Gadd45g and mammalian testis determination

Mammalian sex is determined by the presence or absence of Sry. This gene, located on the Y chromosome, promotes the development of a testis from the biopotential gonad by initiating a transcriptional programme that results in Sertoli cell formation. SRY is required to reach a specific threshold level during a critical time-period in order to induce the testicular differentiation pathway. We have previously shown that XY mouse embryos lacking Growth arrest and DNA damage inducible 45 gamma (Gadd45g) exhibit gonadal sex reversal due to a delay in Sry expression. Efforts to study the role of GADD45γ in sex determination have been stymied by the lack of a working antibody. As an alternative, I have produced a BAC transgene produces a similar expression profile to endogenous Gadd45g. This BAC was also shown to rescue the sex reversal found in B6-YPOS gonads, though further research is required to understand the molecular mechanisms through which this occurs. As a result, this BAC was chosen for modification using BAC recombineering to contain the sequence for a poly-His/FLAG epitope tag. Though this edited BAC was generated successfully, no transgenic lines have yet been produced. Members of the Gadd45 family have previously been associated with active DNA demethylation. Assuch, I have conducted whole genome bisulphite sequencing on DNA from XY wild-type, XX wild-type and XY Gadd45g-/- embryos at the 18 tail somite stage, where Sry expression is at its peak. Gadd45g and Sry are only expressed in the somatic cells of the gonads, which have been purified through fluorescence-activated flow cytometry using the transgenic line Sf1-EGFP. Due to the low number of cells that are purified from the embryonic gonad at this stage, sequencing libraries were generated using post-bisulphite adaptor tagging (PBAT). Analysis of the acquired sequences revealed that there are no significant differences between the methylomes of XY wild-type, XX wild-type and XY Gadd45g-/- mutant gonadal somatic cells. It therefore appears that GADD45γ does not have a role in establishing the methylome at this particular stage of sex determination.