tapestation with high sensitivity D1000 tape (Agilent, 5067-5585). Samples were pooled together to create a 2 nM library and sequenced with 50 bp paired-end (PE) reads on the Next-Seq NovaSeq X Plus 1.5B at the Nancy and Stephen Grand Israel National Center for Personalized Medicine (G-INCPM) facility at Weizmann Institute with ~70 million reads per sample.

RNA-seq mapping and differential expression analysis
FastQ files were processed using the nf-core/rnaseq pipeline v3.12.0. Briefly, read quality controls were performed with FastQC. Sequencing adapters were removed with Trim Galore!. Reads were mapped on the mm10/GRCm38 reference genome from Gencode (M25) with STAR. Gene quantification as read counts and TPM (Transcript Per Million) was obtained using RSEM. Read count matrix was filtered to exclude lowly expressed genes (genes with less than 10 reads and/or TPM value less than 10). Non-protein-coding genes were filtered out in the subsequent analysis. Sample correlation (Spearman) and PCA were performed with R (corr and prcomp functions) using the filtered read counts normalized by library size (sizeFactor) with DESeq2. After inspection of the correlation and the PCA, we excluded the XY WT replicate 3 because it was too different from the other two replicates. The embryo of this sample had 2 more tail somites than the two other replicates.
Differential expression analysis was performed on the filtered read count matrix using DESeq2 using “LRT” (Likelihood Ratio Test). Genes with log2(FoldChange)<−0.5 and log2(FoldChange)>0.5, as well as an adjusted p-value<0.01 were considered as differentially expressed.
Gene expression values (filtered read count matrix normalized by library size) of the differentially expressed genes were transformed as z-scores, clustered into groups according to their expression profiles using hclust and “ward.D2” method, and represented as a heatmap using ComplexeHeatmap. The optimal number of clusters was assessed using the best.cutree function from the Jlutils R package (https://github.com/larmarange/Jlutils), with a minimum of four possible clusters. Samples were ordered according to the hierarchical clustering (“ward.D2”) and split into three groups.

Cloning of Luciferase vectors
The following expression plasmids for luciferase assays were used: pCMV3-hWT1-KTS-C-HA (SINO BIOLOGICAL, Ref: HG12282-CY), pCS2-Flag-FOXL2 (Addgene, Ref 153135), pcDNA3-Flag-hSOX9 (A gift from Gerd Scherer to FP, described in ref. 68, pcDNA3.1-Flag-Runx1-FL (Addgene, Ref: 14585), pCS2-HA-GATA4 (A gift from Segei Tevosian to FP) pcDNA3-2X-MYC-hNR5A1 (a gift from FP as described in ref. 36. Empty pcDNA3 vector was used as a control.
For stimulation of the Wnt pathway, 6 uM CHIR-99021 (Cell signaling, 54290) was added to the media 1 hour before transfection. To confirm stimulation of the Wnt pathway, a commercial TOP/FOP-Flash reporter assay was performed. 2.5 × 10^4 HEK cells were plated on 96-well white chimney cell plates (Greiner, 655098) 24 hr prior to transfection. Cells were transfected with either TOPFlash or FOPFlash reporter plasmids along with pTK-RL plasmid (Promega) with regular media or with media in the presence of 6 µM CHIR. After 24 h, cells were analyzed using the Dual-Glo® Luciferase Assay System (Promega, E2980). Luciferase intensity was normalized to the Renilla to normalize transfection efficiency. Cells transfected with the TOP plasmid, in the presence of 6 µM CHIR, showed 43-fold induction compared to cells transfected without the addition of CHIR, while FOP-transfected cells showed no change upon the addition of CHIR, confirming the ability of 6 µM CHIR to activate the Wnt pathway.
To clone DNA inserts into luciferase plasmids, gDNA was extracted from earpieces of XY Enh13 WT, XX Enh13SOX9*−3bp -/- and XX Enh13SOX9*+1bp-/- homozygous mice using DNeasy Blood & Tissue kit (Qiagen, 69504). Enh13 was amplified along with the addition of restriction enzymes BglII and HindIII sites using 2X Phusion (NEB, M0531L, Primers used in Table S1). DNA inserts were digested using BglII and HindIII enzymes (Thermo, K1991) and ligated into the pGL4.26 plasmid (Promega) between the BglII and HindIII sites in the multiple cloning site using a DNA ligation kit (Takara, 6022). Sanger sequencing was used to validate each ligation product.

Luciferase assay
Luciferase assays were performed in HEK293T (human cells) or COS7 cells (monkey cells). 2.5 × 10^4 cells were plated on 96-well white chimney cell plates (Greiner, 655098) 24 hours prior to transfection. Cells were transfected with either empty or Enh13 WT/Enh13SOX9*−3bp/Enh13SOX9*+1bp pGL4.26 plasmids (100 ng) and expression plasmids for transcription factors, along with Renilla Luciferase pLR-TK vector (Promega) 1 ng for HEK293 cells and 15 ng for COS7 cells. In Fig. 4D, E, Supplementary Fig 8D. 20 ng of each TF was used and topped up with empty pcDNA3 plasmid to 120 ng total. In Fig. 5B and Supplementary Fig. 11A-B, TFs were added in a way that mimics the ratio of TF present at E12.5 granulosa cells according to TPM values from RNA-Seq of E12.5 granulosa cells45. TPM values of E12.5 granulosa cells are as follows: GATA4-385.7, NR5A1-173.6, RUNX1-24.2, SOX9-39.0. Therefore, the following amount was added from each TF: GATA4-61.96 ng, NR5A1-27.88 ng, RUNX1-3.89 ng, SOX9-6.27 ng and topped up to 100 ng total for conditions not including all factors. Transfection was performed using PolyJet transfection reagent (SignaGen, SL100688) at a 3:1 polyJet:DNA ratio. Luciferase activity was measured 24 hours following transfection using the Dual-Glo® Luciferase Assay System according to the manufacturer’s instructions (Promega, E2980).
To calculate relative Luciferase activity, Firefly Luciferase values were normalized to the Renilla Luciferase values and then to the average of the empty pcDNA3 vector. For Luciferase assays in COS7 cells, we normalized the Firefly luciferase values to that of the average of the empty pcDNA3 vector without normalizing first to Renilla, as GATA4, among other TFs, is known to dramatically affect Renilla activity in COS7 cells69. All experiments were performed in duplicate of technical repeats and were repeated at least three independent times or more. Statistical analyses were carried out using the Prism 9 software (GraphPad). Two-way ANOVA was performed to compare the samples, followed by Tukey’s/Dunnett’s. In Luciferase assays comparing only the Enh13 WT plasmid, one-way ANOVA was performed followed by Dunnett’s post-test.

Protein Binding Microarray (PBM) and analysis
Transcription factor (TF) binding characterization was performed using protein binding microarrays (PBMs), as described in refs. 70,71. Briefly, commercial microarrays (Agilent Technologies) containing all possible 10-mer or 9-mer sequences were converted to double-stranded DNA via solid-phase primer extension using Thermo Sequenase DNA Polymerase (Cytiva, Catalogue #: E79000Y) and a deoxynucleotide triphosphate (dNTP) mixture (dATP, dCTP, dGTP, dTTP). Microarrays were then blocked with 2% (wt/vol) non-fat dry milk (Sigma, Catalog #: M7409) and incubated with the TF of interest (pcDNA3-Flag-hSOX9; pcDNA3.1-Flag-Runx1-FL), expressed using the TnT® Quick Coupled Transcription/Translation System (Promega, Catalog #: L1170, L2081). Binding reactions were carried out in different buffers depending on the protein. RUNX1 was incubated in a PBS-based buffer, with 1% (wt/vol) non-fat dry milk, 51.3 ng/µl salmon testes DNA (Sigma, Catalog #: 16-201), 0.2 µg/µl bovine serum albumin (BSA) (NEB, Catalog #: B9200), and 1 mM dithiothreitol (DTT). SOX9 buffer contained 10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 1 mM MgCl₂, 0.01 mM ZnCl₂, 1% glycerol, 1% (wt/vol) non-fat dry milk, 10.4 ng/µl Poly(dI-dC) (Sigma, Catalog #: P4929), 0.2 µg/µl bovine serum albumin (BSA) (NEB, Catalog #: B9200), and 1 mM dithiothreitol (DTT).
Pre-incubated protein binding mixtures were applied to individual microarray chambers and incubated for 1 h at room temperature. Microarrays were then subjected to two sequential washing steps: first with PBS/0.5% (vol/vol) Tween-20 for 3 min, followed by PBS/0.01% Triton X-100 for 2 min. After protein incubation and washing, microarrays were incubated for 1 h at room temperature with fluorescently labelled antibody diluted in protein binding buffer supplemented with 2% milk, according to the epitope tag of the protein. The antibody used was Alexa 488-conjugated DYKDDDDK antibody (FLAG) (Cell Signaling Technology, Catalog #: 15008S; dilution 1:40). Following antibody incubation, microarrays were washed with PBS / 0.05% (vol/vol) Tween-20 for 5 min twice, followed by PBS for 2 min. All washing steps were performed in Coplin jars at room temperature with shaking at 125 rpm.
Fluorescence signals of the bound proteins were recorded at 488 nm using a GenePix® 4400A scanner at 2.5 µm resolution. Signal intensities were extracted using GenePix Pro 7.0 software, and the median pixel intensity was reported for each DNA probe. To identify DNA motifs recognized by the TF of interest, we analyzed all possible 8-base sequences (8-mers) or 7-base sequences (7-mers) as previously described70. In brief, DNA features were grouped into two sets—those containing the 8-mer/7-mer (foreground) and those without (background). We then compare the top half of signal intensities from both sets using a modified Wilcoxon-Mann-Whitney statistic, which helps identify the most enriched 8-mer/7-mer, termed the “seed” of the motif. Next, we assess the contribution of each nucleotide position within this seed by evaluating all possible nucleotide variants at each position, and the motif was further refined by including gaps at positions with high variability. Finally, we convert the derived motif into a position weight matrix (PWM), allowing for a quantitative representation of the TF’s binding specificity. PBM binding affinity visualization along the WT and mutant Enh13 sequence was performed by looking at the E-score, the Z-score and the fluorescent intensity for SOX9 and RUNX1 transcription factors on each 8-mers composing the Enh13 sequence. Plots were generated in R, using ggplot2.

JASPAR Analysis
We extracted Position Probability Matrices (PPMs) for 16 transcription factors (TFs) from the JASPAR 2024 database, selecting a combination of murine and human matrices due to the high conservation of TF binding sites between species. As JASPAR often provides multiple matrices per TF, we manually selected one or two per factor that best represented the core binding motif, minimizing noise in the analyses.
To compare the binding potential of Enh13 in its wild-type (WT) and its mutant forms (1bp insertion and 3bp deletion), we used the TFBSTools R package, as recommended by JASPAR. This package scans genomic sequences for TF binding sites based on PPMs, assigning each putative site a relative score between 0 and 1. We first analyzed the WT sequence, then introduced the mutations and rescanned each sequence.
Since the choice of score threshold significantly impacts TFBS predictions, we adopted an adaptive approach to ensure optimal sensitivity and specificity. While commonly used thresholds range from 0.75 to 0.9, we observed that each PPM exhibited a distinct score distribution across accessible genomic regions. To account for this variability, we tailored the threshold for each TF individually. We first applied TFBSTools to all accessible regions identified by ATAC-seq in mouse embryonic gonads at relevant developmental stages45, using an initial threshold of 0.5. For each PPM, we identified the score cutoff corresponding to the top 1-5% of binding predictions. We then refined this by using known TFBS in Enh13 as a control, selecting the threshold that detected all known binding sites while minimizing the noise. The chosen thresholds were those that identified the top 2% of results. This approach yielded an average threshold of 0.79, aligning with commonly used values. Finally, for each variant, we retained only TFBS prediction that exceeded the selected threshold in either the WT or mutated sequence.

