The specificity and regulation of human histone deacetylase 8 by Carol Ann Pitcairn A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Chemical Biology) in the University of Michigan 2015 Doctoral Committee: Professor Carol A. Fierke, Chair Associate Professor Patrick O’Brien Professor Stephen W. Ragsdale Associate Professor Raymond C. Trievel © Carol Ann Pitcairn 2015 Dedication I dedicate this dissertation to God and to my beloved family. God has given me extraordinary family and friends. I will be forever thankful for the wonderful individuals whose lives have intersected or run parallel with mine for a time. It has been an adventure with marvelous companions on the journey! My parents instilled in me a drive for education, although I don’t think anyone expected me to take my education to the extreme of a doctoral degree. I also learned from my family that when you do something, you give it all you’ve got. Thus, I have completed 22 years of school. Like my mom always says, “I’m accomplishing my goals.” Support and encouragement from my parents have brought me to this place, and I am incredibly thankful. I cannot express my love for my family in words on a page. ii Acknowledgements I thank Dr. Carol A. Fierke for being an excellent mentor and role model. I am overjoyed to have achieved this milestone and Carol was integral to my success. I aspire to be as good a mentor as she is. I am thankful to be a part of the lab group she has assembled, and for her guidance throughout the Ph.D. process. I want to thank my undergraduate research advisor, Dr. Nina V. Stourman for her guidance and support, as well as the faculty in the Chemistry Department at Youngstown State University. I also want to thank my mentors and former lab-mates for their advice, assistance, and support: Dr. Yaru Zhang, Dr. Corissa Lamphear, Dr. Caleb G. Joseph, and Dr. Noah A. Wolfson. Although our protein turns purple (when it bothers to be expressed) and our assays are complicated, the HDAC subgroup is the subgroup that has the most fun. Members of this cluster, from 2011 to present, include Dr. Caleb G. Joseph, Dr. Noah A. Wolfson, Dr. Byungchul Kim, Dr. Ningkun Wang, Dr. Lubomír Dostál, Eric D. Sullivan, Jeffrey E. López, Katherine R. Leng, and George R. Murphy III. I thank them for helpful discussions, collaborations, group protein purifications, and laughter. From the bottom of my heart I thank Katherine R. Leng, Jeffrey E. López, Nancy Wu, Yu Chen, and Dr. Jenna Hendershot for friendship and our snack times, iced tea runs, exercise, matching days, story time, and the occasional, well-timed, much-needed hug. I thank my family in Christ at Crossroads Community Baptist Church for lovingly providing a kind ear to listen, words and smiles of encouragement, food, and fun. Finally, I want to give my deepest thanks to my parents and my family. My family is the epitome of unconditional love. I thank them for: emotional support, long phone calls, lots of food, joyful visits when I was too busy to travel, and for understanding when I skipped family events. Thank you for teaching me to love Jesus and people. Thank you for nurturing my self-confidence and motivation. Thank you for being proud of me. iii Table of Contents Dedication ...................................................................................................................... ii Acknowledgements ........................................................................................................ iii List of Tables ................................................................................................................. vi List of Figures ............................................................................................................... vii List of Acronyms .......................................................................................................... viii Abstract ..................................................................................................................... xi Chapter 1 HDAC8 substrates: Histones and beyond, ..................................................... 1 Overview ............................................................................................................. 1 Introduction of HDACs ......................................................................................... 2 Biological importance of HDAC8 ......................................................................... 7 Known HDAC8 substrates ................................................................................... 8 Candidate non-histone HDAC8 substrates ........................................................ 11 HDAC8 complex formation ................................................................................ 17 Catalytic mechanism and regulation of HDAC8 activity ..................................... 27 HDAC8 localization ........................................................................................... 30 Phosphorylation of HDAC8 ................................................................................ 31 Concluding remarks .......................................................................................... 35 Acknowledgements ........................................................................................... 36 Chapter 2 Histone deacetylase 8 recognizes histone substrates via both long and short range interactions, ....................................................................... 37 Introduction ....................................................................................................... 37 iv Materials and methods ...................................................................................... 39 Results .............................................................................................................. 44 Discussion ......................................................................................................... 53 Acknowledgements ........................................................................................... 57 Chapter 3 Metal switching specificity: A novel regulatory mechanism for HDAC8, ....... 58 Introduction ....................................................................................................... 58 Materials and methods ...................................................................................... 60 Results and discussion ...................................................................................... 64 Acknowledgements ........................................................................................... 76 Chapter 4 Investigation of the effect of HDAC8 phosphorylation on activity, metal dissociation, and substrate specificity ......................................................... 77 Introduction ....................................................................................................... 77 Materials and methods ...................................................................................... 80 Results .............................................................................................................. 88 Discussion ....................................................................................................... 105 Accession Code .............................................................................................. 108 Acknowledgements ......................................................................................... 108 Chapter 5 Conclusions and future directions .............................................................. 109 Overview ......................................................................................................... 109 Substrate recognition: peptide and protein substrates ..................................... 110 Metal switching ................................................................................................ 112 Phosphorylation and HDAC8 ........................................................................... 114 Concluding remarks ........................................................................................ 117 Bibliography ............................................................................................................... 118 v List of Tables Table 2-1: Sequences of peptides used in this study ................................................... 46 Table 2-2: Catalytic efficiencies for deacetylation of histone substrates by HDAC8a .... 46 Table 3-1: Metal dependence of HDAC8 substrate specificity for peptides in solutiona 68 Table 4-1: Data collection and refinement statistics for the S39E-HDAC8-Droxinostat complex ............................................................ 91 Table 4-2: Kinetics of S39E-HDAC8 toward Fluor-de-Lys peptidea .............................. 94 Table 4-3: S39E-HDAC8 apparent catalytic efficiency for unlabeled peptides .............. 97 Table 4-4: Metal ion dissociation rate constants for S39E and WT HDAC8 .................. 98 vi List of Figures Figure 1-1: Select HDAC inhibitors ................................................................................ 6 Figure 1-2: The Fluor-de-Lys® assay (Enzo Life Sciences) ........................................... 9 Figure 1-3: The proposed model of HDAC8 and SMC3 in cohesin dissassembly ........ 13 Figure 1-4: HDAC8 structures ...................................................................................... 21 Figure 1-5: HDAC8 with two bound TSA molecules