PDF(Pubmed)
Abstract：
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 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 seven base pairs apart. However, the mechanistic basis for Gsx DNA binding and cooperativity are poorly understood. Here, we used biochemical, biophysical, structural, and modeling approaches to (1) show that Gsx factors are monomers in solution and require DNA for cooperative complex formation; (2) define the affinity and thermodynamic binding parameters of Gsx2/DNA interactions; (3) solve a high-resolution monomer/DNA structure that reveals Gsx2 induces a 20° bend in DNA; (4) identify a Gsx2 protein-protein interface required for cooperative DNA binding; and (5) 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, thereby providing a deeper understanding of HD specificity.
摘要：
保守的Gsx同源结构域（HD）转录因子指定了动物从苍蝇到哺乳动物的神经细胞命运。像许多HD蛋白一样，Gsx因子结合富含A/T的DNA序列，这引发了一个问题-在体外结合相似DNA序列的HD因子如何在体内调节特定的靶基因？先前的研究表明，Gsx因子结合DNA作为单个A/T富集位点上的单体以及作为合作的同二聚体结合到两个相距精确七个碱基对的位点。然而,对GsxDNA结合和协同作用的机理基础知之甚少。这里,我们使用了生化，生物物理，结构,和建模方法（1）表明Gsx因子是溶液中的单体，需要DNA进行协同复合物的形成；（2）定义Gsx2/DNA相互作用的亲和力和热力学结合参数；（3）解决高分辨率的单体/DNA结构，该结构揭示Gsx2诱导DNA弯曲20°；（4）鉴定协同DNA结合所需的Gsx2蛋白-蛋白界面；（5）确定柔性x2二聚体DNA序列可增强Gsoperacer总之,我们的结果为理解Gsx因子协同DNA结合的蛋白质和DNA结构决定子提供了机制基础，从而提供对HD特异性的更深入的理解。
