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; 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 ( for archiving. Sequence alignments and phylogenetic trees will be uploaded to Dryad ( 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, ARFs

This 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.

National Science Foundation (NSF)
Division of Integrative Organismal Systems (IOS)
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Diane Okamuro
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Morffy Nicholas J
St. Louis
United States
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