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Using "Designer" Nucleosomes to Study Enzymatic Crosstalk Between Histone Ubiquitylation and Histone Methyltransferases Sarah J

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Using Rockefeller University Digital Commons @ RU Student Theses and Dissertations 2012 Using "Designer" Nucleosomes to Study Enzymatic Crosstalk Between Histone Ubiquitylation and Histone Methyltransferases Sarah J. Whitcomb Follow this and additional works at: http://digitalcommons.rockefeller.edu/ student_theses_and_dissertations Part of the Life Sciences Commons Recommended Citation Whitcomb, Sarah J., "Using "Designer" Nucleosomes to Study Enzymatic Crosstalk Between Histone Ubiquitylation and Histone Methyltransferases" (2012). Student Theses and Dissertations. Paper 244. This Thesis is brought to you for free and open access by Digital Commons @ RU. It has been accepted for inclusion in Student Theses and Dissertations by an authorized administrator of Digital Commons @ RU. For more information, please contact [email protected]. USING “DESIGNER” NUCLEOSOMES TO STUDY ENZYMATIC CROSSTALK BETWEEN HISTONE UBIQUITYLATION AND HISTONE METHYLTRANSFERASES A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy by Sarah J. Whitcomb June 2012 © Copyright by Sarah J. Whitcomb 2012 USING “DESIGNER” NUCLEOSOMES TO STUDY ENZYMATIC CROSSTALK BETWEEN HISTONE UBIQUITYLATION AND HISTONE METHYLTRANSFERASES Sarah J. Whitcomb, Ph.D. The Rockefeller University 2012 It is well established that chromatin is a destination and source for signal transduction affecting all types of DNA metabolism. Histone proteins in particular are extensively post-translationally modified (PTM), and many of these individual modifications have been studied in depth. I have been interested in how the complex repertoire of histone PTMs are co-regulated to generate combinations with meaningful physiological outcomes. One important mechanism is “crosstalk” between pre-existing histone PTMs and enzymes that add or remove subsequent modifications on chromatin. It has been previously shown that H3 lysine 4 methyltransferases involved in transcriptional activation are stimulated and repressed by H2Bub and H2Aub, respectively. Here, using chemically-defined “designer” mononucleosomes, I tested whether nucleosomal H2BK120ub and H2AK119ub influence the activity of a well-studied histone methyltransferases complex that is repressive to transcription, Polycomb Repressive Complex 2 (PRC2). I also built upon previous studies of direct enzymatic crosstalk between histone ubiquitylation and H3 lysine 79 methyltransferase, Dot1L, by investigating the plasticity of ubiquitin position in stimulation of Dot1L methyltransferase activity. Finally, using designer mononucleosomes containing H3 phosphoserine mimetics, crosstalk studies with PRC2 methyltransferase uncovered a putative novel methyl/phos switch specific to the histone variant H3.3 These studies address the specificity of crosstalk between pre-existing post-translational modifications and subsequent methylation, and thereby strengthen our understanding of mechanisms to establish and maintain functional combinations of histone modifications in chromatin. ACKNOWLEDGEMENTS First of all, I want to acknowledge my PhD advisor, Dr. C. David Allis. Throughout my thesis, his love for chromatin was infectious, and his remarkable scientific career served as an inspiring example, particularly because he carries it with such humility. I would also like to express my gratitude for the freedom he gave me, his patience and generosity. This thesis would not have been possible without the collaboration, consistent support, and commitment of Dr. Tom Muir and members of his laboratory, especially Drs. Robert McGinty, Champak Chatterjee, and Beat Fierz. Similarly, I want to express my gratitude to the current and past members of Dr. Allis’s lab, for generously sharing their expertise with me as well as providing friendship. And I’d like to specifically thank our laboratory manager, Jamie Winshell, who keeps the lab work humming, despite needing to “save us from ourselves” nearly daily. I want to thank my Faculty Advisory Committee, Drs. F. Nina Papavasiliou, Robert G. Roeder, and Sanford M. Simon, for their guidance throughout my thesis work. I would also like to acknowledge Dr. Yang Shi, of the Children’s Hospital Boston, for serving as the external examiner for my thesis defense. I am blessed to have an extremely supportive immediate family, extended family, and partner. Their unwavering faith in my capacity to solve and to persevere has been a great source of strength. iii TABLE OF CONTENTS ACKNOWLEDGMENTS........................................................................................................ iii TABLE OF CONTENTS ........................................................................................................ iv LIST OF FIGURES .............................................................................................................. vii CHAPTER 1: INTRODUCTION...............................................................................................1 1.1 Chromatin is the physiologically relevant form of eukaryotic genomes............................... 