Electromobility shift assay (EMSA)
Expression Plasmids with the T7 promoter were previously described for pcDNA3 human SOX9 and pcDNA3 human NR5A172. Plasmid pcDNA3 mouse RUNX1 was obtained from Addgene (REF#14585). Plasmid pCS2+mGATA4 was previously described10 and originally obtained by FP from Dr. Sergei Tevosian. Proteins were produced using the TNTTM coupled translation system (Promega) using T7 RNA polymerase for SOX9, RUNX1, NR5A1 and SP6 RNA polymerase for GATA4, according to the manufacturer’s instructions. Negative control was programed with pLuciferase control vector from the TNT Kit.
Probes were annealed by mixing 10 µg of each complementary oligonucleotide and heating at 95°C for three minutes, then at Tm+5° for 5 min in 20 mM Tris pH 7.5, 5 mM NaCl, 1 mM EDTA, slow-cooled to 37°C for 60 minutes and cooled to room-temperature. After annealing, the DNA duplexes were de-salted through a Sephadex G-50 spin column (GE healthcare, 27-5330-01). For radioactive labelling, a 5’ overhang G at both ends of probes was used to fill in with a [32P]-dCTP (Amersham) using Superscript IV reverse transcriptase (Invitrogen). 100 ng of hybridized probes were incubated with 10 µCi of dCTP and 1 µl of Superscript IV for 1 h at 37°C in 1x RT buffer from the manufacturer. Un-incorporated nucleotides were removed through a Sephadex G-50 spin column. DNA binding assay was performed in a final volume of 20 µl with 10 mM Tris pH 8, 100 mM KCl, 5 mM MgCl2, 10 mM ZnSO4, 1 mM Spermidine, 0.075% triton X100, 1 µg BSA, 1 mM DTT, 10% glycerol and 2 µl of in vitro translation mix. As non-specific competitors, for SOX9, 1 µg of poly dG-dC and for NR5A1, GATA4 and RUNX1, 0.1 µg of sonicated salmon sperm DNA were added. 0.5 ng of radio-labeled probe was added, binding reactions were performed on ice for 30 minutes and complexes were resolved on a 5% native acrylamide (37.5:1) gel in 0.5X Tris acetate buffer (10X: 400 mM Tris buffered to pH 7.8 with glacial acetic acid) at 4°C, then dried and revealed using Molecular Dynamics PhosphoImager. Quantifications were performed using ImageQuant software. Statistics were performed using GraphPad Prism 9 with Ordinary one-way ANOVA with Tukey’s multiple comparisons test.

Sequences of oligos for short probes
SOX9 BS Enh13 Wt fw: gGGCTTGGGCAAGCAAACCACAACAATGGTCAGACTGATAA SOX9 BS Enh13 Wt rev: gTTATCAGTCTGACCATTGTTGTGGTTTGCTTGCCCAAGCC SOX9 BS_mut_fw: gGGCTTGGGCAAGCAAACCAGGGGGGGGGTCAGACTGATAA SOX9 BS_mut_rev: gTTATCAGTCTGACCCCCCCCCTGGTTTGCTTGCCCAAGCC SOX9 BS del3bp fw: gGGCTTGGGCAAGCAAACCACAATGGTCAGACTGATAA SOX9 BS del3bp rev: gTTATCAGTCTGACCATTGTGGTTTGCTTGCCCAAGCC SOX9 BS ins1bp fw: gGGCTTGGGCAAGCAAACCACAACAATGGTCAGACTGATAA SOX9 BS ins1bp rev: gTTATCAGTCTGACCATTGTTGTGGTTTGCTTGCCCAAGCC

Sequences of oligos for long probes
Enh13-110-175-WT fw: gTGAGGAATTAGAAGGCCAGGTTGGCTTGGGCAAGCAAACCACAACAATGGTCAGACTGATAAAGCC Enh13-110-175-WT rev: gGGCTTTATCAGTCTGACCATTGTTGTGGTTTGCTTGCCCAAGCCAACCTGGCCTTCTAATTCCTCA Enh13-110-175-del3bp fw: gTGAGGAATTAGAAGGCCAGGTTGGCTTGGGCAAGCAAACCACAATGGTCAGACTGATAAAGCC Enh13-110-175-del3bp rev: gGGGCTTTATCAGTCTGACCATTGTGGTTTGCTTGCCCAAGCCAACCTGGCCTTCTAATTCCTCA Enh13-110-175-ins1bp fw: gTGAGGAATTAGAAGGCCAGGTTGGCTTGGGCAAGCAAACCACAATCAATGGTCAGACTGATAAAGCC Enh13-110-175-ins1bp rev: gGGCTTTATCAGTCTGACCATTGATTGTGGTTTGCTTGCCCAAGCCAACCTGGCCTTCTAATTCCTCA

Statistics & reproducibility
No formal statistical method was used to predetermine sample sizes. Instead, sample sizes were selected based on common practice for comparable assays and on ensuring reproducibility across independent experiments. All key experiments were performed with a minimum of three independent biological replicates. When increased variability was observed between replicates, additional independent repeats were performed to confirm results. Technical replicates where used, are indicated in the Methods/figure legends. Exact n values and what n represents are provided in each figure legend. Statistics were performed using GraphPad Prism 9 with either Ordinary two-way ANOVA or Ordinary one-way ANOVA with Tukey’s multiple comparisons test, as stated in the figure legends.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Testis development is initiated by Sry, which activates Sox9 expression in mice. Loss of Sox9 leads to XY sex reversal, whereas its ectopic activation in XX gonads induces testis formation. The main enhancer of Sox9 is Enh13. Deletion of Enh13 in XY gonads results in a sex reversal phenotype similar to that observed with Sox9 loss. In this study, the authors demonstrate that small mutations (-3bp/+1bp) in Enh13 leads to robust Sox9 expression in XX gonads, resulting in testis development. These mutations could disrupt the binding of key transcription factors including RUNX1, NR5A1 and GATA4. Whereas the mutations described here lead to an abnormal activation of Sox9 in XX ovaries, they do not support a repressive role of non-mutated Enh13. The results can be interpreted as follows: in XX gonads, Enh13 is able to drive low levels of Sox9. This is supported by the transgenic analysis of non-mutated Enh13 in XX gonads, where Enh13-LacZ was able to drive LacZ expression (Gonen, 2018). This low level of expression is not sufficient to allow testis development. In contrast, the mutation described in this article leads to strong Sox9 expression, sufficient to induce Sertoli cell differentiation. Showing Sox9 expression at earlier stages in XX mutant gonads would support that abnormal expression of Sox9 triggers the sex reversal phenotype. Overall, this discovery is both intriguing and clinically relevant, particularly for understanding 46,XX DSD in human patients.\nSeveral points warrant further clarification.\nComments on the presentation\nNature Communications’ guidelines emphasize the importance of accessibility in figure design, particularly regarding color usage. The journal advises avoiding the use of red and green together for contrast, as this combination is not accessible to individuals with color vision deficiencies. Instead, they recommend using high-contrast, color-safe combinations such as green and magenta or other accessible palettes. Additionally, the journal adheres to Web Content Accessibility Guidelines (WCAG 2.1) level AA, ensuring that all readers, regardless of visual abilities, can interpret the figures effectively.\nFurthermore, the figures depicting embryonic gonads would be significantly improved by focusing more closely on the gonadal tissue itself and increasing its relative size within the panels.\nIn the figures, the statistical significance annotations should explicitly indicate which specific samples or groups are being compared to avoid ambiguity.\nThe presentation of the results would benefit from clearer descriptions to make the work more accessible. Some terms and comparisons come across as subjective; providing more explicit definitions and explanations throughout would help ensure that the significance of the findings is more broadly appreciated. Some examples are given below.\nIntroduction\nFirst, genetic sex is defined at fertilization by the provision of an X or a Y chromosome to an X-bearing oocyte. Authors could clarify that the X or Y chromosome is provided by the sperm.\nOnce differentiated, the gonads produce sex hormones necessary for the development of internal and external genitalia, secondary sexual characteristics, and then control puberty and fertility of the individual. The reference has been lost.\nHere we show that XX mice carrying a 3 bp deletion or a 1 bp insertion within the SOX9 binding site (BS) of Enh13 present complete female-to-male sex reversal. The term “complete” may be misleading, since XX mutants exhibit ovotestis development (see Figure 2) and lack germ cells in adulthood.\nAdult XX mice appear externally and internally as males, but with small testes devoid of sperm due to the lack of the Y chromosomes. To illustrate the absence of germ cells in the XX testis, Paul Burgoyne's work on this topic could be cited rather than that of others. See Development (1987) 101 (Supplement): 133–142.\nResults\nPage 4 :Small mutations in the SOX9 BS of Enh13 lead to XX male development in adulthood\nXX mice homozygous for the 3 bp deletion were born fully sex reversed and appeared externally as males. The term 'fully sex reversed' may be overly strong, as 'sex reversed' already conveys a significant change. Consider revising the sentence to simply state: XX mice homozygous for the 3 bp deletion were born sex reversed highlighted by male external genitalia. Adult mice appeared with small testes void of gonocytes, a phenotype known to occur in XX males as they are not able to support spermatogenesis in the absence of Y chromosome genes. I believe the intended word is \"devoid\" instead of \"void\". See my comment above for the reference.\nIn the extended figure 2, there are inconsistencies in the capitalization of annotations (e.g., \"Weeks\" vs. \"weeks\"). To improve clarity and maintain uniformity, I recommend standardizing all annotations in the figures.\nXX WT and heterozygous gonads exhibited no expression of Sox9 and Sox8. No expression is overstated. It has been shown by other groups than the cells of the developing rete ovarii express Sox9, which are difficult to dissect out.\nFigure 1: Are the Mullerian ducts derivatives present in XX mutant mice at 6 weeks ?\nPage 5: The conclusion should be The 3 bp deletion alone allows Sox9 expression to rise above the critical level required to trigger testicular differentiation in XX gonads.\nThere is no evidence for a repressive threshold.\nAltogether, these findings underscore the extraordinary sensitivity of the Sox9 regulatory network to subtle enhancer perturbations, capable of breaching the activation threshold required to initiate testicular development in an XX context. This sentence should be revised to focus more directly on the empirical findings.\nEmbryonic gonads develop as ovotestis upon small mutations in the SOX9 BS of Enh13\nThe statement \"embryos presented variable phenotypes of ovotestis ranging in severity\" requires clarification. The term \"severity\" needs to be defined.\nNormally, at E13.5, ovaries are devoid of clear structural characteristics while the testes contain a coelomic vessel along with testis cords. The use of \"Normally\" seems unnecessary here. Could the authors clarify what is meant by \"structural characteristics\"? What exactly is being compared between ovaries and testes at E13.5? Is the absence of a coelomic vessel in ovaries being contrasted with the presence of a coelomic vessel and testis cords in the testes?\nhalf a testis-half an ovary, please clarify how this is defined.\nIs the ovarian region systematically in the anterior or posterior part of the gonad? What is the orientation of the gonads in Fig 2A-B, Extended Data Figure 1B, Extended Data Figure 4?\nDifferences in severity appearance were also evident between the right and left gonads of the same embryo. This must be explained.\nor the more common view of ovotestis. Unnecessary.\nFOXL2 and SOX9 positive cells were mutually exclusive (Fig. 2B) : this is difficult to appreciate in Figure 2B, may be the authors could show a zoom of the gonad showing the exclusive expression of FOXL2 and SOX9.\nthey are identical to XX WT or XY WT, respectively. Identical? Similar is maybe better.\nXX homozygous mutant gonads from the Enh13SOX9*+1bp-/- (Fig. 2D) and Enh13SOX9*-3bp SRY*-12bp-/- (Extended Data Fig. 4G) strains also presented an ovotestis phenotype, with a more testis-like appearance compared to the Enh13SOX9*-3bp-/- strain. I understand how the +1bp sex reversal could be more complete owing to the presence of an additional potential GATA4 BS, could the authors comment on the reason for a stronger phenotype in the Enh13SOX9*-3bp SRY*-12bp-/- gonads compared to the Enh13SOX9*-3bp-/-.\nHow is the ovotestis phenotype observed at E13.5 resolved into a testis phenotype at 6 weeks? The authors should provide data on an intermediate stage.\nTranscriptomic analysis of E12.5 embryonic gonads demonstrates an ovotestis pattern\nPre-granulosa markers as Foxl2, Fst, Bmp2, Wnt4, Runx1, Emx2 and Lhx9 were highly expressed in XX WT and XX heterozygous gonads, but displayed a low-to-intermediate expression in the XX homozygous mutant gonads of the 3 mouse strains (Fig. 3C and Extended Data Fig. 6B). While Foxl2 and Fst are good markers of pre-granulosa cells, the authors should note that Runx1, Bmp2, Rspo1 and Wnt4 mark pre-supporting cells before their differentiation into pre-granulosa cells. Moreover, Lhx9 and Emx2 are expressed in the CE, pre-supporting cells and stromal/interstitial progenitors.