The conserved Gsx homeodomain (HD) transcription factors specify neural cell fates in animals from flies to mammals. Like many HD proteins, Gsx factors bind A/T-rich DNA sequences prompting the following question: How do HD factors that bind similar DNA sequences in vitro regulate specific target genes in vivo? Prior studies revealed that Gsx factors bind DNA both as a monomer on individual A/T-rich sites and as a cooperative homodimer to two sites spaced precisely 7 bp apart. However, the mechanistic basis for Gsx–DNA binding and cooperativity is poorly understood. Here, we used biochemical, biophysical, structural and modeling approaches to (i) show that Gsx factors are monomers in solution and require DNA for cooperative complex formation, (ii) define the affinity and thermodynamic binding parameters of Gsx2/DNA interactions, (iii) solve a high-resolution monomer/DNA structure that reveals that Gsx2 induces a 20° bend in DNA, (iv) identify a Gsx2 protein–protein interface required for cooperative DNA binding and (v) determine that flexible spacer DNA sequences enhance Gsx2 cooperativity on dimer sites. Altogether, our results provide a mechanistic basis for understanding the protein and DNA structural determinants that underlie cooperative DNA binding by Gsx factors.

中文翻译：

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Gsx2-DNA 协同结合需要 DNA 弯曲和新型 Gsx2 同源结构域界面


保守的 Gsx 同源域 (HD) 转录因子决定从果蝇到哺乳动物等动物的神经细胞命运。与许多 HD 蛋白一样，Gsx 因子结合富含 A/T 的 DNA 序列，引发了以下问题：在体外结合相似 DNA 序列的 HD 因子如何在体内调节特定靶基因？先前的研究表明，Gsx 因子既作为单体结合在富含 A/T 的位点上，又作为协同同二聚体结合到两个精确间隔 7 bp 的位点上。然而，人们对 Gsx-DNA 结合和协同作用的机制基础知之甚少。在这里，我们使用生物化学、生物物理、结构和建模方法来 (i) 表明 Gsx 因子是溶液中的单体，并且需要 DNA 来协同形成复合物，(ii) 定义 Gsx2/DNA 相互作用的亲和力和热力学结合参数，(iii) ) 解析高分辨率单体/DNA 结构，揭示 Gsx2 诱导 DNA 20° 弯曲，(iv) 识别协同 DNA 结合所需的 Gsx2 蛋白-蛋白界面，以及 (v) 确定灵活的间隔 DNA 序列增强 Gsx2 协同性在二聚体位点上。总而言之，我们的结果为理解 Gsx 因子协同 DNA 结合的蛋白质和 DNA 结构决定因素提供了机制基础。