NSF Postdoctoral Fellowship in Biology FY 2019: The Contribution of the AUXIN RESPONSE FACTORS (ARF) Dimerization to ARF DNA-Binding

这项行动为2019财年的NSF国家植物基因组计划生物学博士后研究奖学金提供资金。该研究金支持研究员在东道实验室的研究和培训计划，研究员还提出了扩大生物学参与的计划。Nicholas Morffy博士的研究和培训计划的标题是“ARF二聚化对ARF DNA结合的贡献”。“该研究金的主办机构是圣路易斯的华盛顿大学，赞助科学家是Lucia C.生长素是一种小分子植物信号分子，参与改变生长和发育，对作物生产力有重要影响。生长素通过生长素反应因子（ARF）调节这些过程，ARF是在许多植物物种中发现的转录因子的大家族。转录因子结合DNA并调节基因转录，从而改变植物的生长方式。了解大型转录因子家族（如ARF）如何介导植物基因组中的DNA结合特异性对于研究植物发育非常重要，对于更好地指导作物发育至关重要。已知ARF是成对工作的;然而，科学家们不了解不同的转录因子对如何结合特定的DNA，或者转录因子如何与基因组的不同部分相互作用。该项目将利用ARF转录因子家族的特性来更好地理解不同的转录因子对如何改变整个基因组的DNA结合。这项研究将有助于更好地了解转录因子如何调节基因，并可能为更直接地控制玉米等重要作物的生长和发育奠定基础。更广泛的影响包括通过圣路易斯华盛顿大学的既定计划扩大代表性不足的少数民族学生在STEM领域的参与，例如K12的青年科学家计划（YSP; ysp.wustl.edu/）和NIH支持的研究教育R25和最大限度地获得研究职业（MARC）T34计划。培训目标是为植物生物学家的成功职业生涯做好准备，包括获得蛋白质生物化学，生物物理学，基因组学，植物进化以及科学传播和指导方面的专业知识。植物激素生长素是植物发育各个方面的重要调节剂。许多转录因子以同源和异源二聚体的形式发挥作用，但转录因子二聚化与DNA结合特异性之间的关系尚未研究。生长素反应因子（ARF）蛋白是生长素信号传导的主要转录调节因子，它是研究转录因子二聚化的一个有吸引力的模型，因为最近已经描述了ARF功能的机制，包括ARF DNA结合和ARF二聚化的分子基础。ARF蛋白具有模块化结构，其包括C末端I/II型Phox和Bem 1（PB 1）结构域。PB 1结构域介导ARF之间的蛋白质相互作用的定向方式，但PB 1结构域如何有助于ARF DNA结合和二聚化特异性目前尚不清楚。该项目将结合联合收割机进化，生物物理和遗传/基因组技术，研究PB 1介导的相互作用和它们对DNA结合的影响，在两个不同的陆地植物物种，地钱多形物和玉米。具体目标有三个：（1） 通过鉴定陆地植物谱系中参与ARF-ARF PB 1相互作用的氨基酸残基，确定ARF PB 1相互作用是否有助于ARF二聚化特异性，所述氨基酸残基由结构、生物物理和比较序列数据提供信息。将使用体外和体内蛋白质相互作用技术测试推定的相互作用，以确定不同ARF PB 1结构域的相互作用亲和力; 2）测试特定ARF二聚体是否与Z中的不同基因座结合。mays和M.多形。下一代测序方法，包括DAP-seq，将用于测试和比较来自Z. mays和M. polymorpha; 3）确定M. PB 1结构域有助于染色质相互作用和ARF功能。报告细胞系将用于确定ARF多聚体是否能促进M.多形。项目期间生成的所有基因组数据将在NCBI基因表达综合数据库（https：//www.ncbi.nlm.nih.gov/geo/）中提供，以供存档。序列比对和系统发育树将上传至Dryad（https：datadryad.org）并公开提供。这些研究生成的所有实验材料和样品将储存在华盛顿大学，并根据要求提供。除了基因组和系统发育数据，该项目产生的实验结果将通过使用期刊出版物和科学会议上的演讲（海报演讲和口头演讲）及时传播。该项目产生的数据将为阐明蛋白质相互作用伙伴和DNA结合特异性之间的关系提供一个框架，并增加对ARF和生长素信号传导的理解。关键词：玉米，地钱，DNA结合，生长素，转录因子，ARF该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准。

This action funds an NSF National Plant Genome Initiative Postdoctoral Research Fellowship in Biology for FY 2019. The fellowship supports a research and training plan in a host laboratory for the Fellow who also presents a plan to broaden participation in biology. The title of the research and training plan for this fellowship to Dr. Nicholas Morffy is "The Contribution of the ARF Dimerization to ARF DNA-Binding." The host institution for the fellowship is the Washington University in St. Louis and the sponsoring scientist is Dr. Lucia C. Strader.Auxin is a small plant signaling molecule that is involved in altering growth and development and has a major impact on crop productivity. Auxin regulates these processes through AUXIN RESPONSE FACTORS (ARF), a large family of transcription factors found in many plant species. Transcription factors bind DNA and regulate gene transcription which alters how plants grow. Understanding how large transcription factor families, like the ARFs, mediate DNA-binding specificity across a plant genome is important for the study of plant development and is crucial for better directing crop development. The ARFs are known to work in pairs; however, scientists do not understand how different transcription factor pairs bind specific DNA, or how transcription factors might interact with different parts of the genome. This project will utilize the properties of the ARF transcription factor family to better understand how distinct transcription factor pairs alter DNA binding across a genome. This research will lead to a better understanding of how transcription factors regulate genes and may lay the ground work for more directed control of plant growth and development in important crop species such as maize. Broader impacts include broadening