Departments of Biochemistry, Biomedical Engineering, and Medicine, Case Western Reserve University, Cleveland, OH 44106.
Proc Natl Acad Sci U S A. 2013 Sep 17;110(38):E3567-76. doi: 10.1073/pnas.1300828110. Epub 2013 Sep 3.
Human testis determination is initiated by SRY (sex determining region on Y chromosome). Mutations in SRY cause gonadal dysgenesis with female somatic phenotype. Two subtle variants (V60L and I90M in the high-mobility group box) define inherited alleles shared by an XY sterile daughter and fertile father. Whereas specific DNA binding and bending are unaffected in a rat embryonic pre-Sertoli cell line, the variants exhibited selective defects in nucleocytoplasmic shuttling due to impaired nuclear import (V60L; mediated by Exportin-4) or export (I90M; mediated by chromosome region maintenance 1). Decreased shuttling limits nuclear accumulation of phosphorylated (activated) SRY, in turn reducing occupancy of DNA sites regulating Sertoli-cell differentiation [the testis-specific SRY-box 9 (Sox9) enhancer]. Despite distinct patterns of biochemical and cell-biological perturbations, V60L and I90M each attenuated Sox9 expression in transient transfection assays by twofold. Such attenuation was also observed in studies of V60A, a clinical variant associated with ovotestes and hence ambiguity between divergent cell fates. This shared twofold threshold is reminiscent of autosomal syndromes of transcription-factor haploinsufficiency, including XY sex reversal associated with mutations in SOX9. Our results demonstrate that nucleocytoplasmic shuttling of SRY is necessary for robust initiation of testicular development. Although also characteristic of ungulate orthologs, such shuttling is not conserved among rodents wherein impaired nuclear export of the high-mobility group box and import-dependent phosphorylation are compensated by a microsatellite-associated transcriptional activation domain. Human sex reversal due to subtle defects in the nucleocytoplasmic shuttling of SRY suggests that its transcriptional activity lies near the edge of developmental ambiguity.
人类睾丸的决定是由 SRY（Y 染色体上的性别决定区）启动的。SRY 突变导致性腺发育不良，表现为女性体貌。两个微妙的变体（高迁移率族蛋白盒中的 V60L 和 I90M）定义了 XY 不育女儿和可育父亲所共有的遗传等位基因。虽然在大鼠胚胎前支持细胞系中，特定的 DNA 结合和弯曲不受影响，但由于核输入受损（V60L；由 Exportin-4 介导）或输出（I90M；由染色体区域维持 1 介导），变体表现出选择性的核质穿梭缺陷。穿梭减少限制了磷酸化（激活）SRY 的核积累，进而减少了调节支持细胞分化的 DNA 位点的占据[睾丸特异性 SRY 盒 9（Sox9）增强子]。尽管存在明显的生化和细胞生物学扰动模式，但 V60L 和 I90M 都在瞬时转染试验中使 Sox9 表达降低了两倍。在 V60A 的研究中也观察到了这种衰减，V60A 是一种与卵睾有关的临床变体，因此在不同细胞命运之间存在模糊性。这种两倍的衰减阈值让人想起转录因子单倍不足的常染色体综合征，包括与 SOX9 突变相关的 XY 性别反转。我们的结果表明，SRY 的核质穿梭对于睾丸发育的有力启动是必要的。尽管这种穿梭也存在于有蹄类动物的同源物中，但在啮齿动物中并不保守，啮齿动物中高迁移率族蛋白盒的核输出受损和依赖于磷酸化的输入通过与微卫星相关的转录激活结构域得到补偿。由于 SRY 的核质穿梭细微缺陷导致的人类性别反转表明其转录活性接近发育模糊性的边缘。
