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  • IDENTIFICATION AND CHARACTERIZATION OF MAFA COREGULATORS: MLL3/4

    AND ITS ROLE IN MOUSE AND HUMAN ISLET β-CELLS

    By

    David William Scoville

    Dissertation

    Submitted to the Faculty of the

    Graduate School of Vanderbilt University

    In partial fulfillment of the requirements

    for the degree of

    DOCTOR OF PHILOSOPHY

    In

    Cell and Developmental Biology

    August, 2015

    Nashville, TN

    Approved:

    Maureen A. Gannon, Ph.D.

    Guoqiang Gu, Ph.D.

    Alvin C. Powers, M.D.

    Peter A. Weil, PhD

  • ii

    ACKNOWLEDGEMENTS

    This work would not have been possible without the support from the NIH and

    from the communities of both the Department of Cell and Developmental Biology and the

    Department of Molecular Physiology and Biophysics at the Vanderbilt University Medical

    Center. I am grateful to the various people who have helped me throughout my graduate

    school career, both those included here and those I have not named. I refer to the

    communities of both departments because the collaborative atmosphere that has exists

    within these departments has helped me in too many ways to mention here.

    First, I would like to thank my thesis advisor, Dr. Roland Stein, for allowing me to

    join his lab and for years of mentoring. While we have had many challenging discussion,

    both scientifically and politically, you have pushed me to be more constant in my critical

    assessment of both others work and my own. I have learned a vast array of techniques,

    both in your lab and from collaborators, which will be a boon in my career. You have also

    provided me with several opportunities to review both papers and grants, allowing me to

    obtain a real sense of how the scientific community works.

    Next, I would like to thank the members of the Stein lab, both current and past. I

    initially began working on projects in the lab under the guidance of Shuangli Guo, who

    was critical in inspiring me to choose the thesis project I eventually settled on. Chad

    Hunter and Yan Hang both taught me several techniques, and provided constant insight

    into my project. Min Guo has helped in numerous lab tasks, and Elizabeth Conrad,

    Manisha Gupte, Jason Spaeth, Brian McKenna, Shilpy Dixit, Trey Thompson, and

    Lauren Bonatakis all helped to create a strong lab environment where problems could be

    discussed, solutions formulated, and hypothesis tested, both in science and in life.

    Lastly, I would thank Holly Cyphert, who helped in numerous ways to finish my project,

  • iii

    whether picking islets, editing my manuscript, or providing that extra push of

    encouragement to get things done, she has been invaluable.

    My committee has been an invaluable component of my training. Dr. Maureen

    Gannon, Dr. Guoqiang Gu, Dr. Tony Weil, and Dr. Al Powers have all been constant

    sources of both advice and encouragement during my thesis. I have not only benefitted

    from their thoughtful insight into my project, but from their labs as well. Whether learning

    tamoxifen injections from Rockann Moser and various bits of advice from Maria Golson

    and Kim Gooding Riley in the Gannon lab, or troubleshooting FACS experiments in the

    Gu lab, they have all been invaluable to my research. Thank you so much for the

    support you have offered me, even when I have been too stubborn to ask for it.

    Lastly, this would not have been possible without the love and support of my

    family and friends. My grandparents and parents have always been incredibly supportive

    of my fascination with science. Specifically, my grandfather Dr. Addison B. Scoville Jr.

    was a constant source of encouragement, and my father Charles K. Scoville has always

    pushed me to follow my interests and pursue a career that I love, even when he doesn’t

    understand it. Most importantly, my wife Marisa has been the most supportive pillar, both

    in her ability to understand the trials and tribulations of graduate school, and in her ability

    to continue to support me through thick and thin, through floods and ice-covered trips

    into lab. Without her love and encouragement, I would not be where I am today.

  • iv

    TABLE OF CONTENTS

    Page ACKNOWLEDGMENTS…….......................................………………………………………ii LIST OF TABLES………………………………………………………………………………..v LIST OF FIGURES………………………………………………………………..…………….vi Chapters I. INTRODUCTION………………………………………………………………………..1

    Diabetes Mellitus and the Pancreas………………………………………...……1 Pancreatic cell types and function……………………………………………1 Role of β-cell in Diabetes Mellitus………………………………...………….3 Current Limitations in Treatment…………………………………...………...6 Islet-Enriched Transcription Factors……………...………………………………8 The Maf family of Transcription Factors………………………………..…..11 MafA………………………………………………………………………….…11 MafB……………………………………………………………………….……14

    Pdx-1………………………………………………………………………..….17 Ngn3……………………………………………………………..……………..19 Other Islet-Enriched Transcription Factors……………………………..….20 Coregulators and Transcription…………………….…………………….…...…22 History and Function……………………………………………………...…..22 Transcription Factors and Coregulators…………………………………....25 Coregulators in the Pancreas…………………………………………..……26 The MLL3/4 Complex…………………………………………………..….…30 Overview and Aims of Dissertation…………………………………….……..…35

    II. IDENTIFICATION OF COREGULATORS OF THE MAFA TRANSCRIPTION FACTOR: MLL3/4 METHYLTRANSFERASES ARE LINKED TO ISLET β-CELL ACTIVATION……………………………………………………………………….…..37

    Introduction……………………………………………………………………...……..37 Materials and Methods………………………………………………………….….....39 Results……………………………………………………………………………...…..45 Discussion………………………………………………………………………………64 III. SUMMARY AND FUTURE DIRECTIONS…………………………………………..68 Thesis Summary and Significance…………………………………….…...…….….68 Perspectives of Future Studies………………………………………………………71 Concluding Remarks…………………………………………………………….…….91 REFERENCES……………………………………………………………………………..…..93

  • v

    LIST OF TABLES

    Tables Page

    1. Known coregulators of islet-enriched transcription factors…………………………….28

    2. Antibodies………………………………………………………………………………...…40

    3. Primers used in qPCR experiments……………………………………………………...43

    4. MudPIT results from Re-CLIP……………………………….…………………………....48

    5. Identification of direct targets of MafA………………………………………………..….53

    6. MafABΔendo animals die within 3 weeks of birth…………………………………..……..74

  • vi

    LIST OF FIGURES

    Figures Page

    1. The Pancreas and the Islets of Langerhans………………………………………….2

    2. Development of Type 2 Diabetes over time……………………………………...…..5

    3. The Maf family of transcription factors………………………………………….…….9

    4. Mouse β-cells transition from MafB expression to MafA after birth………..……..16

    5. Transcription factors involved in β-cell differentiation……………………………...21

    6. Model of Chromatin Architecture and Transcription………………………….........23

    7. The MLL family of proteins……..…………………………………………………….31

    8. MafA interacting proteins in β-cells were identified by Re-CLIP/MS……..……...46

    9. Flag-tagged MafA interacts with the MLL3/4 complex………………………….....49

    10. Sucrose gradient sedimentation reveals that MafA comigrates with the

    MLL3/4 complex…………………………………………………………………...…..50

    11. Knockdown of NCoA6 reduces MafA2 target gene expression in βTC-3 cells....52

    12. M