\nIn contrast, XX homozygous gonads from all three mutant strains exhibit a progressive shift from an ovarian transcriptional identity toward a testicular transcriptional identity. The statement should be modulated. The transcriptome at one single developmental stage cannot show a “progressive shift from an ovarian transcriptional identity toward a testicular transcriptional identity” but rather a mixed ovarian and testicular transcriptomic signature at E12.5.\nThe authors should address the expression of Sox9/SOX9 by qPCR and/or IF at an earlier stage, to determine the initial time when Sox9 is abnormally upregulated in XX Enh13 mutant gonads.\nThe SOX9 BS mutations do not abolish the ability of SOX9 to bind and activate Enh13\nElectro mobility shift assays (EMSA) with in vitro–translated SOX9 on synthetic Enh13 sequences (WT, –3bp, +1bp). A negative test with an irrelevant sequence to Enh13 would reinforce the results.\nFig 7C. Can the authors show the Luciferase assay results when individual TF are transfected? This is highly relevant as they conclude from Figure 4 that cooperative binding of TF is altered in Enh13 mutants.\nThe SOX9 BS mutations affect the cooperativity of TFs on Enh13 and enhance its activity\nJASPAR analysis of ~2300 vertebrate TFs, including all those expressed in the gonad at this stage indicated no major gain or loss of binding sites was found between WT and mutant Enh13 sequences to explain the phenotype (Extended data Figure 9). The authors show in Figure 4 the creation of a de novo potential GATA motif in the +1bp mutant. Can they comment on this?\nIn this part, the experiments used materials from human, mouse, and monkey. Are all these reagents equivalent? Please clarify in the Materials and Methods section that they originate from different species.\nTogether, these results indicate that the mutations not only boost enhancer responsiveness but also render it less susceptible to RUNX1/NR5A1/GATA4-mediated repression, thereby facilitating Sox9 activation. The results shown in Fig. 4B and in Extended Data Fig. 10 show boosted enhancer responsiveness to NR5A1/GATA4 of both enhancers in COS7 cells but only of the +1bp enhancer in HEK293T cells. Can the authors comment on this?\nTo better understand how the mutations might affect cooperative binding of the different transcription factors the authors should provide the results of luciferase assays with individual transfections of NR5A1 and GATA4 and SOX9 as well as the results of luciferase assays with the double transfections of NR5A1/RUNX1, GATA 4/RUNX1 and SOX9/RUNX1. The authors state that the distinct responses of the two mutants to NR5A1 and GATA4 alone suggest that they may act through different molecular mechanisms to alter enhancer function but the corresponding luciferase assays of single transfections of NR5A1 and GATA4 are not shown.\nWe also show that the -3bp/+1bp mutations sufficiently modify the 3D structure of Enh13, and hence cooperative binding of TFs. I am not certain that the luciferase assays show this. These experiments show that Enh13 mutations affect the transactivation output of the combined transcription factors, whether this occurs by alteration of the 3D structure and of the TF cooperative binding, or rather by alteration of the 3D structure and the transactivation (positive or negative) abilities of the TF/transcriptional machinery complex is unknown.\nOverall, we demonstrate that Enh13 is used for both the activation and repression of Sox9 expression in the testis and ovary, respectively. The role of Enh 13 is to activate Sox9 in XY gonads. However, the mutations described (-3 bp or +1 bp) appear to cause ectopic up-regulation of Sox9 in XX gonads, rather than reflecting a repressive function of Enh13 in XX gonads. These mutations in XX gonads represent an abnormal activation, and it may be more accurate to describe this as such rather than as Enh13 acting as a repressor in the ovary.\nDiscussion\nThe two mutations described here show that Enh13 has repressive activity in the presence of bipotential factor in the normal ovary, and the subtle alterations in its sequence can overcome this repressive state. In all experiments the transfection of bipotential factors NR5A1 and GATA4 always results in an increase of luciferase activity, both in the context of WT and mutated Enh13. Therefore, I do not agree with the conclusion that Enh13 has repressive activity. Rather Enh13 would have a weak activating activity in the presence of bipotential factors that is further dampened by RUNX1 (RUNX1 being only maintained in XX supporting cells). This activity would not be sufficient to produce the threshold level of SOX9 protein required for the self-amplification loop in the XX WT context. In the XX Enh13 mutant contexts the presence of an additional GATA site and/or the disruption of the enhancer structure would result in an increase of the transactivation activity of the enhancer even in the presence of RUNX1, allowing for sufficient SOX9 production and initiation of self-amplification loop.\nThis conclusion based on activation of Sox9 expression above a given threshold is the one that the authors themselves state in the introduction and in the results:\nIntroduction p3: Our enhancer reporter assays suggest that the mutations we induced do not abolish the SOX9 BS but rather enable the bipotential factors NR5A1, RUNX1, and GATA4 to acquire sufficient activating capacity to induce Sox9 expression above the minimal threshold present in female gonad.\nResults p5 : Altogether, these findings underscore the extraordinary sensitivity of the Sox9 regulatory network to subtle enhancer perturbations, capable of breaching the activation threshold required to initiate testicular development in an XX context.\nOther comments: Yet, KO of individual profemale factors including WT1 (-KTS) 18, FOXL2 24-26, RUNX1 27 and components of the WNT pathway 19-23 do not lead to XX male phenotype in mice. It is only when two pro-female factors or more are deleted together, that XX male phenotype start to appear at embryonic stages 27-30. I'm not sure that I understand what the authors mean. Could they elaborate?", "reviewer_2": "This manuscript is an exciting contribution to the sex determination literature showing that female to male sex reversal can be caused by deletions in a critical enhancer of Sox9. Enh13 has previously been shown to be a principal regulator of Sox9 expression in XY gonadal supporting cells. Several of these authors previously showed that mutations in enh13 disrupt male sex determination and testis development. Surprisingly, they now show that addition of 1 base pair (bp) or deletion of 3 bp in this enhancer show no effect on male sex determination or SOX9 expression in XY gonads but lead to elevated Sox9 expression in XX gonads and female to male sex reversal. This manuscript speaks to the amazing plasticity of the sex determination system at the molecular level and provides an interesting example of cell fate switching hinging on minor changes in an enhancer that may alter the relationship of transcription factors known to bind there. The authors create many new mouse strains to explore this phenotype and each is well characterized. They then turn to a molecular analysis of what could be going on at the locus. Using reporter assays in HEK293 cells and EMSA assays, they demonstrate that SOX9 binding in not inhibited by the indels, but cooperative binding of NR5A1, GATA4, RUNX1 and SOX9 may be affected by changes in spacing. Of course, there is always more to learn but I find this a relatively complete story and have only a few recommendations.\nIn the abstract the authors say, “Mechanistically, these mutations do not eliminate SOX9 binding”. I think a prior sentence to explain that SOX9 binds it’s own promoter would be helpful.\nLater in the manuscript the authors say “These findings rule out increased SOX9 binding or transcriptional activation as the underlying mechanism.” I found this section confusing and felt this conclusion was misleading. After all, the argument is that Sox9 expression is increased by the absence of repression. Have the authors tested whether RUNX1 can repress Sox9 in the presence of low levels of SOX9? Is there a binding competition between these two factors? An assay in HEK cells increasing the dose of SOX9 in the presence of RUNX1 would be ideal.\nI would like to see the wild type ATAC landscape around Enh13 in one of the figures. Would ATAC-seq be a helpful approach in mutants to query how binding is changed?\nI find the annotation confusing. Could the mutants be represented as alleles using the usual annotation? Sox9-Enh13Sox9*-3bp/Sox9*-3bp (denoting a homozygote) ; compared to Sox9-Enh13Sox9+1bp/+ (denoting a het).\nPlease indicate the A/P axis of gonads in Fig.2. Is the ovarian region always anterior?\nFig. 3B – what is the red sample in the XY section of the sample designation line?", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The manuscript by Elisheva Abberbock and colleagues clearly demonstrates that engineered mutations in a key testicular enhancer of the Sox9 gene (named Enhancer13, or Enh13) lead to an absence of Sox9 repression in XX developing ovaries. This is followed by testis differentiation without SRY and XX sex reversal in adulthood. The authors elegantly and convincingly demonstrate that Enh13, which was previously described as the key regulatory region for Sox9 upregulation in XY pre-supporting cells, also plays a pivotal role in Sox9 repression in XX ovarian pre-supporting cells. This work confirms and provides molecular explanations for XX sex reversal observed in other species, where abnormal SOX9 gene upregulation has been shown to play a primary role in some human cases and in pig intersexuality.\nThe major conclusions of the manuscript are stated in the title and throughout the Results section. All of the authors' assertions are supported by clear, statistically validated results from morphological observations of the external and internal genital tracts (including gonads), IHC, gonad transcriptomes by bulk RNA-seq from 45 mice with different genotypes, gene expression levels by qPCR, PBM, EMSA, transfection assays caried onto two different cell types (HEK293T and COS7) and bioinformatic analyses. The main result is impressive: the authors clearly demonstrate that adding one nucleotide or suppressing three in the SOX9 binding site of Enh13 changes the sex of the individual. Additionally, they prove that the molecular mechanism involved is different in each XX sex reversal cases. In conclusion, all of the authors' claims and conclusions are justified by their large number of high-quality, rigorous results.\nIn addition to these general comments, I have two remarks and few minor corrections for the authors.\n1 – My first remark concerns the interpretation of the results proposed by the authors and illustrated in Figure 4E, as well as the binding of RUNX1 and SOX9 to their TFBS, which are very close and overlap by one or two bases in the WT allele, and by an even greater amount in the case of the -3 bp allele. Do the authors not consider that the binding of RUNX1 (expected in XX supporting cells) and SOX9 (expected in XY cells) are mutually exclusive on these TFBS due to their proximity and the steric hindrance of both proteins? In other words, why do they not consider that there is competition between the two transcription factors for binding to this region, with RUNX1 binding resulting in repression (or an absence of activation) and SOX9 binding resulting in activation? When the authors tested the binding capacity of both factors individually using PBM or EMSA, they showed that RUNX1 bound equivalently to all three alleles (WT, +1 and -3 bp), but SOX9 had less affinity for the -3 bp allele. Nevertheless, SOX9 is the dominant factor, since sex reversal occurs. One possible explanation is that, in vivo, the chromatin conformation of this region causes competition between the two TFs for binding to this critical region, and the 3 bp deletion prevents RUNX1 binding and thus promotes SOX9 binding, despite its decreased affinity. For the +1 allele and the creation of a new GATA site in this small region, the competition is now between not only RUNX1 and SOX9, but also GATA4. I leave it up to the authors to decide whether or not to take this comment into account in the discussion of the results.