University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2009-09-11 A Multiparameter Network Reveals Extensive Divergence Between C. elegans bHLH Transcription Factors: A Dissertation Christian A. Grove University of Massachusetts Medical School Worcester Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Amino Acids, Peptides, and Proteins Commons, Animal Experimentation and Research Commons, and the Genetic Phenomena Commons Repository Citation Grove CA. (2009). A Multiparameter Network Reveals Extensive Divergence Between C. elegans bHLH Transcription Factors: A Dissertation. GSBS Dissertations and Theses. https://doi.org/10.13028/d7bb- r923. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/441 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. A Multiparameter Network Reveals Extensive Divergence between C. elegans bHLH Transcription Factors A Dissertation Presented By CHRISTIAN A. GROVE Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY SEPTEMBER 11, 2009 INTERDISCIPLINARY GRADUATE PROGRAM COPYRIGHT INFORMATION The chapters of this dissertation have appeared in separate publications or as part of publications: Grove CA, Walhout AJM. Transcription factor functionality and transcription regulatory networks. Mol Biosyst. 2008 Apr; 4(4):309-314. Grove CA, De Masi F, Barrasa MI, Newburger DE, Alkema MJ, Bulyk ML, Walhout AJM. A multiparameter network reveals extensive divergence between C. elegans bHLH transcription factors. Cell. 2009 Jul 23;138(2):314-327. iii ACKNOWLEDGEMENTS I would first like to thank my parents, Margareta and Jeffrey Grove, for their unwavering support of my choices: career, personal, and otherwise. They have always been 110% behind what I’ve wanted to do, and invested an immense amount of their own personal energy to be sure that I’ve had what I needed. For as long as I’ve been a student, my parents have appreciated my studies as my top priority and have given me the space and opportunity to embrace and nourish my efforts. I would also like to thank my brother, Eric Grove, whose thirst and passion for knowledge inspired me as a young scientist. There have been many late nights where my brother and I have discussed a whole range of topics, none of which were less than profound. I thank my mentor, Marian Walhout, for providing me the opportunity to dive into a field of research that is as cutting edge as her thoughts on the topic. It was my fortune that her arrival at UMass Medical School coincided with my growing curiosity about systems and network biology as well as the field of transcription factors and gene regulatory networks. Marian represents the epitome of hard work, perseverance, and pushing of limits, scientific and personal. My graduate studies would not have been nearly as successful without her guidance and support. I thank Heidi Tissenbaum, who single-handedly introduced me to the nematode Caenorhabditis elegans as a model organism, when she hired me as a iv technician right out of college (eight years ago). Heidi is a very dedicated scientist and an inspiration to those she works with. I thank Jenna Balestrini, whose companionship throughout the six years of my graduate studies has been both wonderful and insightful. Jenna has always provided me with support through difficult times, and has been a peer scientist with whom I could bounce ideas off of and learn things about biology that I had never even thought of. I thank Federico (Fred) De Masi, whose hard work, insight, and endless sense of humor provided a fruitful collaboration of which I am very grateful. I also thank Martha Bulyk for inviting me to her laboratory to learn the techniques of her lab and perform experiments relevant to my thesis. Martha’s knowledge and expertise has been crucial to the success of our work. I thank members of the Walhout and Dekker labs, both past and present, for their help and friendship over the years of graduate school. I would particularly like to thank Bart Deplancke, Natalia Martinez, Efsun Arda, Vanessa Vermeirssen, John Reece-Hoyes, and Inmaculada Barrasa for their years of camaraderie and scientific discussion in the Walhout lab. I would like to express my gratitude to the faculty and staff of the Program in Gene Function and Expression, particularly Nina Bhabhalia, Sharon Briggs, Judy Mondor, Sara Evans, Elizabeth Nourse, and Shanna Spencer for their persistent and friendly administrative and technical support. v I thank the members of my thesis committee, Scot Wolfe, Michael Green, Michelle Kelliher, and Mark Alkema for