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Reading the Histone Code: Methyl Mark Recognition by MBT and Royal Family Proteins

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Reading the Histone Code: Methyl Mark Recognition by MBT and Royal Family Proteins Reading the Histone Code: Methyl Mark Recognition by MBT and Royal Family Proteins by Nataliya Nady A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Medical Biophysics University of Toronto © Copyright by Nataliya Nady 2012 Reading the Histone Code: Methyl Mark Recognition by MBT and Royal Family Proteins Nataliya Nady Doctor of Philosophy, 2012 Department of Medical Biophysics, University of Toronto Abstract The post-translational modifications (PTMs) of histones regulate many cellular processes including transcription, replication, DNA repair, recombination, and chromosome segregation. A large number of combinations of PTMs are possible, with methylation being one of the most complex, since it is found in three states and is recognized in a sequence specific context. Methylation of histones at key lysine residues has been shown to work in concert with other modifications to provide a Histone Code that may determine heritable transcriptional conditions in normal and disease states. On the most basic level it is pivotal to understand how and by which proteins the numerous PTMs are recognized, as well as mechanisms for downstream signal propagation. To address this need we developed a high-throughput method that allows analysis of up to 600 PTMs in a single experiment. This approach was utilized to characterize macromolecules interacting with the specific modifications on histone tails and to screen for the marks that bound to Malignant Brain Tumor (MBT) proteins, important chromatin regulators implicated in cancer. All MBTs recognized either mono- or dimethyllysine histone marks, and using structure-based mutants we identified a triad of residues that were responsible for this discrimination. These results provide the foundation for the rational design of highly selective MBT inhibitors. Additionally, this thesis describes combinatorial recognition of histone modifications, as proposed in the original Histone Code hypothesis. We demonstrate that Tudor domains of UHRF1, a protein involved in epigenetic maintenance of DNA methylation, is able to read a dual modification state of histone H3 in which it is trimethylated at lysine 9 and unmodified at lysine 4. This study provides an elegant example of the combinatorial readout of histone modification states by a single domain. Together, our findings offer mechanistic insights into the recognition of methylated histone tails by MBT domains and Royal Family in general. ii Acknowledgements Looking back, I am very grateful for all I have acquired throughout the years as a graduate student. It has shaped me as a person and a scientist and would not be possible without many great people surrounding me. First and foremost I wish to thank my supervisor, Dr. Cheryl Arrowsmith. She accepted me into her lab as a summer student and continued to support me ever since. I am grateful for her mentorship, guidance, and valuable advice. She has always encouraged my curiosity and ambitious aspirations which were pivotal to the success of my training as a scientist. I would also like to thank my supervisory committee members Dr. Peter Cheung and Dr. Gilbert Privé for their suggestions and encouragement which helped me to stay on track. I am also thankful to Dr. Aled Edwards and the SGC for the plentiful resources and fresh perspective on the issue. I am indebted to past and present members in the Arrowsmith lab for their help, insightful discussions and friendship. I would like to specifically thank my good friend Rob for teaching me how to appreciate good scientific work as well as interesting discussions and occasional beers; Lilia and Shili for their assistance with my project and creating a friendly lab environment; Sasha for teaching me protein NMR; and Liuba for her help over the summers. The years spent as a graduate student would not be as enjoyable without my friends Atoosa Mehrfar, Diana Purushotham, Nadiya Koshtura, Jocelyn Stewart, Fernando Amador, Tharan Srikumar, Alison Aiken. Finally, I wish to thank my family. I am forever indebted to my parents for their unconditional love and invaluable opportunities that allowed me to pursue graduate degree. Lastly, I cannot thank enough my husband, Andrew. Not only he always ensured that my computer is up and running problem-free, but I am above all grateful for his love, endless patience, support, and words of encouragement when it was most required. iii Table of contents Abstract ii Acknowledgements iii Table of Contents iv List of Tables vi List of Figures vii List of Abbreviations viii Chapter 1: Introduction 1 1.1 Thesis overview 2 1.2 Epigenetic mechanisms 2 1.3 Histone post-translational modifications and the Histone Code hypothesis 4 1.4 Histone