\n2 – My second remark concerns the deletion of the SRY BS in addition of the 3-bp deleted in the SOX9 BS (first paragraph of page 5). The authors state that “removal of the SRY BS does not affect the female to male sex reversal of XX homozygous mice”. Why do the authors expect a role of this SRY BS in an XX context where there is no SRY? Moreover, the authors state that deleting SRY BS in addition to the 3-bp aggravate the phenotype of sex reversal (last paragraph of page 6). How could the authors explain this observation since there is no SRY?\n3 – Page 7, last paragraph: the authors write “Analysis of bipotential genes, Nr5a1, Wt1, Gata4…..revealed no major differences in expression between WT and HM mutants”. By contrast Nr5a1 and Wt1 are found differentially expressed in Fig6B (cluster a & f, respectively) and also on Extended data Fig6A, at least between WT males and females with slight differences for HM mutants (male type for Nr5a1, female type for Wt1).\n4 – Page 8, first paragraph. The authors could add Rspo1 in the list of pre-granulosa markers and Aard in those of Sertoli markers, because expression of these two genes are presented on ExtDataFig6 (B & C, respectively).\n5 - Page 10, last paragraph, second sentence: “We also show….even in the absence of male-specific factors”. The authors probably think to SRY. It will be better to write “…even in the absence of SRY”.\n6 – Figure 2C: The number of studied samples for XX+/+ is n=5 (as indicated in the parenthesis), but there are 4 points for Sox9 and 6 points for Sox8 and Foxl2.\n7 – Legend of Figure 2C : “in” is missing between “involved” and “male”.\n8 – Figure 4A: the last base of the consensus sequence for the SOX9 binding site is missing on the Figure (should be A>G=T).\n9 – Extended Data Fig.1B: “oviduct” term is present on the fourth animal (on 6 weeks gonads) and should be suppressed.\n10 – Page 22; Methods; Paragraph “Design and preparation of sgRNAs mRNA”; lane 5 of this paragraph: please write “concentration” instead of “concertation”."}, "author_response": "We want to sincerely thank all four reviewers for their useful, instructive, and supportive comments.\nYour suggestions have been extremely useful and helped us solidify the mechanism mediated by the\nEnh13 mutations, for which we are grateful for.\nWe have experimentally and textually addressed all the comments. We provide here a point-by-point\nreply to all the comments raised by each reviewer and indicate the edits that were done in the manuscript\nand figures. We have added new data and figures and now able to show that pro-female factors, and\nmore so, RUNX1 are able to repress Enh13 activity and hence Sox9.\nThe reviewers’ comments are in black, and our replies are in blue.\nWe also want to apologise for not adding line numbers in the initial submission which made your work\nharder in trying to direct us. We have now included line numbers to the revised manuscript.\nReviewer #1\nTestis development is initiated by Sry, which activates Sox9 expression in mice. Loss of Sox9 leads to\nXY sex reversal, whereas its ectopic activation in XX gonads induces testis formation. The main\nenhancer of Sox9 is Enh13. Deletion of Enh13 in XY gonads results in a sex reversal phenotype similar\nto that observed with Sox9 loss. In this study, the authors demonstrate that small mutations (-3bp/+1bp)\nin Enh13 leads to robust Sox9 expression in XX gonads, resulting in testis development. These\nmutations could disrupt the binding of key transcription factors including RUNX1, NR5A1 and\nGATA4. Whereas the mutations described here lead to an abnormal activation of Sox9 in XX ovaries,\nthey do not support a repressive role of non-mutated Enh13. The results can be interpreted as follows:\nin XX gonads, Enh13 is able to drive low levels of Sox9. This is supported by the transgenic analysis\nof non-mutated Enh13 in XX gonads, where Enh13-LacZ was able to drive LacZ expression (Gonen,\n2018). This low level of expression is not sufficient to allow testis development. In contrast, the\nmutation described in this article leads to strong Sox9 expression, sufficient to induce Sertoli cell\ndifferentiation. Showing Sox9 expression at earlier stages in XX mutant gonads would support that\nabnormal expression of Sox9 triggers the sex reversal phenotype. Overall, this discovery is both\nintriguing and clinically relevant, particularly for understanding 46,XX DSD in human patients.\nWe are thankful for the nice summary of our paper and will address your concerns and suggestion\nbelow.\nSeveral points warrant further clarification.\nComments on the presentation\nNature Communications’ guidelines emphasize the importance of accessibility in figure design,\nparticularly regarding color usage. The journal advises avoiding the use of red and green together for\ncontrast, as this combination is not accessible to individuals with color vision deficiencies. Instead, they\nrecommend using high-contrast, color-safe combinations such as green and magenta or other accessible\npalettes. Additionally, the journal adheres to Web Content Accessibility Guidelines (WCAG 2.1) level\nAA, ensuring that all readers, regardless of visual abilities, can interpret the figures effectively.\nWe thank you for this important comment. Further to your suggestion we have now modified the real\ntime PCR graphs to be presented with Green and Magenta. The new graphs are presented in the revised\nFigure 1E and 2C.\nFurthermore, the figures depicting embryonic gonads would be significantly improved by focusing\nmore closely on the gonadal tissue itself and increasing its relative size within the panels.\nThank you for this point, we have tried to increase the magnification in the brightfield images however,\nthe resolution of the binocular at high magnification is not ideal. To get a closer look at the gonadal\ntissue, we have added X63 confocal images of the immunostaining performed on the gonads in Figures\n2B and 2D.\nIn the figures, the statistical significance annotations should explicitly indicate which specific samples\nor groups are being compared to avoid ambiguity.\nThis is correct. We have added to the figure legends which samples were compared to which.\nThe presentation of the results would benefit from clearer descriptions to make the work more\naccessible. Some terms and comparisons come across as subjective; providing more explicit definitions\nand explanations throughout would help ensure that the significance of the findings is more broadly\nappreciated. Some examples are given below.\nIntroduction\nFirst, genetic sex is defined at fertilization by the provision of an X or a Y chromosome to an X-bearing\noocyte. Authors could clarify that the X or Y chromosome is provided by the sperm.\nPage 2 line 20-22\nThis sentence was amended and now reads as: \"First, genetic sex is defined at fertilization by the\nprovision of an X or a Y chromosome by the sperm, to an X-bearing oocyte.\"\nOnce differentiated, the gonads produce sex hormones necessary for the development of internal and\nexternal genitalia, secondary sexual characteristics, and then control puberty and fertility of the\nindividual. The reference has been lost.\nPage 2 line 26\nA reference was indeed missing. We have now added a suitable reference (Ref No. 4).\nHere we show that XX mice carrying a 3 bp deletion or a 1 bp insertion within the SOX9 binding site\n(BS) of Enh13 present complete female-to-male sex reversal.\nThe term “complete” may be misleading, since XX mutants exhibit ovotestis development (see Figure\n2) and lack germ cells in adulthood.\nPage 3 line 23-24\nThis is correct. We removed the word complete and now describe this sentence as: \"Here we show that\nXX mice carrying a 3 bp deletion or a 1 bp insertion within the SOX9 binding site (BS) of Enh13\npresent female-to-male sex reversal.\"\nAdult XX mice appear externally and internally as males, but with small testes devoid of sperm due to\nthe lack of the Y chromosomes. To illustrate the absence of germ cells in the XX testis, Paul Burgoyne's\nwork on this topic could be cited rather than that of others. See Development (1987) 101 (Supplement):\n133–142.\nThank you for this suggestion. We have now added this reference (Ref No. 39) here and in the other\nplaces that describe the lack of germ cells in these XX sex reversed mice.\nResults\nXX mice homozygous for the 3 bp deletion were born fully sex reversed and appeared externally as\nmales. The term 'fully sex reversed' may be overly strong, as 'sex reversed' already conveys a significant\nchange. Consider revising the sentence to simply state: XX mice homozygous for the 3 bp deletion were\nborn sex reversed highlighted by male external genitalia.\nWe have amended this sentence as suggested and it now reads as: \" XX mice homozygous for the 3 bp\ndeletion were born sex reversed highlighted by male external genitalia and small testes (Fig. 1C).\"\nAdult mice appeared with small testes void of gonocytes, a phenotype known to occur in XX males as\nthey are not able to support spermatogenesis in the absence of Y chromosome genes. I believe the\nintended word is \"devoid\" instead of \"void\". See my comment above for the reference.\nWe have replaced the 'void' to 'devoid' and added the Burgoyne reference also here (Ref No. 39).\nIn the extended figure 2, there are inconsistencies in the capitalization of annotations (e.g., \"Weeks\" vs.\n\"weeks\"). To improve clarity and maintain uniformity, I recommend standardizing all annotations in\nthe figures.\nThank you for spotting this. It appeared in additional figures. We have amended all the figures to have\n6 weeks without capital.\nXX WT and heterozygous gonads exhibited no expression of Sox9 and Sox8. No expression is\noverstated. It has been shown by other groups than the cells of the developing rete ovarii express Sox9,\nwhich are difficult to dissect out.\nThis is very correct. We have amended the sentence to be: \"XX WT and heterozygous gonads exhibited\nlow expression of Sox9 and Sox8 while having high Foxl2 expression (Fig. 1E). XY WT gonads had\nhigh expression of Sox9 and Sox8 and no detectable Foxl2 expression.\"\nFigure 1: Are the Mullerian ducts derivatives present in XX mutant mice at 6 weeks?\nIn all the XX homozygous mice we have dissected from the three mutated lines we could never see any\nMüllerian duct derivatives present.\nThe conclusion should be: The 3 bp deletion alone allows Sox9 expression to rise above the critical\nlevel required to trigger testicular differentiation in XX gonads.\nThere is no evidence for a repressive threshold.\nYou are correct that in the first draft of the paper, there was no strong enough evidence to suggest that\nthe small mutations in Enh13 prevent repression, normally present in ovaries, on Sox9 expression.\nDue to your questions about repression abilities, as well as the suggestions of the other reviewers, we\nhave now added new Luciferase data and figures that prove that RUNX1 has repressive abilities on\nEnh13 and it may compete with SOX9 on the overlapping site (New Fig. 4D-E). We show that when\nwe add factors able to induce the enhancer and then add RUNX1, or other pro-female factors, this\nsignificantly lower enhancer activity (New Fig. 4D-E).\nWe hence believe that normally, Enh13 is repressed in females >>leading to block in Sox9 expression,\nwhereas in males it is strongly activated >> leading to high Sox9 expression. We suggest that the two\nmutants prevent the ability to repress Enh13 and hence, Sox9, and therefore, this triggers auto-\nregulation, elevated Sox9, and testis development (new model in Fig 6).\nBut since this new data is only presented in Figure 4, we have amended the sentence here to be as you\nsuggested. This appears in Page 5, Lines 20-23: \" The 3 bp deletion alone allows Sox9 expression to\nrise above the critical level required to trigger testicular differentiation in XX gonads and XY gonads.\nMoreover, the female-to-male sex reversal is solely due to the 3 bp deletion and does not involve the\nSRY motif, to which SOX9 can bind.