Characterization of CSL Complexes in the Notch Pathway: the Su(H)-NICD Interaction and the RBP-J-DNA Interaction
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Characterization of CSL Complexes in the Notch Pathway: the Su(H)-NICD Interaction and the RBP-J-DNA Interaction Ashley N. Contreras B.A., Earlham College, 2007 Committee Chair: Rhett A. Kovall, Ph.D. A dissertation submitted to the Division of Graduate Studies and Research of the University of Cincinnati In partial fulfillment of the requirements for the degree of Doctor of Philosophy (Ph.D.) In the Department of Molecular Genetics, Biochemistry, and Microbiology of the College of Medicine November 2014 Abstract In metazoans, the Notch pathway is a conserved cell-to-cell signaling mechanism that plays a key role in cellular specification. It is essential for cell fate determination in embryonic development, organogenesis, hematopoiesis, and adult tissue homeostasis. Aberrant Notch signaling has been implicated in various cancers, developmental defects, and cardiovascular disease. Despite these various roles, Notch signaling is relatively simple; extracellular ligand-receptor binding triggers proteolytic processing of the Notch receptor. The newly generated receptor fragment, the Notch intracellular domain (NICD), translocates to the nucleus and interacts with the DNA-binding protein CSL (CBF1/RBPJ in mammals; Suppressor of Hairless [Su(H)] in flies; Lag-1 in worms), to ultimately activate transcription of Notch responsive genes. In the absence of pathway activation, however, CSL also represses transcription of these genes. The ability of CSL to differentially regulate gene expression is determined by its interaction with co-regulators (co-activators or co-repressors). The research described in the following chapters characterizes the molecular details of two different interactions of CSL—the Su(H)-NICD interaction and the RBP-J-DNA interaction. Chapter 1 reviews Notch signaling. In Chapter 2, the thermodynamic details of the Su(H)-NICD interaction are explained, revealing a domain-domain interaction novel to the complex in Drosophila. The interaction between RBP-J and different sequences of DNA is quantified in Chapter 3, which demonstrates variations to the CSL binding sequence can positively or negatively affect the binding affinity of CSL. In Chapter 4, the work presented here is summarized and additional studies are proposed to further elucidate ii the molecular mechanisms of the complexes regulating expression of Notch target genes. iii iv Acknowledgements Science is a difficult path, full of obstacles and failure and fleeting joy. When I chose to go to graduate school for molecular biology, I could not comprehend this truth. Since then, I have experienced my share of obstacles and failure and joy, but I would not have succeeded in reaching this point in my graduate career without a few tremendously important people. I would like to acknowledge them now. First and foremost, I am grateful for my mentor, Dr. Rhett Kovall, and the Kovall lab, who have guided and supported me every step of the way. Rhett, with his passion for science and genuine interest in our scientific development and our well-being, has been the best mentor any graduate student could want. I truly appreciate that he allowed me to pursue options outside the lab, like the Preparing Future Faculty program and being a teaching assistant, and how he has supported me during challenging parts of my graduate career. He has, by word and action, shown me what a scientist should be. As for my lab colleagues, thank you for everything and then some. Zhenyu Yuan and Brad VanderWielen answered a few million questions from me alone, teaching me how to use and even fix equipment, how to troubleshoot a problem, and how to multitask like a pro. Nassif Tabaja and Nate Miller have been equally helpful in developing my lab skills, though I am also grateful they gave me the opportunity to teach them when they first started in the lab. They have gone beyond anything I could teach them now, and I know they will go much further. And for all other members of the Kovall lab, past and present, I will miss our morning coffee discussions, where I learned so much about science and life. Without the Kovall lab, I would not be here. v Next, I would like to thank my family and friends, who have been with me through the roller coaster of graduate school. They have all listened to me drone on about how cool science is or about how frustrating repeated failures in the lab are or about how I will never decide what to do with my life. Because they are incredible, patient, supportive people, they remained with me through it all and have continued to encourage my scientific career. In particular, I would like to thank my parents, Bill and Patsy Reyer, who have always believed in me and helped me with