Abstract
SRY is the master regulator of male sex determination in eutherian mammals. In mice, Sry expression is transcriptionally and epigenetically controlled in a developmental stage-specific manner. The Sry promoter undergoes demethylation in embryonic gonadal somatic cells at the sex-determining period. However, its molecular mechanism and in vivo significance remain unclear. Here, we report that the Sry promoter is actively demethylated during gonadal development, and TET2 plays a fundamental role in Sry demethylation. Tet2-deficient mice showed absence of 5-hydroxymethylcytosine in the Sry promoter. Furthermore, Tet2 deficiency diminished Sry expression, indicating that TET2-mediated DNA demethylation regulates Sry expression positively. We previously showed that the deficiency of the H3K9 demethylase Jmjd1a compromises Sry expression and induces male-to-female sex reversal. Tet2 deficiency enhanced the sex reversal phenotype of Jmjd1a-deficient mice. Thus, TET2-mediated active DNA demethylation and JMJD1A-mediated H3K9 demethylation contribute synergistically to sex determination.
Highlights
Expression of developmental genes is tuned through crosstalk between transcription factors and epigenetic regulation
We discovered that 5hmC was highly enriched in XY gonadal somatic cells at the sex-determining period and that the 5hmC level was increased in Sry promoter concomitantly with Sry expression in these cells
We identified TET2 as the enzyme responsible for active DNA demethylation in Sry promoter
Summary
Expression of developmental genes is tuned through crosstalk between transcription factors and epigenetic regulation. To elucidate whether active DNA demethylation occurs during embryonic gonadal development, we performed double immunostaining analyses on XY embryonic gonad sections at the sex-determining period (E11.5) with antibodies against 5hmC and NR5A1 ( known as AD4BP/SF-1), which is transcription factor expressed in gonadal somatic cells but not in germ cells and mesonephric cells. Quantitative analysis indicated that the average intensity of 5hmC was about two-fold higher in NR5A1-positive gonadal somatic cells compared to that in mesonephric cells (Fig. 1a, right) These data suggest that active DNA demethylation might arise in developing gonads around the sex-determining period. Quantification analysis showed that 5hmC content was about 1.5-fold higher in gonadal somatic cells than that in mesonephric cells (Fig. 1b, right) These findings support the fact that active demethylation occurs preferentially in the gonadal somatic cell population at the sex-determining period
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