\"\nAltogether, these findings underscore the extraordinary sensitivity of the Sox9 regulatory network to\nsubtle enhancer perturbations, capable of breaching the activation threshold required to initiate\ntesticular development in an XX context. This sentence should be revised to focus more directly on the\nempirical findings.\nWe have amended this sentence to now read as: \"Altogether, these findings underscore the extraordinary\nsensitivity of the Sox9 regulatory network to subtle enhancer perturbations, showing that specific\nmutations are capable of inducing Sox9 expression above a critical threshold required to initiate\ntesticular development in an XX context.\"\nThe statement \"embryos presented variable phenotypes of ovotestis ranging in severity\" requires\nclarification. The term \"severity\" needs to be defined.\nWe have removed the term \"ranging in severity\". The sentence is now: \"XX gonads of Enh13SOX9*-3bp-/-\nembryos presented variable phenotypes of ovotestis.\"\nNormally, at E13.5, ovaries are devoid of clear structural characteristics while the testes contain a\ncoelomic vessel along with testis cords. The use of \"Normally\" seems unnecessary here. Could the\nauthors clarify what is meant by \"structural characteristics\"? What exactly is being compared between\novaries and testes at E13.5? Is the absence of a coelomic vessel in ovaries being contrasted with the\npresence of a coelomic vessel and testis cords in the testes?\nWe have rephrased these sentences to a more scientific terms. The revised sentences are in: \"At E13.5,\nWT testis are organised into testis cords, containing Sertoli cells and germ cells, along with an\ninterstitial place where Leydig cell reside and present with a coelomic vessel. WT ovaries on the other\nhand are devoid of clear structural organisation at that stage and germ cells along with somatic cells are\nspread across the ovary area.\"\nhalf a testis-half an ovary, please clarify how this is defined.\nThank you, we have revised that sentence to \"While some XX homozygous gonads appeared mostly as\ntestes with coelomic vessel and testis cords, others had a coelomic vessel without clear testis cords and\nsome presented with a testicular gonadal portion alongside an ovarian portion\".\nIs the ovarian region systematically in the anterior or posterior part of the gonad? What is the orientation\nof the gonads in Fig 2A-B, Extended Data Figure 1B, Extended Data Figure 4?\nIn some gonads we see the ovarian portion in both the anterior and posterior parts, but when it is only\nin one portion, it is always the anterior side. We made sure that all BF and fluorescent images the gonads\nare presented from A-P and added the labelling on the figures.\nDifferences in severity appearance were also evident between the right and left gonads of the same\nembryo.\nWe have amended this sentence accordingly: \"Variability in ovotestis phenotypes were also evident\nbetween the right and left gonads of the same embryo where in one the anterior portion was ovarian\nand the posterior was testicular, whereas the other gonad presented an anterior and posterior ovarian\nportions and a centralised testicular portion (Fig. 2A, first and second mutant gonads are the right and\nleft gonads of the same embryo).\"\nor the more common view of ovotestis. Unnecessary.\nWe have amended this sentence, and it now reads as: \"XX Enh13SOX9*-3bp-/- gonads presented with\novotestis with an anterior ovarian portion alongside a testicular posterior portion, or an ovotestis where\nthe testicular tissue is centrally located, flanked by ovarian domains at both poles (Fig. 2B).\"\nFOXL2 and SOX9 positive cells were mutually exclusive (Fig. 2B) : this is difficult appreciate in Figure\n2B, may be the authors could show a zoom of the gonad showing the exclusive expression of FOXL2\nand SOX9.\nWe have now added a bigger magnification (X63) of these gonads in Fig. 2B, D, focusing on the border\nregions between the ovarian and testicular portions to show there is no overlap between SOX9 and\nFOXL2 expression.\nthey are identical to XX WT or XY WT, respectively. Identical? Similar is maybe better.\nwe changed to 'similar'.\nXX homozygous mutant gonads from the Enh13SOX9*+1bp-/- (Fig. 2D) and Enh13SOX9*-3bp\nSRY*-12bp-/- (Extended Data Fig. 4G) strains also presented an ovotestis phenotype, with a more\ntestis-like appearance compared to the Enh13SOX9*-3bp-/- strain. I understand how the +1bp sex\nreversal could be more complete owing to the presence of an additional potential GATA4 BS, could\nthe authors comment on the reason for a stronger phenotype in the Enh13SOX9*-3bp SRY*-12bp-/-\ngonads compared to the Enh13SOX9*-3bp-/-.\nThis is a good point that you raised, also raised by reviewer 4, and we do not have any explanation to\nthat. The 3 bp deletion in the Enh13SOX9*-3bp is completely identical to that in the Enh13SOX9*-3bp SRY*-12bp\nstrain. Perhaps by removing the SRY BS we also removed a repressive BS located nearby that slightly\nelevated Sox9 or maybe we altered cooperative binding of other factors in that region in a way that\nfurther enhanced Sox9 expression. We cannot know for sure without going into much experimental\ndetail and we feel this is beyond the scope of the current study which is big and elaborative as is.\nHow is the ovotestis phenotype observed at E13.5 resolved into a testis phenotype at 6 weeks? The\nauthors should provide data on an intermediate stage.\nThis is an excellent point. To address this, we have experimentally analysed by bright field and\nimmunostaining gonads of E15.5 and P1 mutant and WT embryos/ mice. This now appears in page 7,\nlines 18-30\nWe see that at E15.5, the -3bp line still appear as ovotestis with FOXL2 expression in one or two poles.\nOn the contrary, the +1bp line is mostly expressing SOX9 and very few FOXL2 expression is present,\nif at all.\nIn P1 gonads, we see the same pattern where in the -3bp line we were still able to observe some FOXL2\npositive cells, while in the +1bp line we observe only a small number of FOXL2 positive cells, if at all.\nThis now appears in the new Extend data Fig. 5 and the text.\nPre-granulosa markers as Foxl2, Fst, Bmp2, Wnt4, Runx1, Emx2 and Lhx9 were highly expressed in\nXX WT and XX heterozygous gonads, but displayed a low-to-intermediate expression in the XX\nhomozygous mutant gonads of the 3 mouse strains (Fig. 3C and Extended Data Fig. 6B).\nWhile Foxl2 and Fst are good markers of pre-granulosa cells, the authors should note that Runx1, Bmp2,\nRspo1 and Wnt4 mark pre-supporting cells before their differentiation into pre-granulosa cells.\nMoreover, Lhx9 and Emx2 are expressed in the CE, pre-supporting cells and stromal/interstitial\nprogenitors.\nThank you for that correct comment. We have modified the sentence to say: \"Markers expressed in pre-\ngranulosa cells as Foxl2, Fst, Rspo1, Bmp2, Wnt4, Runx1, Emx2 and Lhx9 were highly expressed in XX\nWT and XX heterozygous gonads, but displayed a low-to-intermediate expression in the XX\nhomozygous mutant gonads of the 3 mouse strains (Fig. 3C and Extended Data Fig. 7B)\"\n\"In contrast, XX homozygous gonads from all three mutant strains exhibit a progressive shift from an\novarian transcriptional identity toward a testicular transcriptional identity\".\nThe statement should be modulated. The transcriptome at one single developmental stage cannot show\na “progressive shift from an ovarian transcriptional identity toward a testicular transcriptional identity”\nbut rather a mixed ovarian and testicular transcriptomic signature at E12.5.\nThis is correct and we have amended the sentence as you suggested. The new sentence reads as: \"In\ncontrast, XX homozygous gonads from all three mutant strains exhibit a progressive shift from an\novarian transcriptional identity towards a testicular transcriptional identity.\"\nThe authors should address the expression of Sox9/SOX9 by qPCR and/or IF at an earlier stage, to\ndetermine the initial time when Sox9 is abnormally upregulated in XX Enh13 mutant gonads.\nSince E11.5 is a very dynamic stage where even a few TS can make a dramatic change (describe my\nScience paper), we decided to perform WM immunostaining at E11.5 (18-21TS) to analyse SOX9\nexpression.\nThank you for this excellent suggestion. According to your suggestion we performed wholemount\nimmunostaining of E11.5 gonads (18-21 TS) of XX WT, XX Enh13SOX9*-3bp-/-, XX Enh13SOX9*+1bp-/-, and\nXY. This appears in the new Ext Data Fig. 12. We did co-staining of SF1 to label the gonad region,\nDAPI that also labels the mesonephros, and SOX9. We can see SOX9 staining in WT XY gonads, and\nno staining in WT XX gonads. The XX Enh13SOX9*+1bp-/- gonads clearly express SOX9 whereas the XX\nEnh13SOX9*-3bp-/- do not yet. This is consistent with the stronger testicular appearance in the +1bp mutant\ncompared with the ovotestis appearance in the - 3bp mutants at E13.5.\nThis figure and accompanying text is now included in the revised manuscript (Page 12, lines 18-26).\nElectro mobility shift assays (EMSA) with in vitro–translated SOX9 on synthetic Enh13 sequences\n(WT, –3bp, +1bp). A negative test with an irrelevant sequence to Enh13 would reinforce the results.\nThis was added in the revised extended Data Fig. 8B. We have added an EMSA experiment using either\nthe WT SOX9 BS region in Enh13 or the mutated version used in the Ogawa paper (PMID: 35921234)\nand also in our NAR paper (PMID: 38499491) where the SOX9 BS was replaced by a poly G sequence.\nIndeed, the mutant probe shows complete elimination of SOX9 binding.\nSupp Fig 7C. Can the authors show the Luciferase assay results when individual TF are transfected?\nThis is highly relevant as they conclude from Figure 4 that cooperative binding of TF is altered in Enh13\nmutants.\nThank you for this suggestion. We have performed Luciferase experiments with individual TFs and this\nis now included in the new Extended Data Fig. 8D. Individual TFs are not able to induce enhancer\nactivity, whereas combination of several TFs is inducing enhancer activity.\nPage 9 line 16-19\nJASPAR analysis of ~2300 vertebrate TFs, including all those expressed in the gonad at this stage\nindicated no major gain or loss of binding sites was found between WT and mutant Enh13 sequences\nto explain the phenotype (Extended data Figure 9).\nThe authors show in Figure 4 the creation of a de novo potential GATA motif in the +1bp mutant. Can\nthey comment on this?\nWhen we did the JAPSAR analysis, we used a very strict analysis where we only kept binding sites\nwith high Position Probability Matrices (PPM) score. The JAPSAR analysis did not identify the creation\nof the Neo GATA4 site as it fell below the cutoff score we were using. We went to look for the new\nGATA4 BS only after the GATA4 EMSA indicated the presence of another complex in the 1 bp mutant\nand this is how we spotted the new site created upon the T insertion.\nIn this part, the experiments used materials from human, mouse, and monkey. Are all these reagents\nequivalent? Please clarify in the Materials and Methods section that they originate from different\nspecies.\nWe have added to the methods the following sentence: \" Luciferase assays were performed in HEK293T\n(human cells) or COS7 cells (monkey cells)\".\nPage 10, lines 4-6\n\"Together, these results indicate that the mutations not only boost enhancer responsiveness but also\nrender it less susceptible to RUNX1/NR5A1/GATA4-mediated repression, thereby facilitating Sox9\nactivation\".\nThe results shown in Fig. 4B and in Extended Data Fig. 10 show boosted enhancer responsiveness to\nNR5A1/GATA4 of both enhancers in COS7 cells but only of the +1bp enhancer in HEK293T cells.\nCan the authors comment on this?\nThere is indeed a slight difference in enhancer activation between HEK293 and COS7 cell. When we\nadd GATA4 and NR5A1, we see in HEK cells significant higher activation in the +1bp but not the -\n3bp, whereas in COS7 cells, both show very strong activation compared to WT. These two cell lines\nrepresent an optimal system to explore these questions as they are naturally devoid of these factors and\nhence represent \"a neutral system\" to explore the effect of adding the factors in the reconstruction\nexperiments we did. While addition of only GATA4 and NR5A1was not statistically significant in\nHEK293, addition of RUNX1, GATA4 and NR5A1 together showed a strong statistical significance in\nboth cell lines, suggesting that it is the combination of the 3 factors that is mediating the difference\nbetween the WT and mutants.