Hirata Yurie, Mizushima Shusei, Mitsukawa Shoichiro, Kon Masafumi, Kuroki Yoko, Jogahara Takamichi, Shinohara Nobuo, Kuroiwa Asato
Department of Renal and Genitourinary Surgery, Graduate School of Medicine, Hokkaido University, Sapporo, Japan.
Division of Reproductive and Developmental Biology, Department of Biological Sciences, Faculty of Science, Hokkaido University, Sapporo, Japan.
Cytogenet Genome Res. 2023;163(5-6):307-316. doi: 10.1159/000536408. Epub 2024 Jan 20.
Testis differentiation is initiated by the SRY gene on the Y chromosome in mammalian species. However, the Amami spiny rat, Tokudaia osimensis, lacks both the Y chromosome and the Sry gene and acquired a unique Sox9 regulatory mechanism via a male-specific duplication upstream of Sox9, without Sry. In general mammalian species, the SRY protein binds to a testis-specific enhancer to promote SOX9 gene expression. Several enhancers located upstream of Sox9/SOX9 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.
Sequences of T. osimensis homologues of three Sox9 enhancers that were previously reported in mice, Enh8, Enh14, and Enh13, were determined. We performed in vitro assays to confirm enhancer activity involved in Sox9 regulation in T. osimensis.
T. osimensis Enh13 showed enhancer activity when co-transfected with NR5A1 and SOX9. Mouse Enh13 was activated by NR5A1 and SRY; however, T. osimensis 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 T. osimensis, we performed reporter gene assays using vectors in which partial sequences of T. osimensis Enh13 were replaced with mouse sequences. For T. osimensis 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.
We showed that there are unknown sequences responsible for the interaction between NR5A1 and SRY and mEnh13 based on comparative analyses of Sry-dependent and Sry-independent species. Our comparative analyses revealed new molecular mechanisms underlying mammalian sex determination.
在哺乳动物物种中，睾丸分化由Y染色体上的SRY基因启动。然而，奄美刺鼠（Tokudaia osimensis）既没有Y染色体也没有Sry基因，并且在没有Sry的情况下，通过Sox9上游的雄性特异性重复获得了独特的Sox9调控机制。在一般的哺乳动物物种中，SRY蛋白与睾丸特异性增强子结合以促进SOX9基因表达。在小鼠和人类中，已经报道了位于Sox9/SOX9上游的几个增强子。特别是，SRY与高度保守的增强子Enh13的结合被认为是哺乳动物睾丸分化和性别决定的共同机制。
确定了先前在小鼠中报道的三个Sox9增强子（Enh8、Enh14和Enh13）的奄美刺鼠同源物序列。我们进行了体外试验，以确认参与奄美刺鼠Sox9调控的增强子活性。
奄美刺鼠Enh13与NR5A1和SOX9共转染时显示出增强子活性。小鼠Enh13被NR5A1和SRY激活；然而，奄美刺鼠Enh13对SRY没有反应，尽管SRY和NR5A1的结合位点是保守的。为了鉴定小鼠中存在但奄美刺鼠中不存在的关键序列，我们使用了将奄美刺鼠Enh13的部分序列替换为小鼠序列的载体进行报告基因试验。对于后半部分（约430 bp）被相应小鼠序列替换的奄美刺鼠Enh13，其对NR5A1和SRY的反应活性得以恢复。此外，报告基因试验表明，小鼠Enh13序列后半部分的多个区域对于对NR5A1和SRY的反应是必需的。后49 bp尤为重要，包含三个转录因子POU2F1、HOXA3和GATA1的四个结合位点。
基于对依赖Sry和不依赖Sry物种的比较分析，我们表明存在负责NR5A1与SRY和mEnh13之间相互作用的未知序列。我们的比较分析揭示了哺乳动物性别决定的新分子机制。