their years of guidance, support, and encouragement. I also thank Keith Blackwell for agreeing to take time from his busy schedule and travel to Worcester to be an external examiner for my thesis defense. And last, but not least, I’d like to thank my community of friends in and around Worcester and beyond, whose friendship, music, knowledge, and interests have enriched my life outside of school. vi ABSTRACT It has become increasingly clear that transcription factors (TFs) play crucial roles in the development and day-to-day homeostasis that all biological systems experience. TFs target particular genes in a genome, at the appropriate place and time, to regulate their expression so as to elicit the most appropriate biological response from a cell or multicellular organism. TFs can often be grouped into families based on the presence of similar DNA binding domains, and these families are believed to have expanded and diverged throughout evolution by several rounds of gene duplication and mutation. The extent to which TFs within a family have functionally diverged, however, has remained unclear. We propose that systematic analysis of multiple aspects, or parameters, of TF functionality for entire families of TFs could provide clues as to how divergent paralogous TFs really are. We present here a multiparameter integrated network of the activity of the basic helix-loop-helix (bHLH) TFs from the nematode Caenorhabditis elegans. Our data, and the resulting network, indicate that several parameters of bHLH function contribute to their divergence and that many bHLH TFs and their associated parameters exhibit a wide range of connectivity in the network, some being uniquely associated to one another, whereas others are highly connected to multiple parameter associations. We find that 34 bHLH proteins dimerize to form 30 bHLH dimers, which are expressed in a wide range of tissues and cell types, particularly during the vii development of the nematode. These dimers bind to E-Box DNA sequences and E-Box-like sequences with specificity for nucleotides central to and flanking those E-Boxes and related sequences. Our integrated network is the first such network for a multicellular organism, describing the dimerization specificity, spatiotemporal expression patterns, and DNA binding specificities of an entire family of TFs. The network elucidates the state of bHLH TF divergence in C. elegans with respect to multiple functional parameters and suggests that each bHLH TF, despite many molecular similarities, is distinct from its family members. This functional distinction may indeed explain how TFs from a single family can acquire different biological functions despite descending from common genetic ancestry. viii TABLE OF CONTENTS TITLE PAGE i SIGNATURE PAGE ii COPYRIGHT PAGE iii ACKNOWLEDGEMENTS iv ABSTRACT vii TABLE OF CONTENTS ix LIST OF FIGURES xiv LIST OF TABLES xvii LIST OF ABBREVIATIONS xviii PREFACE TO CHAPTER I 1 CHAPTER I: Transcription Regulatory Networks, Transcription Factor Parameters, and the Divergence of Transcription Factor Families 2 Introduction 3 Transcription Factors and Transcription Regulatory Networks 4 Transcription Factor Predictions 4 DNA Binding Domains 5 Emerging Properties of Transcription Regulatory Networks 5 Parameters of TF Function 8 Alternative Splicing 8 Dimerization 10 DNA-Binding Specificity 11 Spatiotemporal Expression 12 Post-Translational Modifications 13 Ligands 14 Co-Factors 15 Transcription Factor Variants and Disease 16 Integrating Multiple Parameters to Visualize and Understand TRNs 17 ix Divergence of TF Families 17 The C. elegans bHLH TFs 20 Classification of C. elegans bHLH Proteins 20 An Experimental Approach to Integrate Multiple Functional Parameters of the C.elegans bHLH TFs 21 Synopsis 24 PREFACE TO CHAPTER II 33 CHAPTER II: The C. elegans bHLH Dimerization Network 34 Abstract 35 Introduction 36 Results 38 Updates to Various C. elegans bHLH Gene Models 38 Auto-Activation of C. elegans bHLH Proteins in Yeast 39 The C. elegans bHLH Dimerization Network 40 Dimerization Interactions are Class Specific 40 Two Dimerization Modules: The HLH-2 and AHA-1 Modules 41 HLH-3, HLH-4, and HLH-10 vs. the TF-Array 42 Discussion 44 Yeast Two-Hybrid: Data Quality 44 Dimerization Hub Proteins May Confer Transcriptional Activation Activity 45 bHLH Structural Components that Potentially Contribute to Dimerization Specificity 46 bHLH Proteins with no Yeast Two-Hybrid Data 49 Known Human bHLH Network 49 HLH-33: A Novel bHLH-PAS Protein? 51 Summary 52 Materials