\nTo better understand how the mutations might affect cooperative binding of the different transcription\nfactors the authors should provide the results of luciferase assays with individual transfections of\nNR5A1 and GATA4 and SOX9 as well as the results of luciferase assays with the double transfections\nof NR5A1/RUNX1, GATA4/RUNX1 and SOX9/RUNX1. The authors state that the distinct responses\nof the two mutants to NR5A1 and GATA4 alone suggest that they may act through different molecular\nmechanisms to alter enhancer function but the corresponding luciferase assays of single transfections\nof NR5A1 and GATA4 are not shown.\nThis is an excellent suggestion, and we are grateful for this. We have taken your advice and now\nperformed Luciferase assays with either individual TFs (NR5A1, GATA4, RUNX1, SOX9) or\ncombination of RUNX1 with each of the TFs- NR5A1, GATA4 and SOX9. We see that neither\nindividual TFs nor combination of two TFs (when one is RUNX1) is able to induce enhancer activity.\nIt is only combination of NR5A1 and GATA4 that is inducing the WT enhancer but more so the mutated\nversions. Addition of RUNX1 lowers the WT Enh13 but not the two mutants. This further supports our\nhypothesis that the two mutants prevent RUNX1-mediated repression that should normally happen.\nThis new data appears in the new Extended Data Fig. 11A.\nWe also show that the -3bp/+1bp mutations sufficiently modify the 3D structure of Enh13, and hence\ncooperative binding of TFs. I am not certain that the luciferase assays show this. These experiments\nshow that Enh13 mutations affect the transactivation output of the combined transcription factors,\nwhether this occurs by alteration of the 3D structure and of the TF cooperative binding, or rather by\nalteration of the 3D structure and the transactivation (positive or negative) abilities of the\nTF/transcriptional machinery complex is unknown.\nWe indeed do not provide evidence that the mutations have an effect on the 3D structure of Enh13 and\nwe hence modified the sentence to: \" We also show that the –3bp/+1bp mutations sufficiently modify\nthe cooperative binding of TFs, to allow significantly higher activation of Sox9 even in the absence of\nSRY.\"\n\"Overall, we demonstrate that Enh13 is used for both the activation and repression of Sox9 expression\nin the testis and ovary, respectively\". The role of Enh 13 is to activate Sox9 in XY gonads. However,\nthe mutations described (-3 bp or +1 bp) appear to cause ectopic up-regulation of Sox9 in XX gonads,\nrather than reflecting a repressive function of Enh13 in XX gonads. These mutations in XX gonads\nrepresent an abnormal activation, and it may be more accurate to describe this as such rather than as\nEnh13 acting as a repressor in the ovary.\nYou are correct that in the first draft of the paper we did not have strong enough evidence for a\nrepressive role of Enh13 in the ovary. Yet, the new data we present in the new Fig. 4D and 4E shows\nthat when we activate the WT Enh13 with either just bipotential factors (NR5A1, GATA4 and WT1)\nor also with SOX9 (NR5A1, GATA4, WT1 and SOX9), and then we add pro-female factors, this\nstrongly repress enhancer activity. With the bipotential mix it drops from 10 to 5 (50% reduction) and\nin the mix with SOX9 it goes from 133 fold to 32 fold induction (75% reduction).\nWe believe that this new added data does allow us to conclude that normally in XY gonads, the enhancer\nis activated by bipotential factors and SRY, leading to elevated SOX9, that then replaces SRY and\nfurther activates its own expression leading to testis development.\nOur revised model is now presented in the new Figure 6 and is describes as such: \" In XY gonads, SRY,\nparticularly through its functional interaction with NR5A1 7, activates Enh13 and induces Sox9\nexpression, which subsequently reinforces its own expression through auto-activation, leading to testis\ndevelopment (Fig. 6, XY WT). In XX gonads, bipotential factors, including RUNX1, bind Enh13 and\nmediate repression of Sox9. Around E12.0, upon the onset of FOXL2 expression, pro-female factors\nact cooperatively to fully repress Sox9, thereby allowing ovary development (Fig. 6 XX WT). In the −3\nbp mutant, the mutation alters the conformation of Enh13 by bringing RUNX1 and GATA4 proteins\ncloser together on chromatin. Under these conditions, NR5A1-, RUNX1- and GATA4-containing\ncomplexes acquire sufficient activating capacity to weakly induce Sox9 expression, reaching the\nthreshold required for its auto-activation, at which point SOX9 replaces RUNX1 on Enh13.\nThis\ninitiates ovotestis development, which is subsequently resolved into a testis (Fig. 6, XX −3bp). In the\n+1 bp mutant, the mutation creates an additional GATA4 binding site in close proximity to RUNX1.\nThis results in higher levels of SOX9 protein, which more efficiently reinforces Sox9 autoregulation,\nleading to a milder ovotestis phenotype that more closely resembles a testis and ultimately resolves into\na testis (Fig. 6, XX +1bp). Taken together, both mutations share a common feature: they bring RUNX1\nand GATA4 proteins closer together on chromatin: either directly, in the −3 bp mutant, by shortening\nthe distance between their binding motifs, or indirectly, by creating a de novo motif that allows GATA4\nto bind in very close proximity to RUNX1 on chromatin.\" This appears in the discussion page 15, lines\n1-17.\nDiscussion\n\"The two mutations described here show that Enh13 has repressive activity in the presence of bipotential\nfactor in the normal ovary, and the subtle alterations in its sequence can overcome this repressive state\".\nIn all experiments the transfection of bipotential factors NR5A1 and GATA4 always results in an\nincrease of luciferase activity, both in the context of WT and mutated Enh13. Therefore, I do not agree\nwith the conclusion that Enh13 has repressive activity. Rather Enh13 would have a weak activating\nactivity in the presence of bipotential factors that is further dampened by RUNX1 (RUNX1 being only\nmaintained in XX supporting cells). This activity would not be sufficient to produce the threshold level\nof SOX9 protein required for the self-amplification loop in the XX WT context. In the XX Enh13\nmutant contexts the presence of an additional GATA site and/or the disruption of the enhancer structure\nwould result in an increase of the transactivation activity of the enhancer even in the presence of\nRUNX1, allowing for sufficient SOX9 production and initiation of self-amplification loop.\nThis conclusion based on activation of Sox9 expression above a given threshold is the one that the\nauthors themselves state in the introduction and in the results:\nIntroduction p3: Our enhancer reporter assays suggest that the mutations we induced do not abolish the\nSOX9 BS but rather enable the bipotential factors NR5A1, RUNX1, and GATA4 to acquire sufficient\nactivating capacity to induce Sox9 expression above the minimal threshold present in female gonad.\nResults p5 : Altogether, these findings underscore the extraordinary sensitivity of the Sox9 regulatory\nnetwork to subtle enhancer perturbations, capable of breaching the activation threshold required to\ninitiate testicular development in an XX context.\nWe agree that the original paper submitted did not have a good evidence of repression apart from\nRUNX1. Thanks to all the reviewer's suggestions we have now added new Luciferase experiments\nhighlighting RUNX1 repression abilities and pro-female factor repression abilities of the enhancer (Fig\n4D-E).\nWe have amended this sentence in the discussion to: \"Our reporter assay experiments suggest that\nRUNX1, and later on, additional pro-female factors, are able to repress the enhancer, mediating Sox9\nrepression in the ovary that is mandatory for proper ovary development. The two mutations described\nhere show that subtle alterations in Enh13 sequence can overcome this repressive state, allowing low-\nlevel activation of Sox9 independently of the SRY protein.\" This appears in page 13 lines 18-22.\nOther comments: Yet, KO of individual pro-female factors including WT1 (-KTS) 18, FOXL2 24-26,\nRUNX1 27 and components of the WNT pathway 19-23 do not lead to XX male phenotype in mice. It\nis only when two pro-female factors or more are deleted together, that XX male phenotype start to\nappear at embryonic stages 27-30.\nI'm not sure that I understand what the authors mean. Could they elaborate?\nWe apologise that the original sentence was not clear. We have amended the sentence to be: \"While in\nthe male pathway, there are two strong players: SRY and SOX9, where loss of either would result in\nXY sex reversal 5, in the female pathway, loss of an individual pro-female factor including WT1 (-KTS)\n20, FOXL2 26, 27, 28, RUNX1 29 and components of the WNT pathway 21, 22, 23, 24, 25 do not lead to XX sex\nreversal in mice. It is only when two pro-female factors or more are deleted together, that the XX male\nphenotype start to appear at embryonic stages 29, 30, 31, 32. Therefore, it is striking that single nucleotide\ninsertion within a non-coding regulatory element, located more than 500 kb upstream of the Sox9 gene,\ncan lead to a robust XX female-to-male phenotype, not generated by ablation of any known pro-female\ngene.\"\nReviewer #2 (Remarks to the Author):\nThis manuscript is an exciting contribution to the sex determination literature showing that female to\nmale sex reversal can be caused by deletions in a critical enhancer of Sox9. Enh13 has previously been\nshown to be a principal regulator of Sox9 expression in XY gonadal supporting cells. Several of these\nauthors previously showed that mutations in enh13 disrupt male sex determination and testis\ndevelopment. Surprisingly, they now show that addition of 1 base pair (bp) or deletion of 3 bp in this\nenhancer show no effect on male sex determination or SOX9 expression in XY gonads but lead to\nelevated Sox9 expression in XX gonads and female to male sex reversal. This manuscript speaks to the\namazing plasticity of the sex determination system at the molecular level and provides an interesting\nexample of cell fate switching hinging on minor changes in an enhancer that may alter the relationship\nof transcription factors known to bind there. The authors create many new mouse strains to explore this\nphenotype and each is well characterized. They then turn to a molecular analysis of what could be going\non at the locus. Using reporter assays in HEK293 cells and EMSA assays, they demonstrate that SOX9\nbinding in not inhibited by the indels, but cooperative binding of NR5A1, GATA4, RUNX1 and SOX9\nmay be affected by changes in spacing. Of course, there is always more to learn but I find this a\nrelatively complete story and have only a few recommendations.\nWe thank you for the nice summary of our paper and results.\nIn the abstract the authors say, “Mechanistically, these mutations do not eliminate SOX9 binding”. I\nthink a prior sentence to explain that SOX9 binds it’s own promoter would be helpful.\nThank you for that. This is very correct as we did not even mention in the abstract that the mutations\nare in a SOX9 BS. We have removed this from the abstract.\nLater in the manuscript the authors say “These findings rule out increased SOX9 binding or\ntranscriptional activation as the underlying mechanism.” I found this section confusing and felt this\nconclusion was misleading. After all, the argument is that Sox9 expression is increased by the absence\nof repression.\nThis is very correct and we have amended this sentence. This original sentence appeared after we show\nvia PBM and EMSA that the two mutations do not create a more potent SOX9 BS. We amended the\nsentence accordingly: \"These findings rule out that the mutation have created a more potent SOX9\nbinding site that would lead to higher Sox9 transcriptional activation as the underlying mechanism.\"\nHave the authors tested whether RUNX1 can repress Sox9 in the presence of low levels of SOX9?\nIs there a binding competition between these two factors? An assay in HEK cells increasing the dose of\nSOX9 in the presence of RUNX1 would be ideal.