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

这一行动为NSF国家植物基因组计划2019财年生物学博士后研究奖学金提供了资金。该奖学金支持在东道主实验室为该研究员制定的研究和培训计划，该研究员还提出了扩大生物学参与度的计划。尼古拉斯·莫菲博士的这项研究和培训计划的标题是“ARF二聚化对ARF DNA结合的贡献”。该奖学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.Strader博士。Auxin是一种小的植物信号分子，参与改变生长和发育，对作物生产力有重大影响。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一个转录因子大家族。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。了解大的转录因子家族，如ARF，如何在植物基因组中介导DNA结合特异性，对于植物发育的研究是重要的，对于更好地指导作物发展至关重要。众所周知，ARF是成对工作的；然而，科学家们不知道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。这个项目将利用ARF转录因子家族的特性来更好地了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控基因，并可能为更直接地控制玉米等重要作物物种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现有的项目，如K12青年科学家计划(ysp；ysp.wustl.edu/)和NIH支持的研究教育R25，扩大未被充分代表的少数族裔学生在STEM领域的参与，以及最大限度地增加本科生获得研究职业(MARC)T34计划的机会。培训目标包括获得蛋白质生物化学、生物物理学、基因组学、植物进化以及科学交流和指导方面的专业知识，为该研究员成为一名成功的植物生物学家做好准备。植物激素生长素对植物发育的各个方面起着至关重要的调节作用。许多转录因子以同源和异源二聚体的形式发挥作用，但转录因子二聚体与DNA结合特异性之间的关系尚不清楚。生长素信号的主要转录调节因子生长素反应因子(ARF)蛋白是研究转录因子二聚化的一个很有吸引力的模型，因为ARF的作用机制，包括ARF DNA结合和ARF二聚化的分子基础，最近已经被描述。ARF蛋白具有模块化结构，包括一个C-末端的I/II型Phox和Bem1(PB1)结构域。PB1结构域以定向方式介导ARF之间的蛋白质相互作用，但PB1结构域如何促进ARF DNA结合和二聚化特异性目前尚不清楚。本项目将结合进化、生物物理和遗传/基因组技术，研究PB1介导的相互作用及其对两种不同陆地植物--多态地钱和玉米--DNA结合的影响。有三个特定的目标：1)通过鉴定整个陆地植物谱系中参与ARF-ARF PB1相互作用的氨基酸残基，根据结构、生物物理和比较序列数据，确定ARF PB1相互作用是否有助于ARF二聚特异性。将使用体外和体内蛋白质相互作用技术来测试可能的相互作用，以确定不同ARF PB1结构域的相互作用亲和力；2)测试特定的ARF二聚体是否与Z.Mays和M.Polya中的不同位置结合。下一代测序方法，包括DAP-SEQ，将被用来测试和比较来自玉米红曲霉和多态红曲霉的不同ARF同源和异源二聚体的DNA结合模式；以及3)确定多态红曲霉ARF1 PB1结构域是否有助于染色质相互作用和ARF功能。报告系将被用来确定ARF多聚体是否能促进多态分枝杆菌的染色体相互作用。项目期间产生的所有基因组数据将在NCBI基因表达总览(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被上传到Dryad(https://datadryad.org))并公开提供。为这些研究生成的所有实验材料和样本将存储在华盛顿大学，并根据要求提供。除了基因组和系统发育数据外，该项目产生的实验结果将通过使用期刊出版物和在科学会议上的陈述(海报陈述和口头陈述)及时传播。这个项目产生的数据将为阐明蛋白质相互作用伙伴和DNA结合专一性之间的关系提供一个框架，并增加对ARF和生长素信号的总体理解。关键词：玉米，Marchantia Polya，DNA结合，生长素，转录因子，ARF该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.