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

转录因子 f 中新型 DNA 特异性的进化机制

• 批准号: 8734454
• 负责人: Jamie T Bridgham
• 金额: $ 32.39万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-17 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8734454
• 关键词: Accounting; Androgen Receptor; Androgens; Anisotropy; Architecture; Area; Binding; Binding Sites; Biochemical; Biophysical Process; Biophysics; Calorimetry; Cardiovascular Diseases; Case Study; Cell physiology; Comparative Study; Computational Technique; Crystallography; DNA; DNA Binding; DNA Sequence; Data; Deuterium; Development; Disease; Engineering; Estrogen Receptors; Evolution; Family; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Epistasis; Genetic Processes; Genetic Variation; Glucocorticoids; Goals; Homeostasis; Hormone Receptor DNA-Binding Domain; Hydrogen; Immune System Diseases; Immunity; Knowledge; Libraries; Link; Malignant Neoplasms; Maps; Metabolic; Methods; Mineralocorticoids; Modeling; Mutation; Organism; Phenotype; Phylogenetic Analysis; Physiology; Play; Process; Protein Biochemistry; Protein Family; Proteins; Recording of previous events; Regulator Genes; Relative (related person); Reporter; Reproduction; Role; Specificity; Steroid Receptors; Structure; System; Techniques; Thermodynamics; Time; Transcriptional Regulation; Variant; Work; base; biophysical techniques; deep sequencing; driving force; experimental analysis; genetic analysis; genetic manipulation; high throughput screening; molecular dynamics; novel; protein function; public health relevance; receptor; receptor binding; reconstruction; reproductive; research study; steroid hormone receptor; success; synthetic biology; transcription factor

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.

描述（由申请人提供）： 我们首次对转录因子 (TF) 进化出新 DNA 结合位点特异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体能够精确调节细胞发育和生理过程；转录因子作用的调节也是生物体进化的关键手段。但人们对 TF 进化其 DNA 特异性的机制和动态知之甚少。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，因此需要历史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，我们将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变异蛋白质库的高通量筛选结合起来，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 TF 多样化的绝佳模型。 SR 在发育、生殖、体内平衡、癌症和许多疾病中发挥着关键作用。两类 SR（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 (APGMR) 受体）识别不同的 DNA 结合位点。初步数据表明，这种多样性是通过所有类固醇受体的祖先 (AncSR1) 和 APGMR 的祖先 (AncSR2) 之间发生的 DNA 识别的急剧转变而进化的。我们的目标是：1）通过将系统发育推断与功能和生物化学/生物物理技术相结合来剖析这种进化转变，以“复活”AncSR1和AncSR2并通过实验表征它们； 2) 使用有针对性的遗传操作和祖先背景的实验分析，识别改变 DNA 特异性的历史突变并描述其机制； 3) 确定 AncSR1 耐受改变其 DNA 识别的突变所需的允许突变，并确定其影响机制； 4) 开发一种新的高通量方法来识别AncSR1和AncSR2之间所有历史突变的功能效应和相互作用。我们的实验将为新型 TF 特异性的进化建立一个完整的机制解释，将历史遗传变化与蛋白质功能和生物化学的变化联系起来， 产生了新的基因调控系统。这个完整的因果链将阐明现有蛋白质的生物物理结构是如何进化的，以及该结构如何构建进化遗传过程。作为转录因子功能机制演化的第一个案例研究，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，因此我们的工作将建立可能适用于其他 TF 家族的进化过程的基线知识。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。