Identification of candidate transcription factors that regulate Sox9 expression through the testis-specific enhancer in the Amami spiny rat (Tokudaia osimensis), an XO/XO mammal.

Abstract

Testicular differentiation of undifferentiated gonads is generally triggered by the sex-determining region of chromosome Y (SRY/Sry) gene on the Y chromosome in most mammals. SRY and nuclear receptor subfamily 5, group A, member 1 (NR5A1) proteins regulate transcription of the autosomal SRY-box9 (SOX9/Sox9) gene in XY embryonic gonads, inducing testicular differentiation. One exception, the Amami spiny rat (Tokudaia osimensis), lacks the Y chromosome and Sry. We previously reported that this species has a male-specific duplication upstream of Sox9, and an enhancer (tosEnh14) in the duplication regulates Sox9 transcription without Sry. However, tosEnh14 is not activated by NR5A1 alone, suggesting another transcription factor(s) that binds to tosEnh14 is necessary. Although this species is endangered and heavily protected, it presents many challenges for genetic studies. Therefore, we explored novel transcription factors that regulate Sox9 via tosEnh14 using mouse samples. To detect proteins that bind to tosEnh14 DNA, Southwestern blotting analysis was performed using mouse embryonic gonad extracts. Bands of a similar molecular weight but prominent in males and faint in females were subjected to mass spectrometry analysis. Peptides derived from 174 genes were identified, and eight genes associated with gene ontology terms such as "DNA binding" and "regulation of transcription by RNA polymerase II" were selected. For further screening, the expression level of each gene was examined using single-cell RNA-sequencing data of mouse progenitor cells, which differentiate into Sertoli cells in mouse embryonic testes and granulosa cells in embryonic ovaries. Finally, five genes (Elf2, Etv6, Fiz1, Gtf2f1, and Trim27) whose expression was confirmed in seminiferous tubules of E13.5 XY embryos by whole-mount in situ hybridization were selected as candidates. Binding sites of ELF2 and ETV6 are present in tosEnh14 DNA sequence. Our study contributes to understanding of the molecular mechanisms underlying sex determination in mammals.