ROLE OF THE MAIZE TRANSCRIPTION FACTOR R IN THE REGULATION OF ANTHOCYANIN BIOSYNTHESIS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Antje Christin Feller Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2010 Dissertation Committee: Professor Erich Grotewold, Advisor Professor David Bisaro Professor Venkat Gopalan Professor JC Jang Professor Bernd Weisshaar Copyright by Antje Christin Feller 2010 ABSTRACT The maize flavonoid biosynthetic pathway is one of the best characterized plant model systems to study the combinatorial regulation of gene expression. Flavonoids are important secondary metabolites and important for the plant, such as in the case for anthocyanins as attractants to pollinators as well as for humans due to a number of biological activities. Anthocyanins in maize are regulated by the cooperation of the R2R3-MYB domain protein C1 and the basic helix-loop-helix (bHLH) protein R. In contrast, phlobaphene pigments, derived from a separate flavonoid biosynthetic branch, are regulated by the R2R3-MYB domain protein P1, which can activate transcription in the absence of a (known) bHLH. Our laboratory has established that the bHLH transcription factor R plays a key role in determining the biological specificity of C1. I describe here how R might contribute to regulatory specificity, on one hand by forming a platform for protein-protein interactions, and on another, by binding to different sites in the regulatory regions of its target genes depending on the interacting partners. Using a variety of techniques I have investigated three protein-protein interacting regions of R. I demonstrate that the highly conserved bHLH domain of R is involved in transcriptional regulation and histone functions. I show that R interacts with the EMSY-like protein RIF1 specifically via the bHLH domain and that this interaction is required for the ii regulation of endogenous flavonoid genes. RIF1 is part of the C1/R regulatory complex and I discuss how RIF1 links transcriptional regulation of flavonoid biosynthetic genes with chromatin function. In addition, I show that the region adjacent to the bHLH domain has structural similarity to a leucine zipper and that the extended bHLH-LZ-like region is able to homodimerize. The bHLH-LZ-like mediated dimerization is required for activation of a synthetic pG-box::Luc promoter::reporter construct in transient expression studies and for binding to a synthetic G-box probe, as well as to the Bz1 and C2 promoter in vitro. Furthermore, I demonstrate that the ACT domain at the C-terminus of R homodimerizes. This domain is necessary for anthocyanin pigment formation, for transcriptional activation of at least four anthocyanin biosynthetic genes and important for DNA-binding to the A1 and Bz1 promoters. I show that interplay between the functional domains described here is necessary for transcriptional activation and DNA- binding. I am also characterizing R-interacting partners which possibly tether R to as yet unknown target genes and therefore might show the involvement of R in other cellular processes. Taken together, these studies emphasize the importance of the bHLH transcription factor R in combinatorial regulation of gene expression of anthocyanin biosynthetic genes and open new possibilities for R to function in other cellular processes. Moreover, these studies highlight the complexity of biochemical pathway regulation and show novel mechanisms of how one TF can participate in several regulatory complexes. iii Dedication This document is dedicated to my family. iv Acknowledgments This work would not have been possible without the continuous guidance, support and friendship of my advisor Prof. Erich Grotewold. Thank you Boss for believing in me. I would like to thank my collaborator Dr. Ling Yuan and his lab members Que Kong and Sitakanta Pattanaik for starting a highly interesting project and for letting me be a part of it. I am grateful to all my lab mates throughout the years, undergraduate students (Kenneth Frame, Ali Azad, Carmen Perrino and Julia Muntean), visiting scholars (Margarita Barros, Andrea Gonzalez-Conca, Lina Palacio), visiting professors (Rivka Barg and Lijun Wang), graduate students (Zidian Xie, George Heine, Marcela Hernandez, Anusha Diaz, Robert Lockwood, Laura Martz and MinGab Kim) and postdoctoral fellows (Vinod Malik, Frantisek Poustka and Yuhua Lu). A super special thanks to former graduate student Niloufer Irani for her tremendous help in the lab, for her friendship and for making the time spent in the lab so much more fun. I especially want to thank my current colleagues, visiting scholars Andres Bohorquez and Katherine Mejia-Guerra, graduate students (Katja Machemer and Daniel Arango), postdoctoral fellows (Lucille Pourcel, Kengo Morohashi, Asela Wijeratne, v Chenglin Chai, Yongqin Wang, Waka Omata, Alper Yilmaz, Xinli Sun and Dan Siegal- Gaskin). Thank you for all the discussions and for helping me making my research a success. I want to express my great appreciation to my lab mate and graduate student Isabel Casas. Thank you Isa for scientific and non-scientific conversations and for being a wonderful friend. Thank you Isabel, Katja, Chenglin and Yongqin for carefully reviewing my dissertation. A special thanks goes to all faculty, postdoctoral fellows and students in the Plant Biotech Center for letting me borrow lab supplies and use equipment and for many helpful discussions. Thanks also to the staff in Rightmire Hall; Melinda Parker, Diane Furtney, Scott Hines, Dave Long and Joe Takayama. Thank you for taking care of “things”, which I have no clue about. I am grateful to my committee members, Prof. David Bisaro, Prof. J.C. Jang, Prof. Venkat Gopalan and Prof. Bernd Weisshaar for their time and guidance. A special thanks goes to Oliver Voss, for a very unique friendship, for his help with any scientific question and for keeping me company while writing my dissertation. Thank you John Bruzzese for flowers, dinners, movies and for wonderful times. Finally I would like to thank my family, for sending packages with my favorite chocolate, for hour-long phone calls and for believing in me. vi Vita March, 1st 1976……….…… born in Brandenburg a. d. H., Germany 1998…………………………B.S. Chemistry, GeoForschungsZentrum , (National Research Center for Geosciences), Potsdam, Germany 2002…………………………Diploma in Biotechnology, University of Applied Sciences, Berlin, Germany 2004 to present ……………..Graduate Teaching and Research Associate, Department of Plant Cellular and Molecular Biology, The Ohio State University Publications Falcone Ferreyra M, Rius S, Emiliani J, Feller A, Pourcel L, Morohashi K, Casati P, Grotewold E. 2010. Cloning and Characterization of a UV-B Inducible Maize Flavonol Synthase. Plant J. 62: 77-91 Poustka F, Irani NG, Feller A, Lu Y, Pourcel L, Frame K, Grotewold E (2007) A Trafficking Pathway for Anthocyanins Overlaps with the Endoplasmic Reticulum-to- Vacuole Protein Sorting Route in Arabidopsis and Contributes to the Formation of Vacuolar Inclusions. Plant Physiol. 145: 1323-1335 *Hernandez, JM, *Feller A, *Morohashi K, Frame K, Grotewold E. (2007) The Basic- helix-loop-helix Domain of Maize R Links Transcriptional Regulation and Histone Modifications by Recruitment of an EMSY-related Factor. Proc. Natl. Acad. Sci. USA 104: 17222-17227 (*these authors contributed equally to the work) Feller A, Hernadez JM, Grotewold E. (2006) An ACT-like Domain Participates in the Dimerization of Several Plant Basic-helix-loop-helix Transcription Factors. J. Biol. Chem. 281: 28964-28974 Hernandez JM, Heine GF, Irani NG, Feller A, Kim MG, Matulnik T, Chandler VL, Grotewold E. (2004) Different Mechanisms Participate in the R-dependent Activity of the R2R3 MYB Transcription Factor C1. J. Biol. Chem. 279: 48205-48213 vii Fields of Study Major Field: Molecular, Cellular and Developmental Biology viii Table of Contents Abstract ............................................................................................................................... ii Dedication ......................................................................................................................... iiii Acknowledgments…………………………………………………………………………v Vita………………………………………………………………………………………vii List of Tables .................................................................................................................... xv List of Figures…………………………………………………………………………...xvi Abbreviations……………………………………………………………………….…xviii CHAPTER 1 ....................................................................................................................... 1 INTRODUCTION .............................................................................................................. 1 1.1 Overview ................................................................................................................... 1 1.2 Combinatorial regulation of gene expression............................................................ 2 1.2.1. Aspects of Combinatorial Gene Regulation ...................................................... 3 1.2.2. The Role of Chromatin Structure in Transcriptional Regulation ...................... 6 1.3 The Flavonoid Biosynthetic Pathway ....................................................................... 8 1.3.1. The Regulators of Anthocyanin and Phlobaphene Biosynthesis ....................... 8 1.3.1.1 The MYB Domain Regulators ........................................................................