Identification of a New Enhancer That Promotes <i>Sox9</i> Expression by a Comparative Analysis of Mouse and <i>Sry</i>-Deficient Amami Spiny Rat

Abstract

<b><i>Introduction:</i></b> Testis differentiation is initiated by the <i>SRY</i> gene on the Y chromosome in mammalian species. However, the Amami spiny rat, <i>Tokudaia osimensis</i>, lacks both the Y chromosome and the <i>Sry</i> gene and acquired a unique <i>Sox9</i> regulatory mechanism via a male-specific duplication upstream of <i>Sox9</i>, without <i>Sry</i>. In general mammalian species, the SRY protein binds to a testis-specific enhancer to promote <i>SOX9</i> gene expression. Several enhancers located upstream of <i>Sox9</i>/<i>SOX9</i> have been reported in mice and humans. In particular, the binding of SRY to the highly conserved enhancer Enh13 is thought to be a common mechanism underlying testis differentiation and sex determination in mammals. <b><i>Methods:</i></b> Sequences of <i>T. osimensis</i> homologues of three <i>Sox9</i> enhancers that were previously reported in mice, Enh8, Enh14, and Enh13, were determined. We performed in vitro assays to confirm enhancer activity involved in <i>Sox9</i> regulation in <i>T. osimensis</i>. <b><i>Results:</i></b> <i>T. osimensis</i> Enh13 showed enhancer activity when co-transfected with NR5A1 and SOX9. Mouse Enh13 was activated by NR5A1 and SRY; however, <i>T. osimensis</i> Enh13 did not respond to SRY, even though the binding sites of SRY and NR5A1 were conserved. To identify the key sequence that is present in mouse but absent from <i>T. osimensis</i>, we performed reporter gene assays using vectors in which partial sequences of <i>T. osimensis</i> Enh13 were replaced with mouse sequences. For <i>T. osimensis</i> Enh13 in which the second half (approximately 430 bp) was replaced with the corresponding mouse sequence, activity in response to NR5A1 and SRY was recovered. Further, reporter assays revealed that multiple regions in the second half of the mouse Enh13 sequence are required for the response to NR5A1 and SRY. The latter 49 bp was particularly important and contained four binding sites for three transcription factors, POU2F1, HOXA3, and GATA1. <b><i>Conclusion:</i></b> We showed that there are unknown sequences responsible for the interaction between NR5A1 and SRY and mEnh13 based on comparative analyses of <i>Sry</i>-dependent and <i>Sry</i>-independent species. Our comparative analyses revealed new molecular mechanisms underlying mammalian sex determination.