Combining ATAC-seq and RNA-seq reveals key genes for gonadal abnormalities in one-month-old XX-DSD pigs

Disorders of Sex Development (DSD) are caused by congenital abnormalities in the chromosomes, and subsequent development of gonads or sexual anatomy. XX-DSD pigs exhibit a series of adverse symptoms such as sterility, genital infections, and decline in meat quality, leading to significant economic losses in the breeding industry. However, the understanding of the etiology and pathogenesis of XX-DSD in pigs remains limited. To investigate the molecular mechanisms underlying abnormal gonadal development in XX-DSD pigs, we analyzed the gonads of 1-month-old XX-DSD pigs, normal females, and normal males using RNA-seq and ATAC-seq techniques.

From RNA-seq, we identified potential genes involved in gonadal development in XX-DSD pigs, including SOX9, HSD3B1, CYP19A1, CCNB2, CYP11A1, DMRT1, and MGP. Following this, we analyzed ATAC-seq data and identified 14,820 differential accessible chromatin peaks. Then, by integrating the ATAC-seq and RNA-seq analysis results, we identified several candidate genes (SOX9, COL1A1, COL1A2, FDX1, COL6A1, HSD3B1, FSHR, and CYP17A1) that might be associated with sex development. Through PPI (Protein-Protein Interaction Networks) analysis, we found that SOX9 gene was the top hub gene. Furthermore, we confirmed the effect of the open chromatin region on SOX9 gene expression by a dual-luciferase reporter assay, thus further validating the critical role of this open region in regulating SOX9 expression.

This study elucidates the critical regulatory role of specific open chromatin structures in the SOX9 gene promoter region (8647563-8648475) in gonadal development of XX-DSD pigs. Additionally, we identify that genes such as SOX9, HSD3B1, and CYP19A1 act in concert to participate in gonadal development. These findings provide molecular evidence for the dynamic chromatin regulatory network underlying gonadal dysgenesis in XX-DSD and lay the foundation for subsequent mechanistic studies.