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Structural and Biochemical Mechanisms of Notch Signal Activation and Inhibition Structural and Biochemical Mechanisms of Notch Signal Activation and Inhibition The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Jarrett, Sanchez Mario. 2019. Structural and Biochemical Mechanisms of Notch Signal Activation and Inhibition. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:42013090 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Structural and Biochemical Mechanisms of Notch Signal Activation and Inhibition A dissertation presented by Sanchez Mario Jarrett to The Division of Medical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Biomedical and Biological Sciences Harvard University Cambridge, Massachusetts August 2019 © 2019 Sanchez Mario Jarrett All rights reserved. Dissertation Advisor: Stephen Blacklow Sanchez Mario Jarrett Structural and Biochemical Mechanisms of Notch Signal Activation and Inhibition Abstract Cell signaling encompasses the fundamental processes which enable an organism to perceive and respond to its environment. Productive cell signaling occurs in a series of tightly regulated steps that require stimulus detection, signal transmission, and a downstream response. The Notch signaling pathway is an essential cell-cell communication system in metazoans that has critical roles in cell fate determination, growth and development. In mammals, there are two families of Notch-activating ligands, Jagged and Delta-like. The Delta- like paralogues, DLL1 and DLL4, have high structural similarity but exhibit context-dependent functional differences. Notch pathway activation induces transcription of Notch-responsive genes including the Notch regulated ankyrin repeat protein (NRARP) which acts as a feedback inhibitor of the Notch response and is among the most commonly Notch-induced genes in many cell types. In this thesis, I describe the molecular basis for functional divergence of the highly similar ligands DLL1 and DLL4 and a molecular mechanism for NRARP-mediated feedback inhibition of Notch signaling. First, I analyze the functional divergence of DLL1 and DLL4 using cellular assays and biochemical studies. DLL1 and DLL4 activate NOTCH1 and NOTCH2 differently in cell-based assays and this discriminating potential lies in the region between the N-terminus and EGF repeat three. Our results reveal that DLL4 preferentially activates NOTCH1 over NOTCH2, whereas DLL1 exhibits no preference for either receptor. These studies establish that ligand iii ectodomains dictate selective function and features outside the receptor-binding interface contribute to functional differences. Next, I show that NRARP exerts its inhibitory effect by binding directly to the Notch transcriptional activation complex (NTC), requiring both the RBPJ transcription factor and Notch intracellular domain, but not Mastermind-like proteins or DNA. The X-ray structure of a NRARP/RBPJ/NOTCH1/DNA complex reveals that assembly of NRARP/RBPJ/NOTCH1 complexes relies on simultaneous engagement of RBPJ and NOTCH1 in a non-canonical binding mode involving the extension of the NOTCH1 ankyrin repeat stack by the ankyrin repeats of NRARP. Interface mutations of NRARP disrupt entry into NTCs and abrogate feedback inhibition of Notch signaling. These studies establish the structural basis for NTC engagement by NRARP and provide insights into a critical negative feedback mechanism that regulates Notch signaling. iv Table of Contents Abstract………………………………………………………………..………………………….iii Table of Contents………………………………………….……………………………………....v List of Figures…………………………………………………………………………………….vi List of Tables…………………………………………………………………………………….vii Acknowledgments…………………………………………………………...………………….viii Chapter 1 – Introduction……………………………………………………………….……….1 Chapter 2 – Refining Notch – Delta like ligands’ interaction and ligand in vivo functions..30 Chapter 3 – Extension of the NICD Ankyrin Repeat Stack by NRARP Promotes Feedback Inhibition of Notch Signaling……………………………..……………………………………58 Chapter 4 – Conclusions and Future Directions………………….…………………………102 Appendix 1 – Ligand ectodomains determine function in vivo and selectivity of DLL1 and DLL4 toward NOTCH1 and NOTCH2 in vitro………………………..……………………113 Appendix 2 – Supplemental figures for Chapter 3………………………………………….141 Appendix 3 – Bispecific Forkhead Transcription Factor FoxN3 Recognizes Two Distinct Motifs with Different DNA Shapes……………………………...