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

本行动资助NSF国家植物基因组计划2019财年生物学博士后研究奖学金。该奖学金支持奖学金获得者在主办实验室的研究和培训计划，该奖学金获得者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，赞助科学家是露西亚·c·斯特拉德博士。生长素是一种小的植物信号分子，参与改变植物的生长发育，对作物产量有重要影响。生长素通过生长素反应因子（Auxin RESPONSE FACTORS， ARF）调控这些过程，这是一个存在于许多植物物种中的转录因子大家族。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。了解大型转录因子家族（如arf）如何在植物基因组中介导dna结合特异性，对于研究植物发育和更好地指导作物发育至关重要。已知arf是成对工作的；然而，科学家们并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了解不同的转录因子对如何改变基因组中的DNA结合。这项研究将使人们更好地理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物种的植物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建立的项目，扩大代表性不足的少数民族学生在STEM领域的参与，如针对K12的青年科学家计划（YSP; ysp.wustl.edu/），以及美国国立卫生研究院支持的针对本科生的研究教育R25和最大化研究职业机会（MARC） T34项目。培养目标包括获取蛋白质生物化学、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，以使研究员成为一名成功的植物生物学家。植物激素生长素是植物发育各个方面的重要调节因子。许多转录因子作为同源二聚体和异源二聚体起作用，但转录因子二聚体与dna结合特异性之间的关系尚未研究。生长素信号传导的主要转录调控因子——生长素反应因子（auxin RESPONSE FACTORS， ARF）蛋白提供了一个很有吸引力的模型来研究转录因子二聚化，因为最近已经描述了包括ARF DNA结合和ARF二聚化的分子基础在内的ARF功能机制。ARF蛋白具有模块化结构，包括C端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结构域如何参与ARF的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——多态玉米（Marchantia polymorpha）和玉米（Zea mays） DNA结合的影响。有三个具体目标：1)通过结构、生物物理和比较序列数据，确定参与ARF-ARF PB1相互作用的氨基酸残基，确定ARF PB1相互作用是否有助于ARF二聚化特异性。假定的相互作用将使用体外和体内蛋白质相互作用技术进行测试，以确定不同ARF PB1结构域的相互作用亲和力；2)检测特定的ARF二聚体是否结合到Z. maays和M. polymorpha的不同位点。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的不同ARF同源和异源二聚体的dna结合模式；3)确定M. polymorpha ARF1 PB1结构域是否参与染色质相互作用和ARF功能。报告细胞系将用于确定ARF多聚体是否能促进多态霉的染色体相互作用。项目期间产生的所有基因组数据将在NCBI基因表达综合数据库（https://www.ncbi.nlm.nih.gov/geo/）中存档。序列比对和系统发育树将上传到Dryad （https://datadryad.org）并公开提供。为这些研究产生的所有实验材料和样品将存储在华盛顿大学，并在要求时提供。除了基因组和系统发育数据外，本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报告（海报报告和口头报告）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与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，多形玉米，dna结合，生长素，转录因子，arfs该奖项反映了美国国家科学基金会的法定使命，并通过基金会的智力价值和更广泛的影响审查标准进行评估，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.