\nYou have raised an excellent question, which we addressed\nexperimentally. Following your suggestion, we first tried to 6\nincrease the dosage of SOX9 in HEK cells and then explore the\neffect of RUNX1 addition. We were suspecting though that adding\nSOX9 alone will not induce the enhancer, and this was indeed the 4\ncase. We tried an increasing array of concentrations of SOX9\nalone and could not see any enhancer activation. Hence, it did not\n2\nmake sense to add RUNX1 as we will not be able to see repression.\nSee the graph below.\nNormally the concentration we were using of SOX9 were 40 ng\n0\nbut adding even higher concentration did not activate the\ng g g g g g\nn n n n n n\nenhancer. 0 2 0 4 0 6 0 8 0 0 0\nThen, we took a different strategy where we used the WT Enh13 and applied the two combinations that\nbest activate the enhancer: either just bipotential factors (NR5A1, GATA4 and WT1) or the bipotential\nnoisserpxE\nesareficuL\nevitaleR\nfactors along with SOX9. We then added RUNX1 into the mix to explore its repressive abilities. We\nsee that addition of RUNX1 dramatically repress Luciferase expression, much more so in the presence\nof SOX9. This figure now appears as the new Fig 4D-E, and we believe it suggests that RUNX1\nnormally repress the enhancer by competing with SOX9 on a partially overlapping site. This was added\nto the text in the result part and discussion.\nI would like to see the wild type ATAC landscape around Enh13 in one of the figures. Would ATAC-\nseq be a helpful approach in mutants to query how binding is changed?\nYou have raised another excellent suggestion that we were also thinking of. We have recently performed\nATAC-seq on sorted Sertoli and pre-granulosa cells from E11.5, E12.5, E13.5 and E15.5 gonads. Enh13\nis open and accessible in Sertoli cells from E11.5 whereas it is closed in pre-granulosa cells (PMID:\n40712010- Figure 6E, see below).\nSince in the mutants we have no ability to fluorescently sort Sertoli and pre-granulosa cells, we thought\nof using whole XY and XX gonads. Normally since WT XX gonads have no signal in Enh13, we were\nexpecting to see accessibility in the XX mutant gonads.\nTo that aim we performed a preliminary experiment (n=1) and took 100K cells from E12.5 WT XX\ngonad (separated from the m/s) and 100K cells from E12.5 Enh13 -3bp homozygous gonad.\nWe did whole gonad ATAC-seq for those two samples and did a low coverage sequencing to get an\nidea of how it looks.\nYou can see in the preliminary figure below the results.\nThe top track is E12.5 sorted pre-granulosa cells from our previous paper (PMID: 40712010).\nThe second track is E12.5 sorted Sertoli cells from our previous paper (PMID: 40712010).\nThe third track is E12.5 XX whole gonad\nThe last track is E12.5 XX Enh13SOX9*-3bp-/- whole gonad\nFirst, looking at the TESCO region, located upstream to the Sox9 gene you can see it is not accessible\nin pre-granulosa cells, highly accessible in Sertoli cells, not accessible in XX whole ovary and becoming\naccessible in the XX homozygous mutant ovaries.\nWe then looked at the Enh13 region- we see a similar picture of opening of the enhancer in the mutant\nXX gonads.\nThese results supported the hypothesis that due to the mutations, there is activation of the enhancer, it\nbecomes accessible, and mediates activation of Sox9. However, this is not highly surprising, and it is\nwhat we would expect if we saw that Sox9 is expressed. We would expect that the +1bp mutant will\nhave even more accessibility\nDespite these promising preliminary results, we eventually decided to not conduct the full experiment\nas we would need 3-5 gonads of XX WT, XY WT, XX 3 bp mutant and XX 1 bp mutants. Harvesting\nthe embryos, purchasing the ATAC-seq kit and sequencing all of that turned out to be a substantial\namount of money and time that we felt was not proportional for the input it would bring as this is quite\nan expected outcome. So, we ended up not doing it.\nIf you were to insist on that, we will do that, but we would hope that these preliminary results will be\nenough to convince you. Of course, since it is n=1, shallow sequencing and preliminary data, this was\nnot included in the revised manuscript.\nWe have added a paragraph to the discussion regarding the accessibility of the enhancer. Page 16, lines\n1-15.\nTESCO Enh13\nI find the annotation confusing. Could the mutants be represented as alleles using the usual annotation?\nSox9-Enh13Sox9*-3bp/Sox9*-3bp (denoting a homozygote) ; compared to Sox9-Enh13Sox9+1bp/+ (denoting a\nhet).\nThank you for this comment. There are currently no guidelines in place of how to refer to enhancers.\nAnd in addition, we have different mutations within the same enhancer. We considered your option but\ndecided to stay with our original as it would create very long titles that will not fit the boxes labelling\nthe mutants in the figures, so aesthetic wise, it will look less good. In our labelling like \"Enh13SOX9*-3bp-\n/-\", we mention the enhancer, followed by the specific mutation and then either +/+ for WT, +/- for\nheterozygous and -/- for homozygous mutant.\nPlease indicate the A/P axis of gonads in Fig.2. Is the ovarian region always anterior?\nThis was also raised by reviewer 1. Yes, when there is FOXL2 expression observed at only one pole,\nthe ovarian portion is always at anterior. We have now added A-P labelling to all BF pictures\nFig. 3B – what is the red sample in the XY section of the sample designation line?\nThe red sample that appears in the XY section is one XX Enh13SOX9*+1bp-/- gonad. This gonad was more\nmasculinised/ testicular than the others and in the PCA plot appeared closer to all the XY gonads. You\ncan see it here labelled with a red circle.\nIn the heatmap it clusters with the XY samples.\nReviewer #3 (Remarks to the Author)\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nWe thank you for your review and welcome you to the reviewing arena.\nReviewer #4 (Remarks to the Author):\nThe manuscript by Elisheva Abberbock and colleagues clearly demonstrates that engineered mutations\nin a key testicular enhancer of the Sox9 gene (named Enhancer13, or Enh13) lead to an absence of Sox9\nrepression in XX developing ovaries. This is followed by testis differentiation without SRY and XX\nsex reversal in adulthood. The authors elegantly and convincingly demonstrate that Enh13, which was\npreviously described as the key regulatory region for Sox9 upregulation in XY pre-supporting cells,\nalso plays a pivotal role in Sox9 repression in XX ovarian pre-supporting cells. This work confirms and\nprovides molecular explanations for XX sex reversal observed in other species, where abnormal SOX9\ngene upregulation has been shown to play a primary role in some human cases and in pig intersexuality.\nThe major conclusions of the manuscript are stated in the title and throughout the Results section. All\nof the authors' assertions are supported by clear, statistically validated results from morphological\nobservations of the external and internal genital tracts (including gonads), IHC, gonad transcriptomes\nby bulk RNA-seq from 45 mice with different genotypes, gene expression levels by qPCR, PBM,\nEMSA, transfection assays caried onto two different cell types (HEK293T and COS7) and\nbioinformatic analyses.\nThe main result is impressive: the authors clearly demonstrate that adding one nucleotide or suppressing\nthree in the SOX9 binding site of Enh13 changes the sex of the individual. Additionally, they prove that\nthe molecular mechanism involved is different in each XX sex reversal cases. In conclusion, all of the\nauthors' claims and conclusions are justified by their large number of high-quality, rigorous results.\nWe are grateful for your review and the good suggestions.\nIn addition to these general comments, I have two remarks and few minor corrections for the authors.\n1 – My first remark concerns the interpretation of the results proposed by the authors and illustrated in\nFigure 4E, as well as the binding of RUNX1 and SOX9 to their TFBS, which are very close and overlap\nby one or two bases in the WT allele, and by an even greater amount in the case of the -3 bp allele.\nDo the authors not consider that the binding of RUNX1 (expected in XX supporting cells) and SOX9\n(expected in XY cells) are mutually exclusive on these TFBS due to their proximity and the steric\nhindrance of both proteins? In other words, why do they not consider that there is competition between\nthe two transcription factors for binding to this region, with RUNX1 binding resulting in repression (or\nan absence of activation) and SOX9 binding resulting in activation? When the authors tested the binding\ncapacity of both factors individually using PBM or EMSA, they showed that RUNX1 bound\nequivalently to all three alleles (WT, +1 and -3 bp), but SOX9 had less affinity for the -3 bp allele.\nNevertheless, SOX9 is the dominant factor, since sex reversal occurs. One possible explanation is that,\nin vivo, the chromatin conformation of this region causes competition between the two TFs for binding\nto this critical region, and the 3 bp deletion prevents RUNX1 binding and thus promotes SOX9 binding,\ndespite its decreased affinity. For the +1 allele and the creation of a new GATA site in this small region,\nthe competition is now between not only RUNX1 and SOX9, but also GATA4. I leave it up to the authors\nto decide whether or not to take this comment into account in the discussion of the results.\nWe respectfully differ from Reviewer 4’s interpretation suggesting that the −3 bp mutant would lead to\na competition in favour of SOX9 as a consequence of reduced RUNX1 binding. Several observations\nargue against this view. First, our EMSA experiments do not reveal any decrease in RUNX1 binding\nwhen using the −3 bp probe. Second, at E11.5, SOX9 is not present in the nucleus of XX gonads,\nas a robust nuclear export mechanism actively excludes SOX9 from the nucleus at this stage.\nInstead, we favour an alternative model, which is developed in the revised version of the manuscript.\nIn the −3 bp mutant, the primary change consists of a 3 bp shortening between the RUNX1 and GATA\nbinding motifs. Remarkably, the spacing between these motifs is highly conserved across mammals\n(Fig. 5A), with only a single base-pair variation observed in rabbit. We propose that this shortening\nbrings RUNX and GATA proteins into closer proximity on chromatin and alters their relative\norientation compared with the wild-type configuration, as a consequence of an approximately one-third\nturn rotation of the DNA double helix. This configuration may facilitate physical interactions between\nRUNX and GATA proteins, thereby conferring activating potential. Consistent with this idea, RUNX\nand GATA proteins are known to interact in hematopoietic cells, and this interaction is evolutionarily\nconserved up to Drosophila.\nIn the +1 bp mutant, a conceptually similar mechanism is at play: the mutation generates a neo-GATA\nbinding site in very close proximity to the RUNX site, further favouring functional interactions between\nthese factors. Accordingly, the +1 bp mutant displays a stronger phenotype than the −3 bp mutant.\nConceptually, a +1 bp mutation that does not create a GATA binding site would be expected to have\nlittle or no effect. Importantly, the Enh13 mutants described here cannot be explained solely by changes\nin transcription factor binding affinity. Rather, our data support a model in which these mutations\nprimarily induce conformational changes of the enhancer itself, thereby reshaping the spatial\norganization and cooperative interactions of transcription factors bound to Enh13. Looking ahead, our\nstudy provides a framework for future conformational analyses of Enh13, calling for three-dimensional\nmodelling and structural biology approaches to determine how subtle sequence changes translate into\ndistinct enhancer architectures and regulatory outcomes.\nThis model is described in the discussion (pages 14-15) and presented in the new Figure 6.\n2 – My second remark concerns the deletion of the SRY BS in addition of the 3-bp deleted in the SOX9\nBS (first paragraph of page 5). The authors state that “removal of the SRY BS does not affect the female\nto male sex reversal of XX homozygous mice”. Why do the authors expect a role of this SRY BS in an\nXX context where there is no SRY?\nMoreover, the authors state that deleting SRY BS in addition to the 3-bp aggravate the phenotype of\nsex reversal (last paragraph of page 6). How could the authors explain this observation since there is no\nSRY?