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

IF 1.7 4区 生物学 Q4 CELL BIOLOGY
Cytogenetic and Genome Research Pub Date : 2023-01-01 Epub Date: 2024-01-20 DOI:10.1159/000536408
Yurie Hirata, Shusei Mizushima, Shoichiro Mitsukawa, Masafumi Kon, Yoko Kuroki, Takamichi Jogahara, Nobuo Shinohara, Asato Kuroiwa
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引用次数: 0

Abstract

Introduction: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

通过对小鼠和 Sry 缺陷奄美刺鼠的比较分析,确定了促进 Sox9 表达的新增强子。
引言哺乳动物的睾丸分化是由Y染色体上的SRY基因启动的。然而,奄美刺鼠(Tokudaia osimensis)缺乏Y染色体和Sry基因,并通过Sox9上游的雄性特异性复制获得了独特的Sox9调控机制,而没有Sry基因。在一般哺乳动物物种中,SRY 蛋白与睾丸特异性增强子结合,促进 SOX9 基因的表达。据报道,在小鼠和人类中,有多个增强子位于 Sox9/SOX9 的上游。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:方法:我们测定了T. osimensis同源的三个Sox9增强子的序列,这三个增强子以前曾在小鼠中报道过,即Enh8、Enh14和Enh13。我们进行了体外实验,以确认在 T. osimensis 中参与 Sox9 调节的增强子的活性:结果:当与 NR5A1 和 SOX9 共同转染时,T. osimensis Enh13 显示出增强子活性。小鼠Enh13被NR5A1和SRY激活;然而,尽管SRY和NR5A1的结合位点是保守的,但T. osimensis Enh13对SRY没有反应。为了确定在小鼠中存在而在T. osimensis中不存在的关键序列,我们使用载体进行了报告基因检测,在载体中,T. osimensis Enh13的部分序列被小鼠序列取代。对于后半部分(约 430 bp)被相应的小鼠序列替换的 T. osimensis Enh13,对 NR5A1 和 SRY 的反应活性得以恢复。进一步的报告测定显示,小鼠 Enh13 序列后半部分的多个区域对 NR5A1 和 SRY 的反应是必需的。后 49 bp 尤为重要,它包含三个转录因子(POU2F1、HOXA3 和 GATA1)的四个结合位点:结论:根据对依赖 Sry 和不依赖 Sry 的物种的比较分析,我们发现 NR5A1 与 SRY 和 mEnh13 之间的相互作用存在未知序列。我们的比较分析揭示了哺乳动物性别决定的新分子机制。
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来源期刊
Cytogenetic and Genome Research
Cytogenetic and Genome Research 生物-细胞生物学
CiteScore
3.10
自引率
5.90%
发文量
25
审稿时长
1 months
期刊介绍: During the last decades, ''Cytogenetic and Genome Research'' has been the leading forum for original reports and reviews in human and animal cytogenetics, including molecular, clinical and comparative cytogenetics. In recent years, most of its papers have centered on genome research, including gene cloning and sequencing, gene mapping, gene regulation and expression, cancer genetics, comparative genetics, gene linkage and related areas. The journal also publishes key papers on chromosome aberrations in somatic, meiotic and malignant cells. Its scope has expanded to include studies on invertebrate and plant cytogenetics and genomics. Also featured are the vast majority of the reports of the International Workshops on Human Chromosome Mapping, the reports of international human and animal chromosome nomenclature committees, and proceedings of the American and European cytogenetic conferences and other events. In addition to regular issues, the journal has been publishing since 2002 a series of topical issues on a broad variety of themes from cytogenetic and genome research.
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