participation of underrepresented minority students in STEM fields through established programs at Washington University in St. Louis such as the Young Scientist Program (YSP; ysp.wustl.edu/) for K12 and the NIH-supported Research Education R25 and Maximizing Access to Research Careers (MARC) T34 programs for undergraduate students. Training objectives to prepare the Fellow for a successful career as a plant biologist include acquiring expertise in protein biochemistry, biophysics, genomics, plant evolution as well as in science communication and mentorship. The phytohormone auxin is a crucial regulator of all aspects of plant development. Many transcription factors work as homo- and heterodimers, but the relationship between transcription factor dimerization and DNA-binding specificity remains unstudied. The primary transcriptional regulators of auxin signaling, the AUXIN RESPONSE FACTORS (ARF) proteins present an appealing model to study transcription factor dimerization because the mechanisms of ARF function, including the molecular basis of ARF DNA binding and ARF dimerization, have recently been described. ARF proteins have a modular structure that includes a C- terminal type I/II Phox and Bem1 (PB1) domain. PB1 domains mediate protein interactions between ARFs in a directional manner, but how the PB1 domain contributes to ARF DNA-binding and dimerization specificity is currently unknown. This project will combine evolutionary, biophysical, and genetic/genomic techniques to study PB1-mediated interactions and their impact on DNA binding in two divergent land plant species, Marchantia polymorpha and Zea mays. There are three specific aims: 1) determine if ARF PB1 interactions contribute to ARF dimerization specificity by identifying amino acid residues participating in ARF-ARF PB1 interactions across the land plant lineage, informed by structural, biophysical, and comparative sequence data. Putative interactions will be tested using in vitro and in vivo protein interaction techniques to determine the interaction affinities of different ARF PB1 domains; 2) test if specific ARF dimers bind to distinct loci in Z. mays and M. polymorpha. Next generation sequencing approaches, including DAP-seq, will be used to test and compare DNA-binding patterns of different ARF homo- and heterodimers from both Z. mays and M. polymorpha; and, 3) determine if the M. polymorpha ARF1 PB1 domain contributes to chromatin interactions and ARF function. Reporter lines will be used to determine if ARF multimers can promote chromosomal interactions in M. polymorpha. All genomic data generated during the project will be made available in the NCBI Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) for archiving. Sequence alignments and phylogenetic trees will be uploaded to Dryad (https://datadryad.org) and made publicly available. All experimental material and samples generated for these studies will be stored at Washington University and made available upon request. In addition to the genomic and phylogenetic data, experimental results generated from this project will be disseminated through the use of journal publications and presentations at scientific meetings (poster presentations and oral presentations) in a timely fashion. The data generated by this project will provide a framework for elucidating the relationship between protein interaction partners and DNA-binding specificity, as well increasing understanding of the ARFs and auxin signaling generally.Keywords: Maize, Marchantia polymorpha, DNA-binding, Auxin, Transcription Factors, ARFsThis award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.