methylation 8 1.5 Methyl-reader modules 10 1.6 Royal family 12 1.6.1 Identification 12 1.6.2 Architecture 13 1.6.3 Biological function 16 1.7 Rationale and Intensions of the Thesis 18 1.8 References 19 Chapter 2: A SPOT on the chromatin landscape? Histone peptide arrays as a tool for epigenetic research 24 2.1 Summary 25 2.2 Introduction 25 2.3 Results 28 2.3.1 SPOT blotting 28 2.3.2 Application 1: Characterization of reagents for chromatin research 30 2.3.3 Application 2: Mapping the specificity of histone binding domains 32 2.3.4 Application 3: Demonstration of non-sequence specific interactions 36 2.4 Discussion 38 2.5 Materials and Methods 40 2.6 References 50 Chapter 3: Histone Substrate Recognition by Human MBT Domains 53 3.1 Summary 54 3.2 Introduction 54 3.3 Results 58 3.3.1 MBT domains recognize a variety of methylated lysines on core histones 58 3.3.2 Recognition mechanism for mono- and dimethyllysine 60 3.3.3 Lack of sequence specific binding by L3MBTL1 and L3MBTL3 67 3.3.4 Potential recognition of non-histone proteins 70 3.3.5 Recognition of specific histone sequences 70 3.4 Discussion 79 3.5 Materials and Methods 83 iv 3.6 References 89 Chapter 4: Recognition of multivalent histone states associated with heterochromatin by Tandem Tudor Domains of UHRF1 93 4.1 Summary 94 4.2 Introduction 94 4.3 Results 97 4.3.1 UHRF1 contains a Tandem Tudor Domain that binds H3K9me3 in vitro 97 4.3.2 TTD recognizes hallmarks of heterochromatin 100 4.3.3 Structural basis for the recognition of the H3K4me0/K9me3 signature 103 4.3.4 Reorientation of TTD subdomains upon histone H3 binding 107 4.3.5 Mutational analysis confirms localization of TTD to heterochromatin 110 4.4 Discussion 113 4.5 Materials and Methods 115 4.6 References 125 Chapter 5: Conclusions and Future Directions 130 5.1 Conclusions 131 5.2 Future Directions 132 5.3 Concluding remarks 137 5.4 References 138 v List of Tables Chapter 1 Table 1.1. Examples of the protein domains, the histone marks they recognize and their biological effect. 11 Chapter 2 Table 2.1. List of peptides with known modifications used in SPOT-blot synthesis. 42 Table 2.2. List of peptides used in SPOT-blot synthesis. 44 Chapter 3 Table 3.1. Overview of the highest affinity histone substrates for MBT domains within human proteins. 61 Table 3.2. Thermal stability of the wild type and mutant proteins as indicated by aggregation temperatures. 65 Table 3.3. Overview of the ability of MBT mutants to bind lower lysine methylation states. 66 Table 3.4. Crystallographic data collection and refinement statistics. 75 Chapter 4 Table 4.1. NMR data and refinement statistics. 105 Table 4.2. Intra- and intermolecular distance restraints used for TTD-H3K4me0/K9me3 calculations. 121 iv List of Figures Chapter 1 Figure 1.1. Sites of histone post-translational modifications (PTMs). 5 Figure 1.2. Model representation of lysine and arginine methylation. 9 Figure 1.3. Recognition of methyllysine by the Royal family protein domains. 14 Chapter 2 Figure 2.1. Schematic representation of the SPOT binding assay. 29 Figure 2.2. Confirmation of complete synthesis of modified histone peptides. 31 Figure 2.3. Characterization of reagents for chromatin research. 33 Figure 2.4. Mapping the specificity of histone-binding domains. 35 Figure 2.5. Demonstration of non-sequence-specific interactions. 37 Chapter 3 Figure 3.1. Domain architecture and analysis of the MBT-containing proteins. 56 Figure 3.2. Interactions of the MBT domains with histones in SPOT arrays. 59 Figure 3.3. Influence of the MBT pocket architecture on the recognition of the lysine methylation state. 63 Figure 3.4. L3MBTL1 and L3MBTL3 are promiscuous binders. 69 Figure 3.5. Poor recognition of histone substrates by MBTs. 71 Figure 3.6. Recognition of specific histone sequences. 72 Figure 3.7. Structural and functional analysis of the SCML2 binding. 74 Figure 3.8. Binding of SCML2 to nucleosomes. 77 Figure 3.9. SCML2 binds to DNA with its basic region. 78 Figure 3.10. SCML2 interacts specifically with the H2AK36me1-containing nucleosomes. 80 Chapter 4 Figure 4.1. A novel evolutionary conserved domain within UHRF1 corresponds to TTD. 96 Figure 4.2. A novel TTD domain within UHRF1 binds H3 histone tail with H3K9me3. 98 Figure 4.3. UHRF1 recognizes multivalent histone signatures associated with heterochromatin. 101 Figure 4.4. Minimal interactions between TTD and the short H3K9me3-containing peptide in solution. 103 Figure 4.5. Recognition of multivalent sites at the interface between the two Tudor subdomains. 106 Figure 4.6. Tandem tudor domain behaves as a single rigid body. 108 Figure 4.7. Structural re-adjustment of TTDC in order to accommodate the histone tail. 109 Figure 4.8. Residual dipolar couplings (RDCs) collected on the TTD/H3K4me0K9me3 complex fit poorly the apo crystal structure. 111 Figure 4.9. Mutational analysis confirms localization of TTD to heterochromatin in mouse ES cells.
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