Mechanisms for the evolution of novel DNA specificity in a transcription factor f

描述(由申请人提供)： 我们首次对转录因子(TF)进化为新DNA结合位点的专一性的机制进行了实验分析。转录因子的多样性和特异性使生物能够精确地调节发育和生理过程中的细胞过程；调控转录因子的作用也是生物进化的重要手段。但人们对TF进化其DNA特异性的机制和动力学知之甚少。对现存蛋白质的比较研究取得的成功有限，因为蛋白质多样性的原因发生在遥远的过去，因此需要用历史的方法将它们与自那时以来发生的许多其他变化区分开来。在这里，我们结合了分析进化机制和过程的强大策略-祖先蛋白质重建-与先进的生物物理分析和高通量筛选变异蛋白质库相结合，以分析类固醇激素受体(SR)蛋白质家族中DNA特异性的进化，这是转移因子多样化的极好模型。SRS在发育、生殖、动态平衡、癌症和许多疾病中起着关键作用。这两类SRS--一方面是雌激素受体，另一方面是雄激素、孕激素、糖皮质激素和盐皮质激素受体(APGMR)--识别不同的DNA结合部位。初步数据表明，这种多样性是通过发生在所有类固醇受体的祖先(AncSR1)和APGMR的祖先(AncSR2)之间的DNA识别的急剧转变而演变的。我们的目标是：1)通过结合系统发育推断与功能和生化/生物物理技术来“复活”AncSR1和AncSR2来剖析这种进化转变，并对它们进行实验表征；2)利用有针对性的遗传操作和祖先背景中的实验分析，识别转换DNA特异性的历史突变并表征它们的机制；3)确定AncSR1容忍改变其DNA识别的突变所需的允许突变，并确定其影响机制；以及4)开发一种新的高通量方法来确定AncSR1和AncSR2之间所有历史突变的功能影响和相互作用。我们的实验将建立一个新的转铁蛋白特异性进化的完整机制，将历史上的遗传变化与蛋白质功能和生化的变化联系起来 产生了一个新的基因调控系统。这一完整的因果链将阐明现存蛋白质的生物物理结构是如何进化的，以及该结构是如何构建进化遗传过程的。作为第一个关于Tf函数机制演化的个案研究，该项目将为未来的研究建立一个方法论范例。由于SR与DNA结合的架构是经典的，我们的工作将建立进化过程的基线知识，这可能适用于其他TF家族。由此产生的结构-功能知识将有助于在合成生物学和生物医学中设计具有新的DNA结合特异性的转录因子。

DESCRIPTION (provided by applicant): We propose the first experimental analysis of the mechanisms by which transcription factors (TFs) evolved specificity for new DNA binding sites. The diversity and specificity of TFs allows organisms to precisely regulate cellular processes in development and physiology; modulation of TF action is also a critical means by which organisms evolve. But little is known about the mechanisms and dynamics by which TF's evolved their DNA specificities. Comparative studies of extant proteins have had limited success, because the causes of protein diversity occurred in the deep past, so historical approaches are required to distinguish them from the many other changes that have accrued since that time. Here we combine a powerful strategy for analyzing evolutionary mechanisms and processes-ancestral protein reconstruction-with advanced biophysical analysis and high-throughput screening of variant protein libraries to analyze the evolution of DNA specificity in the steroid hormone receptor (SR) protein family, a superb model of TF diversification. SRs play key roles in development, reproduction, homeostasis, cancer, and many diseases. The two classes of SRs-estrogen receptors on one hand and the receptors for androgens, progestagens, glucocorticoids, and mineralocorticoids (APGMRs) on the other-recognize different DNA binding sites. Preliminary data indicate that this diversity evolved via a sharp shift in DNA recognition that occurred between the ancestor of all steroid receptors (AncSR1) and the ancestor of the APGMRs (AncSR2). Our goals are to: 1) Dissect this evolutionary shift by combining phylogenetic inference with functional and biochemical/ biophysical techniques to "resurrect" AncSR1 and AncSR2 and experimentally characterize them; 2) Identify the historical mutations that switched DNA specificity and characterize the mechanisms by which they did so, using targeted genetic manipulations and experimental analysis in ancestral backgrounds; 3) Identify permissive mutations that were required for AncSR1 to tolerate the mutations that shifted its DNA recognition and determine the mechanisms for their effects; and 4) Develop a new high-throughput method to identify the functional effects and interactions of all historical mutations between AncSR1 and AncSR2. Our experiments will establish a complete mechanistic account for the evolution of novel TF specificity, linking historical genetic changes to shifts in protein function and biochemistry that generated a new gene regulatory system. This complete causal chain will elucidate how the biophysical architecture of extant proteins evolved and how that architecture structured the evolutionary genetic process. As the first-of-its-kind case study of the mechanistic evolution of TF function, this project will establish a methodological exemplar for future studies. Because the architecture of SR binding to DNA is classical, our work will establish baseline knowledge of evolutionary processes that is likely to apply to other TF families. The resulting structure-function knowledge will facilitate efforts to engineer TFs with new DNA-binding specificities in synthetic biology and biomedicine.