The Regulatory Consequences of Transcription Factor Evolution

这个项目将解释动物DNA序列的进化变化如何导致形态的进化，并将更全面地解释基因组是如何工作的。基因组广泛地由编码蛋白质的基因和非基因、非编码区组成。这些非编码DNA序列的一个重要功能是结合蛋白质，这反过来将控制基因被激活或表达的时间、地点和水平。这些DNA：蛋白质相互作用是如何进化的，以及基因表达的直接变化几乎完全未知。这里的研究将有助于理解蛋白质如何进化DNA结合强度或亲和力的变化，以及这如何导致基因表达的进化。该项目还将为博士后研究员提供培训机会，包括指导更多初级科学家的培训机会。本科生将有意义地从事在基因组学、遗传学、生物信息学和进化论方面的实践和自我指导的研究培训。这种跨学科的培训对于培养下一代生物学家至关重要。实验室小组还将通过提供动物多样性、进化以及基因组和生物信息学工具的使用模块来接触当地的高中生，从而扩展现有课程，并让学生及早参与生物教育。这项研究产生的测序数据将保存在一个在线的、可公开访问的数据库中。该项目将提供第一个比较的、广泛的体内实验验证，即低亲和力和高亲和力的转录因子结合位点在基因表达中提供功能差异，并将有助于了解这种差异是如何演变的。最近开发的技术可以评估转录因子的DNA结合偏好和相关亲和力的范围。这些技术的使用表明，三个同源转录因子--小鼠Eomesodermin、海胆TBrain和海星TBrain--保持了几乎相同的高亲和力结合基序偏好，但进化出了对低亲和力基序的偏好变化。这里的项目直接扩展了这项工作，以检查转录因子DNA结合亲和力在调控基因表达中可能发挥的作用，以及这对表型的影响。以棘皮动物胚胎为模型，该项目使用全基因组芯片-Seq和RNA-Seq，以及一种新的、中等吞吐量的“纳米标签”方法，来评估体内的调节功能。目的是确定基序使用与对TBrain及其假定的辅因子Smad2/3水平变化的反应之间的关联。这将测试高亲和力基序或低亲和力基序对TBR蛋白水平变化的响应，如果关联取决于位点的数量，以及这种反应在两个物种之间有何不同。这项工作有望为基因表达如何通过使用高亲和力和低亲和力结合位点进化提供一个新的概念框架。

This project will explain how evolutionary changes in an animal's DNA sequence lead to evolution of morphology, and will more fully explain how genomes work. Genomes are broadly comprised of genes which encode proteins, and the non-gene, noncoding regions. An important function of these noncoding DNA sequences is to bind proteins, which in turn will control when, where, and to what level the genes are activated, or expressed. How these DNA:protein interactions evolve and direct changes in gene expression is almost entirely unknown. The research here will contribute to an understanding of how proteins can evolve changes in the strength, or affinity, of DNA binding, and how this leads to the evolution of gene expression. The project will also provide training opportunities to a postdoctoral fellow, including opportunities for training in mentoring more junior scientists. Undergraduates will be meaningfully engaged in hands-on, self-directed research training in genomics, genetics, bioinformatics and evolution. Such cross-disciplinary training is critical for preparing the next generation of biologists. The lab group will also reach out to local area high school students by providing a module in animal diversity, evolution, and the use of genome and bioinformatics tools, thereby extending the existing curriculum and engaging students early in their biology education. The sequencing data generated from this research will be housed in an online, openly accessible database. This project will provide the first comparative, broad-scale, in vivo experimental validation that low- and high-affinity transcription factor binding sites confer functional differences in gene expression and will contribute to an understanding of how this has evolved. Recent technologies have been developed that allow assessment of the range of DNA binding preferences and associated affinities for transcription factors. Use of these technologies demonstrated that three orthologous transcription factors--mouse Eomesodermin, sea urchin Tbrain and sea star Tbrain--have maintained almost identical high-affinity binding motif preferences but have evolved changed preferences for a low-affinity motif. The project here directly extends this work to examine the roles that transcription factor DNA binding affinity may have in regulating gene expression, and the consequences that this has for phenotype. Using echinoderm embryos as a model, the project uses whole-genome ChIP-Seq and RNA-Seq, together with a new, medium-throughput "nanotag" method, to assess regulatory function in vivo. The aim is to determine the association between motif use and response to changing levels of Tbrain and its putative cofactor Smad2/3. This will test whether high-affinity or low-affinity motifs are most responsive to changing levels of Tbr protein, if association depends on the number of sites, and how this response will differ between the two species. The work is expected to provide a new conceptual framework for how gene expression can evolve through use of high- and low-affinity binding sites.