\nThis is a good question. We will first address the first part of it. The SOX9 protein is known to be able\nto bind (better) to a putative SOX9 BS but it can also bind to other SOX binding sites including the\nputative SRY BS. Indeed, this was also shown in our recent paper (PMID: 38499491) where we had to\ndelete both the SOX9 and SRY BS of Enh13 to get XY sex reversal. Although females do not have\nSRY, as they lack the Y chromosome, we expected that the SOX9 protein can mediate its function via\neither binding to the SOX9 BS or the SRY BS in Enh13, or both. To test this, we generated mice with\nthe same gain-of-function 3 bp deletion in the SOX9 BS but full elimination of the SRY BS. These XX\nhomozygous mice also presented XX male sex reversal, suggesting that the action in this case is not\nmediated via binding of SOX9 to the SRY BS located 200 bp downstream.\nSince this point was not fully clear to you, we have amended the wording in the text. It now reads as\nsuch: \"Since SOX9 protein can bind to both SRY and SOX9 transcription factor binding sites, we tested\nwhether the elevated Sox9 expression observed in the XX Enh13SOX9*-3bp-/- is due to SOX9 binding on\nboth the mutated SOX9 BS as well as the SRY BS. To that aim we generated mice carrying the same 3\nbp deletion in the SOX9 BS along with a deletion removing the entire SRY BS (Extended Data Fig.\n3A, termed Enh13SOX9*-3bp SRY*-12bp).\"\nWe also amended the wording in the same page, slightly below to be as such: \"Homozygous XX mice\npresented as males (Extended Data Fig. 3B-C), demonstrating that removal of the SRY BS, to which\nSOX9 can bind, does not affect the female-to-male sex reversal phenotype, suggesting that the mutated\nSOX9 BS is still functional and able to induce Sox9 expression\"\nAs to the second part of your question of why do the Enh13SOX9*-3bp SRY*-12bp embryos present a more\ntestis phenotype compared to the ovotestis of the Enh13SOX9*-3bp, This we do not have a good answer.\nThis was also raised by Reviewer 1.\nThe 3 bp deletion in the Enh13SOX9*-3bp is completely identical to that in the Enh13SOX9*-3bp SRY*-12bp strain.\nPerhaps by removing the SRY BS we also removed a repressive BS located nearby that elevated Sox9\nor maybe we altered cooperative binding of other factors in that region in a way that further enhanced\nSox9 expression. We cannot know for sure without going into much experimental detail and we feel\nthis is beyond the scope of the current study which is big and elaborative as is.\n3 – Page 7, last paragraph: the authors write “Analysis of bipotential genes, Nr5a1, Wt1,\nGata4…..revealed no major differences in expression between WT and HM mutants”. By contrast\nNr5a1 and Wt1 are found differentially expressed in Fig6B (cluster a & f, respectively) and also on\nExtended data Fig6A, at least between WT males and females with slight differences for HM mutants\n(male type for Nr5a1, female type for Wt1).\nThis is correct, we have amended the sentence as such: \"Analysis of bipotential genes, Gata4 and Zfpm2\n(Fog2), which are expressed in supporting cell precursors, and later maintained in both Sertoli and pre-\ngranulosa cells, revealed no major differences in expression between WT, heterozygous, and\nhomozygous gonads from the Enh13SOX9*-3bp, Enh13SOX9*+1bp and Enh13SOX9*-3bp SRY*-12bp mutants\n(Extended Data Fig. 6A). Nr5a1 expression in the XX homozygous mutants was more similar to the\nmale expression levels, whereas Wt1 expression in the homozygous mutants resembles more that of the\nWT XX gonads.\"\n4 – Page 8, first paragraph. The authors could add Rspo1 in the list of pre-granulosa markers and Aard\nin those of Sertoli markers, because expression of these two genes are presented on Ext Data Fig6\n(B & C, respectively).\nThank you, we have added these genes to the description.\n5 - Page 10, last paragraph, second sentence: “We also show….even in the absence of male-specific\nfactors”. The authors probably think to SRY. It will be better to write “…even in the absence of SRY”.\nWe amended this and replaced male-specific factors with SRY.\n6 – Figure 2C: The number of studied samples for XX+/+ is n=5 (as indicated in the parenthesis), but\nthere are 4 points for Sox9 and 6 points for Sox8 and Foxl2.\nYou are correct, we first had a typo in the legend and there were originally 6 samples of XX WT\nembryos. All these 6 points appear in the Sox8 and Foxl2 graphs but in the Sox9 graph we now have 5\npoints and not 6 as one of the points appeared with a value of 10 and was omitted by Prism GraphPad\nas being an outlier.\n7 – Legend of Figure 2C : “in” is missing between “involved” and “male”.\nThank you for spotting this typo. We amended this.\n8 – Figure 4A: the last base of the consensus sequence for the SOX9 binding site is missing on the\nFigure (should be A>G=T).\nThe SOX9 consensus BS is AACAATGG. The AACAATG are highly conserved, but the last\nnucleotide is a G/ A/ C in different mammals and hence the software does not place a letter there, but\nthe dashed lines do label all 8 nucleotides of the binding site.\n9 – Extended Data Fig.1B: “oviduct” term is present on the fourth animal (on 6 weeks gonads) and\nshould be suppressed.\nMany thanks for spotting this typo. We removed this.\n10 – Page 22; Methods; Paragraph “Design and preparation of sgRNAs mRNA”; lane 5 of this\nparagraph: please write “concentration” instead of “concertation”.\nThank you for spotting this typo. We amended this.\nResponse letter to reviewers Second Round\nWe are grateful to all four reviewers for their constructive comments and for recommending\nour manuscript for publication.\nThe reviewers’ comments are in black, and our replies are in blue.\nReviewer #1 (Remarks to the Author):\nThe authors have satisfactorily addressed my questions and have strengthened the manuscript\nthrough their revisions and the inclusion of additional results. Overall, this study provides a\ncompelling demonstration of how subtle alterations in regulatory DNA can lead to major\ndevelopmental effects, using Enh13, a regulatory element of Sox9, as an example. It delivers\nnovel and clinically meaningful insights into the genetic mechanisms underlying disorders of\nsex development (DSD). While a few minor typographical and editorial corrections can still be\naddressed, the manuscript is suitable for publication in Nature Communications.\nMany thanks\nFew comments\nThe figures currently use red and green together. Nature Communications recommends high-\ncontrast, accessible color palettes (e.g., green and magenta) for clarity and inclusivity. No\nchanges are required if the current colors are essential, but alternatives can be used if possible.\nThis is correct, and this comment was raised at the first stage of the revision. We hence\namended all real time graphs to be presented with magenta and green. The only thing that\nstayed red and green are the immunostaining, and for a good scientific reason. When using red\nand green, if an overlap in protein location exists, it created the very well spot colour of yellow.\nGreen and magenta combination are less clear when overlapping. Since an important scientific\nquestion is whether in the ovotestis, it is the same cells that express SOX9 and FOXL2 or\ndifferent cells, we feel it is important to use green and red to show that we have no yellow cells,\nmeaning although the gonad contain ovarian and testicular portions, these do not overlap at the\ncellular level.\nA typo appears in Reference 4.\nThank you for spotting this. We are unsure of how this happened, but we have now amended\nthe typo.\n\"In contrast, XX homozygous gonads from all three mutant strains exhibit a progressive shift\nfrom an ovarian transcriptional identity toward a testicular transcriptional identity\".\nAlthough the authors state that the sentence has been amended, the revised version is\nunchanged from the original and continues to imply a progressive temporal shift.\nWe apologise; it was our mistake. This is now amended to: \"In contrast, XX homozygous\ngonads from all three mutant strains exhibit a mixed ovarian and testicular transcriptome\nsignature at E12.5\"\nThe authors have now examined Sox9 expression at earlier developmental stages to determine\nwhen Sox9 first becomes abnormally upregulated in XX Enh13 mutant gonads. To allow\naccurate comparison between samples, it would be helpful if the authors could clarify the\ndevelopmental staging used for these analyses, in particular by indicating the number of tail\nsomites, if available.\nThis was now added to Extended Data Figure 12. All gonads were 17-19 tail somites.\nReviewer #2 (Remarks to the Author):\nThe authors have carefully responded to the reviewers’ comments, performed new\nexperiments, and improved many aspects of the manuscript.\nI think it would be helpful if the model in Figure 6 showed that Sox9 is initially expressed at\nlow levels in the XX gonad, where SOX9 is cytoplasmic, as the authors explain on line 6 and\n26 (Gasco). The question becomes whether it is upregulated or downregulated as gonads\ndevelop. This is a subtle but important difference in the way we think about it.\nAs we know, first SRY and later SOX9 upregulate Sox9.\nThank you for this suggestion. We have now added to the scheme in Figure 6 the presence of\nlow levels of SOX9 in both XY and XX early gonads\nIn this manuscript, the authors convince me that RUNX1 is an important repressor of SOX9\n(although I am puzzled as to why in its absence, XX Runx1-/- animals do not develop testes –\npresumably SOX9 levels are not high enough without up-regulation by SRY). I admire the in\nvitro TF competition assays and model as an explanation for why these 1 or 3 bp mutations\ncause sex reversal.\nThis is indeed puzzling. We hence delved into the Nicol et al., 2019 paper who first described\nthe role of RUNX1 in the gonads. We also had personal communications with Humphrey Yao\nand Barbara Nicol about that. Full KO of RUNX1 is embryonic lethal, and hence Nicol et al.,\nused cKO using the Sf1-cre line. Data from the lab of Marie-Christine Chaboissier shows that\nalthough Nr5a1 is expressed at E9.0 in the gonads, the Sf1-cre line from Keith Parker only\nstarts to work from ~E11.0-E11.5 in the gonads. We believe that this stage is too late, and\nknocking out RUNX1 at that stage misses the window to prevent Sox9 repression and lead to\nXX males. We are hoping that Barbara and Humphrey will use the Wt1-cre, which is earlier to\ndrive gonadal expression, in order to do cKO of RUNX1. We anticipate this may lead to XX\nsex reversal and support our model/ hypothesis.\nThis will be as part of a follow up work, but we fully agree that this role of RUNX1 is striking\nand require future investigation.\nThis manuscript moves the field in the important direction of understanding the interaction of\nTFs on chromatin during the process of cell fate determination and sex determination. Although\nthere is a lot more to do, that’s for another manuscript. The authors have made significant\nprogress in this work.\nThank you\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nThank you\nReviewer #4 (Remarks to the Author):\nThe revised version proposed by the authors is even more convincing. I fully agree with both\nthe new model and the interpretation of the results set out in this version and I strongly\nrecommend the publication of this Ms.\nThank you\nOne final detail regarding the sentence on lines 8–11 of page 13: it is not surprising, in my\nopinion, that a gain of function of Sox9 induces a very robust XX female to male sex-reversal\n(Vidal et al., 2001). What is striking is that such subtle modifications of a TFBS can induce\nsuch a change in the transcriptional expression of the Sox9 gene. In my opinion, XX sex\nreversal through loss of function of pro-ovarian genes is a completely different story.\nWe fully agree with you on this statement, but what we are comparing in these sentences is the\nfact that KO of each pro-female factor that we know of cannot induce XX sex reversal and yet\na single insertion in a non-coding genome can do that.\nAnd a last typo on line 15 page 10: suppress one “on the”.\nThank you for that. This was amended on Page 10, line 15. We removed the duplicated \"on\nthe\""}]

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