everything beyond the lab. I would also be remiss if I did not thank my close friends Maureen and Megan as well as the other ladies in my year of graduate school—Kayleigh, Christine, and Fabiola—who made life fun, encouraged me, and commiserated with me. Last, I owe endless gratitude to my husband, Eric Contreras. We met during my first year of graduate school, and my life has been infinitely better because of it. I cannot say thank you enough for listening to me prattle on about my research or school issues or searching for a career. Eric listens, then tells me to calm down, points out it is not that big a deal, gives me encouragement, and makes me laugh. Without his strength, calm, and humor, I could never have survived the obstacles of graduate school. vi Table of Contents ABSTRACT…………………………………………………………………………….. ii ACKNOWLEDGEMENTS…………………………………………………………….. v TABLE OF CONTENTS………………………………………………………………. vii LIST OF ABBREVIATIONS…………………………………………………………... x CHAPTER 1: AN OVERVIEW OF THE NOTCH SIGNALING PATHWAY…….. 2 Signaling Through the Notch Pathway………………………………………….. 3 Figure 1: Overview of the Notch Signaling Mechanism…………………… 6 Regulation of Notch Signaling……………………………………………………. 7 Biological Role of Notch Signaling and Associated Disease States…………. 12 Notch Pathway Components……………………………………………………… 21 Figure 2: Domain Schematics of Notch Signaling Components………….. 24 CSL and DNA Binding………………..………………..………………..…………. 32 Figure 3: CSL Binds Two Distinct Sites in the Hes1 Target Gene………… 35 Figure 4: Structures of Corepressor Complexes in the Notch Pathway….. 43 Figure 5: The Ternary Complex………………………………………………. 45 CHAPTER 2: THERMODYNAMIC BINDING ANALYSIS OF NOTCH TRANSCRIPTION COMPLEXES FROM D. MELANOGASTER………………….. 49 Abstract..………………..………………..………………..………………………... 50 Introduction..………………..………………..……………………………………… 51 Figure 1: Overview of CSL-Mediated Transcription Regulation……………. 53 Results………………..………………..………………..…………………………… 55 Analysis of Su(H)-NICD Interactions……………….………………………….. 55 Cross-species Binding Studies of CSL-NICD Interactions…………………...56 Table 1: Calorimetric Data for the Binding of Drosophila NICD to Su(H)…. 57 Figure 2: Thermodynamic Binding Analysis of Notch Proteins from Drosophila………..………………..………………..…………. 58 Table 2: Calorimetric Data for NICD-CSL Binding Between Mouse and Drosophila Components…………………………………………….. 59 Figure 3: Cross-Species Binding Experiments (RBP-J + dNICD)…………. 60 Figure 4: Cross-Species Binding Experiments (Su(H) + mNICD)…………. 61 Binding Analysis of Su(H)-dRAMANK Point Mutations……………………… 62 Characterizing the Effect RAM Binding Has on Su(H)-Hairless Interactions 63 Table 3: Calorimetric Data for Su(H) and dNICD Points Mutants…………. 64 Table 4: Calorimetric Data for Competition ITC Between Su(H)-RAM and Hairless…………………………………………….. 64 Discussion………………………..………………..………………..……………….. 65 vii Figure 5: Characterizing the Effect RAM has on Su(H)-Hairless Interactions………………………………………… 66 Figure 6: Revised Model of Ternary Complex Assembly for Drosophila Notch Proteins……………………………………… 70 Materials and Methods……………………………………………………………... 71 CHAPTER 3: QUANTITATIVE BINDING ANALYSIS OF THE CSL-DNA INTERACTION IN THE NOTCH PATHWAY………………………………………… 74 Figure 1: CSL Binds two Distinct Sites in the Hes1 Gene………………….. 78 Figure 2: Diagram of the Contacts Made Between CSL and the Hes1 Consensus Sequence DNA………………………………….. 83 Figure 3: CSL-Hes1 DNA ITC Binding Assays………………………………. 86 Putative CSL Binding Sites in the Mouse Math5 Gene…………………………. 89 Thermodynamic Characterization of Computationally Derived CSL Binding Sites…………………………………………………………. 90 Figure 4: ITC Binding Data for RBP-J and Putative CSL Binding Sites in the Math5 Gene……………………………………………………………. 93 Additional DNA Sequences………………………………………………………… 95 Figure 5: ITC Binding Data for RBP-J and Computationally Derived Sites…………………………………….... 97 Table 1: Additional Sequences of DNA Analyzed for RBP-J Binding by ITC…………………………………………….. 99 CHAPTER 4: CONCLUSIONS AND FUTURE DIRECTIONS…………………….. 100 Hairless and NICD Competition for Binding to Su(H)…………………………… 101 The Effect of Sequence Variation on the CSL-DNA Interaction………………. 105 Figure 1: Species-Specific Models of Ternary Complex Assembly………. 107 Figure 2: Sequence Alignments of CSL, NICD, and the ANK Domain for Mouse and Fly Orthologs……………………….. 108 Future Directions……………………………………………………………………. 110 Figure 3: Comparison of CTD Binding Site for Hairless and ANK Domain 114 BIBLIOGRAPHY………………………………………………………………………… 117 APPENDIX A: A COMBINATION OF COMPUTATIONAL AND EXPERIMENTAL APPROACHES IDENTIFIES DNA SEQUENCE CONSTRAINTS ASSOCIATED WITH TARGET SITE BINDING SPECIFICITY OF THE TRANSCRIPTION FACTOR CSL……………………………………………………………………………. 130 Figure 3: CSL-DNA ITC Binding Experiments………………………………. 138 Table 2: Calorimetric Data for Various DNA Sequences Binding to CSL… 141 APPENDIX B: STRUCTURAL