Racca Joseph D, Chen Yen-Shan, Maloy James D, Wickramasinghe Nalinda, Phillips Nelson B, Weiss Michael A
From the Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106.
From the Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106
J Biol Chem. 2014 Nov 21;289(47):32410-29. doi: 10.1074/jbc.M114.597526. Epub 2014 Sep 24.
Human testis determination is initiated by SRY, a Y-encoded architectural transcription factor. Mutations in SRY cause 46 XY gonadal dysgenesis with female somatic phenotype (Swyer syndrome) and confer a high risk of malignancy (gonadoblastoma). Such mutations cluster in the SRY high mobility group (HMG) box, a conserved motif of specific DNA binding and bending. To explore structure-function relationships, we constructed all possible substitutions at a site of clinical mutation (W70L). Our studies thus focused on a core aromatic residue (position 15 of the consensus HMG box) that is invariant among SRY-related HMG box transcription factors (the SOX family) and conserved as aromatic (Phe or Tyr) among other sequence-specific boxes. In a yeast one-hybrid system sensitive to specific SRY-DNA binding, the variant domains exhibited reduced (Phe and Tyr) or absent activity (the remaining 17 substitutions). Representative nonpolar variants with partial or absent activity (Tyr, Phe, Leu, and Ala in order of decreasing side-chain volume) were chosen for study in vitro and in mammalian cell culture. The clinical mutation (Leu) was found to markedly impair multiple biochemical and cellular activities as respectively probed through the following: (i) in vitro assays of specific DNA binding and protein stability, and (ii) cell culture-based assays of proteosomal degradation, nuclear import, enhancer DNA occupancy, and SRY-dependent transcriptional activation. Surprisingly, however, DNA bending is robust to this or the related Ala substitution that profoundly impairs box stability. Together, our findings demonstrate that the folding, trafficking, and gene-regulatory function of SRY requires an invariant aromatic "buttress" beneath its specific DNA-bending surface.
人类睾丸决定由SRY启动，SRY是一种Y染色体编码的结构转录因子。SRY突变会导致46,XY性腺发育不全并伴有女性体细胞表型（Swyer综合征），并具有较高的恶性肿瘤（性腺母细胞瘤）风险。此类突变聚集在SRY高迁移率族（HMG）框中，这是一个特定DNA结合和弯曲的保守基序。为了探索结构 - 功能关系，我们在一个临床突变位点（W70L）构建了所有可能的替换。因此，我们的研究聚焦于一个核心芳香族残基（保守HMG框共识序列的第15位），该残基在SRY相关的HMG框转录因子（SOX家族）中是不变的，并且在其他序列特异性框中保守为芳香族（苯丙氨酸或酪氨酸）。在对特定SRY - DNA结合敏感的酵母单杂交系统中，变体结构域表现出活性降低（苯丙氨酸和酪氨酸）或无活性（其余17种替换）。选择具有部分或无活性的代表性非极性变体（酪氨酸、苯丙氨酸、亮氨酸和丙氨酸，按侧链体积递减顺序）进行体外和哺乳动物细胞培养研究。通过以下方式分别检测发现，临床突变（亮氨酸）明显损害多种生化和细胞活性：（i）特异性DNA结合和蛋白质稳定性的体外测定，以及（ii）基于细胞培养的蛋白酶体降解、核输入、增强子DNA占据和SRY依赖性转录激活测定。然而，令人惊讶的是，DNA弯曲对这种或严重损害框稳定性的相关丙氨酸替换具有抗性。总之，我们的研究结果表明，SRY的折叠、运输和基因调控功能在其特定DNA弯曲表面下方需要一个不变的芳香族“支撑”。