1 1.1.1 Variation and complexity in chromatin ..................................................................... 2 1.2 Post-translational modification of histones............................................................................ 5 1.2.1 General mechanisms of histone PTM function.......................................................... 5 1.2.2 Histone methylation ................................................................................................... 9 1.2.2.1 SET-mediated methylation of H3K27 by PRC2 ............................................ 11 1.2.2.2 Non-SET-mediated methylation of H3K79 by Dot1...................................... 13 1.2.3 Histone ubiquitylation.............................................................................................. 15 1.2.3.1 Functions of H2BK120ub............................................................................... 21 1.2.3.2 Functions of H2AK119ub .............................................................................. 24 1.2.4 “Designer” nucleosomes to study enzymatic crosstalk............................................ 27 CHAPTER 2: PREPARATION OF “DESIGNER” AND ENDOGENOUS NUCLEOSOMES ............29 2.1 Expressed protein ligation to generate “nearly native” H2Bub(G76A) .............................. 31 2.2 Application of expressed protein ligation to generate “nearly native” H2Aub(G76A)....... 35 2.2.1 Traceless EPL at alanine to generate hsH2Aub(G76A) .................................... 35 2.2.2 Traceless EPL at valine to generate xlH2Aub(G76A) ...................................... 40 2.3 Disulfide-directed ubiquitylation of H2B and H2A ............................................................ 40 iv 2.4 Designer octamer generation ............................................................................................... 45 2.5 Designer mononucleosome generation................................................................................ 51 2.6 Endogenous HeLa nucleosomes .......................................................................................... 65 CHAPTER 3: SPECIFICITY OF ENZYMATIC CROSSTALK BETWEEN HISTONE UBIQUITYLATION H3K79 METHYLATION ........................................................................61 3.1 Dot1, evolutionarily conserved methyltransferase for H3K79............................................ 61 3.2 DNA binding is critical for Dot1 methyltransferase activity............................................... 64 3.3 Indirect in vivo crosstalk between H4K16Ac and H3K79methylation................................ 65 3.4 Direct Enzymatic crosstalk between H2Bub and H3K79methylation................................. 65 3.5 Preparation of recombinant hDot1L methyltransferase ...................................................... 66 3.6 Ubiquitin stimulates Dot1L activity in cis .......................................................................... 67 3.7 Histone ubiquitylation positional plasticity in Dot1L MTase stimulation .......................... 68 3.8 Dot1L activity is stimulated by Mg2+ and insensitive to DTT............................................. 70 3.9 Use of ssUb nucleosomes to investigate potential crosstalk between H2Aub and Dot1L .. 71 CHAPTER 4: TESTING FOR CROSSTALK BETWEEN HISTONE UBIQUITYLATION AND H3K27 METHYLATION ......................................................................................................76 4.1 Evolutionary conservation of Polycomb Repressive Complex 2 ........................................ 78 4.2 Mammalian PRC2 core complex composition .................................................................... 78 4.3 In vitro PRC2 HMTase activity........................................................................................... 81 4.4 Recombinant PRC2 complex preparation............................................................................ 82 4.5 PRC2 activity is sensitive to the concentration of Mg2+, Li+, and reducing agents ............. 84 4.6 In vitro enzymatic ubiquitylation of H2A for PRC2 crosstalk studies................................ 85 4.7 H2A ubiquitylation machinery inhibits PRC2 methylation of H3K27................................ 90 v 4.8 H2Aub modestly inhibits PRC2 activity ............................................................................. 91 4.9 Presence of H2Bub in nucleosome doesn’t appreciably influence PRC2 activity .............
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