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

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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,

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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.

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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

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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

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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