……………………………145 v List of Figures Figure 1.1 Notch mutant female………………………………………………………………….5 Figure 1.2 Notch signaling pathway……………………………………………………………...9 Figure 2.1. Contributions of DLL4 contact residues to NOTCH1 activation…………………...37 Figure 2.2. DLL4 binding to NOTCH1 on the cell surface……………………………………..39 Figure 2.3. DLL1 and DLL4 differentially activate NOTCH1 and NOTCH2………………….42 Figure 2.4. Schematic representation of DLL1 and DLL4 and variant proteins………………..44 Figure 2.5. Contributions of the MNNL-EGF3 portion and contact amino acids to ligand selectivity towards N1 and N2…………………………………………………………………...47 Figure 3.1. NRARP is a negative feedback regulator of Notch signaling………………………66 Figure 3.2. Requirements for complexation of NRARP with NOTCH1 and RBPJ…………….70 Figure 3.3. Structure of an NRARP-NOTCH1-RBPJ complex on DNA, and comparison with the human Notch1-RBPJ-MAML1…………………………………………………………………..74 Figure 3.4. Interface between NRARP and NICD1-RBPJ……………………………………...76 Figure 3.5. A. Effect of NRARP interface mutations on Notch-dependent luciferase reporter gene activity……………………………………………………………………………………...79 Figure 3.6. Effect of NRARP on the degradation of activated NOTCH1 complexes…………..82 vi List of Tables Table 1. Tandem immunoprecipitation and mass spectrometry results from Jurkat cells using tagged NRARP as bait………………………………………………………………………….100 Table 2. Data collection, structure determination and refinement statistics…………………...101 vii Acknowledgements Pursuing a PhD has been an arduous but satisfying endeavor and for those who have helped me along this journey you have my undying gratitude. To my advisor Steve, I would like to express my utmost appreciation for your support over the years. Steve’s passion for science has always been inspiring and his guidance has shaped the way I think as a scientist. His leadership and unwavering efforts have created a haven for scientific pursuits in the form of the Blacklow laboratory. Thank you for your mentorship and the freedom and opportunity to explore. To my lab members, the Blacklow lab has been an exceptional place to develop as a scientist and I am grateful to every member, past and present, for thoughtful discussions, experimental advice and for always being available for a laugh or a drink to celebrate our victories and to commiserate lessons learned. Thanks in particular to Tom and Brandon, for all your advice and teachings whether directly or by example – I am forever grateful. Many thanks to Jon Aster, Joe Loparo and Mike Eck for serving on my Dissertation Advisory Committee over the years. Your advice throughout my dissertation work has truly been invaluable and I thank you for guiding me to good ideas and away from bad ones. And thanks to my Examination Committee for reading this dissertation. To all my friends and BBS classmates, you have taught me so much and helped me grow as a scientist as well as a person. Thank you for enriching my life, I hope I have enriched yours. viii Finally, I would like to thank my family for the love and support you have always given me. Thanks to my parents who have always supported me in my pursuits. I will cherish the lessons learned from observing your persistence and sacrifice no matter where life takes me. To my late grandmother, I am not a fraction of the person I am today without you. Thank you for your love and support when I needed it most. ix CHAPTER 1 Introduction 1 1.1 Introduction to Intercellular Signaling Pathways The ability of cells to perceive and respond to their environment is fundamental to the development of multicellular organisms. Cells are constantly exposed to signals that may arrive in the form of soluble factors generated locally or distantly, ligands expressed on other cells, or components of the extracellular matrix. The capacity to perceive these signals and coordinate a physiological response facilitates the formation and maintenance of the specialized tissues of multicellular organisms. Specialized tissues that arise during embryogenesis are characterized by the careful regulation of cellular behaviors, so that cells proliferate, migrate, differentiate and form tissues in the proper place and at the proper time. These processes are genetically programmed and depend on cell history, lineage, and the action of the signaling pathways which coordinate the cellular interactions that lead to organogenesis. A limited number of signaling pathways regulate many of the key events in normal development. Among the most important systems for intercellular communication are the Notch, Wnt, Hedgehog, FGF, and TGF β signaling systems. These pathways operate repeatedly in various contexts and elicit diverse signaling outcomes. Not only do they control cell fate decisions in development, they also function throughout the lifetime
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