Abstract
SRY (sex-determining region Y)-box 9 (SOX9) is a transcription factor regulating both chondrogenesis and sex determination. Among vertebrates, SOX9’s functions in chondrogenesis are well conserved, while they vary in sex determination. To investigate the conservation of SOX9’s regulatory functions in chondrogenesis and gonad development among species, we performed chromatin immunoprecipitation sequencing (ChIP-seq) using developing limb buds and male gonads from embryos of two vertebrates, mouse and chicken. In both mouse and chicken, SOX9 bound to intronic and distal regions of genes more frequently in limb buds than in male gonads, while SOX9 bound to the proximal upstream regions of genes more frequently in male gonads than in limb buds. In both species, SOX palindromic repeats were identified more frequently in SOX9 binding regions in limb bud genes compared with those in male gonad genes. The conservation of SOX9 binding regions was significantly higher in limb bud genes. In addition, we combined RNA expression analysis (RNA sequencing) with the ChIP-seq results at the same stage in developing chondrocytes and Sertoli cells and determined SOX9 target genes in these cells of the two species and disclosed that SOX9 targets showed high similarity of targets in chondrocytes, but not in Sertoli cells.
Highlights
Www.nature.com/scientificreports lethal at the embryonic stage[7]
To identify the genomic locations of SOX9 binding regions in developing chondrocytes and Sertoli cells, we applied ChIP followed by deep sequencing (ChIP-seq) to developing limb buds and male gonads collected from embryonic day (E) 13 mice (Fig. 1A)
We identified 6,728 significantly enriched ChIP-seq peak regions bound by SOX9 in E13 limb buds and 1,308 regions in E13 male gonads, among which 755 regions overlapped between the two tissues (Fig. 1D, Supplementary Tables S1 and S2)
Summary
Www.nature.com/scientificreports lethal at the embryonic stage[7]. Sox[9] inactivation in limb buds using the Cre/loxP recombination system revealed that SOX9 has an essential function on mesenchymal condensation and subsequent cartilage formation[8]. Ectopic expression of Sox[9] in XX mice gonads induced testis formation, while XY gonad-specific inactivation of Sox[9] resulted in the absence of testes[9,10] These data clearly indicated that SOX9 is a critical regulator of chondrogenesis and sex determination. Studies using chromatin immunoprecipitation sequencing (ChIP-seq) have precisely determined the functions of SOX9 in chondrogenesis, mainly in mice[23,24] Another ChIP-seq study using male gonads of mouse and cattle showed that SOX9 could bind to and regulate a similar set of genes in these two mammals[25]. The present study provided new insights into cell type-specific binding preferences of SOX9 and their conservation in chondrocytes and Sertoli cells between mouse and chicken
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