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Profiling Dynamic Chromatome Reveals HP1BP3 As a Key Regulator of Cell Cycle and Hypoxia Cancer Progression

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Profiling Dynamic Chromatome Reveals HP1BP3 As a Key Regulator of Cell Cycle and Hypoxia Cancer Progression This document is downloaded from DR‑NTU (https://dr.ntu.edu.sg) Nanyang Technological University, Singapore. Profiling dynamic chromatome reveals HP1BP3 as a key regulator of cell cycle and hypoxia cancer progression Bamaprasad Dutta 2014 Bamaprasad Dutta. (2014). Profiling dynamic chromatome reveals HP1BP3 as a key regulator of cell cycle and hypoxia cancer progression. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/59912 https://doi.org/10.32657/10356/59912 Downloaded on 06 Oct 2021 14:05:24 SGT PROFILING DYNAMIC CHROMATOME REVEALS HP1BP3 AS A KEY REGULATOR OF CELL CYCLE AND HYPOXIA CANCER PROGRESSION BAMAPRASAD DUTTA SCHOOL OF BIOLOGICAL SCIENCES 2014 Profiling dynamic chromatome reveals HP1BP3 as a key regulator of cell cycle and hypoxia cancer progression Bamaprasad Dutta School of Biological Sciences A thesis submitted to the Nanyang Technological University in partial fulfillment of the requirement of the degree of Doctor of Philosophy 2014 Acknowledgements Acknowledgements I would like to express my sincere gratitude to my supervisor Dr. Siu Kwan Sze, for his expert guidance and warm encouragement. He shared his wealth of experience on numerous occasions and this has provided me with a great learning experience. Special thanks go to Dr. Ren Yan for her invaluable discussions and suggestions, which helped me a lot in this project and also for helping me to learn the basic experiments. I am indebted to Dr. Sunil Shankar Adav for sharing his experience and helping. I would also like to acknowledge the kind support of my current and former laboratory collogues, especially Dr. Park Jung Eun, Tiannan Gao, Li Xin, Arnab Datta, Hao Piliang, Sim Kae Hwan, and Cheow Sok Hwee Esther for extending their helping hand anytime I needed. I would also like to express my sincere gratitude to my friends Sandip, Barindra, Bhabwana, Manisha and Phat for their kind help and support and also for their encouragement during long period of my PhD study in Singapore. I would also like to convey deep appreciation for my thesis advisory committee, Dr. Li Hoi Yeung and Dr. Su I Hsin for their excellent advice and constructive criticism during the annual committee meetings and conformation of my PhD candidature. I would also like to express my sincere thanks to our collaborator Dr. Koh Cheng Gee and her lab member for their help in fluorescent microscopy studies I gratefully acknowledge the financial support rendered by the Nanyang Technological University of Singapore in the form of Research Scholarship. I am also grateful to the academic and technical staffs at the School of Biological Sciences, who have helped me in one way or the other in my research work. Last but not least, my heartedly gratitude goes to my parents for selfless support, love and understanding during and before my PhD study in Singapore. I thank all my family members who are always proud of my academic achievements and have taken all the troubles to inspire, encourage and support me to reach this stage. i Contents Contents Acknowledgements i Contents iii List of figures vi List of tables viii Abbreviations ix Abstract xii Chapter 1 1 1 General introduction 2 1.1 Introduction 2 1.1.1 Chromatin 2 1.1.1.1 Chromatin structure and function 2 1.1.1.2 Chromatin structural regulation 3 1.1.2 Chromatin proteomics 4 1.1.2.1 Chromatin protein and its importance 4 1.1.2.2 Nuclease digestion and chromatin protein extraction 5 1.1.3 Mass spectrometry-based quantitative proteomics 6 1.1.4 Cell cycle progression and chromatin proteomics 9 1.1.4.1 Cell cycle clock 9 1.1.4.2 Cell cycle regulation 10 1.1.4.3 Cell cycle checkpoints 11 1.1.4.4 Cell cycle and cell proliferation 12 1.1.5 Chromatin proteomics and hypoxia induced cancer progression 13 1.1.5.1 Tumor hypoxia and cancer progression 13 1.1.5.2 Hypoxia and radio and chemo-resistance 15 1.1.5.3 Hypoxia and cell migration and invasion 15 1.1.5.4 Hypoxia and cancer steam cell 16 1.1.6 Objectives and the overview of the thesis 18 Chapter 2 20 2 Developing quantitative proteomics approach to elucidate the chromatin protein binding 20 topology in the chromatome 2.1 Summary 21 2.2 Introduction 21 2.3 Materials and methods 24 2.3.1 Reagents 24 2.3.2 Experimental design 24 2.3.3 Chromatin isolation from rat liver and nuclease treatment 24 2.3.4 One dimensional gel electrophoresis and in-gel digestion 26 2.3.5 LC-MS/MS analysis 27 2.3.6 Database searching 28 2.3.7 Data analysis 28 ii Contents 2.3.8 HEPG2 Cell culture 29 2.3.9 Immunofluorescence analysis 29 2.4 Results and discussion 30 2.4.1 LC-MS/MS based chromatin associated protein profile 30 2.4.2 Temporal release profiles of chromatin associated proteins 34 2.4.3 Temporal release dynamics of histones upon nuclease digestion 34 2.4.4 Temporal release dynamics of non-histones proteins 37 2.4.5 Novel chromatin associated proteins and immunofluorescence analysis 49 2.5 Conclusion 51 Chapter 3 52 3 Proteomic and functional studies of chromatome dynamic in cell cycle progression 52 3.1 Summary 53 3.2 Introduction 53 3.3 Materials and methods 56 3.3.1 Reagents 56 3.3.2 Experimental methodology 56 3.3.2.1 293F cell culture and Cell cycle synchronization 56 3.3.2.2 Propidium iodide staining and flow cytrometry analysis 57 3.3.2.3 Chromatin isolation and digestion 57 3.3.2.4 In-gel Digestion 58 3.3.2.5 iTRAQ Labeling 59 3.3.2.6 Electrostatic Repulsion- Hydrophilic Interaction Chromatography (ERLIC) 59 3.3.2.7 LC-MS/MS Analysis 60 3.3.2.8 Mass Spectrometric Data Analysis 60 3.3.2.9 Western blot analysis 61 3.3.2.10 Preparation HP1BP3 knockdown cell phenotypes 61 3.3.2.11 Chromatin compaction measurement 62 3.3.2.12 Quantitative proteomic profiling of HP1BP3 depleted cells 62 3.3.2.13 Cell proliferation assay 63 3.3.2.14 Cell cycle phase duration measurement 63 3.3.2.15 Clonogenic assay 64 3.3.2.16 Immunofluorescence analysis 64 3.4 Result and discussion 65 3.4.1 Mass spectrometric identification and quantification of the chromatin proteome 65 3.4.2 Interphase progression and DNA repair 68 3.4.3 Chromatin organization and transcription regulations during interphase phase progression 71 3.4.4 HP1BP3 novel regulator of heterochromatin structure 77 3.4.5 Effect of HP1BP3 depletion upon protein expression 79 3.4.6 HP1BP3 novel regulator of cell proliferation 79 3.4.7 HP1BP3 novel regulator of nuclear size 80 3.5 Conclusions 82 Chapter 4 84 iii Contents 4 Studies of hypoxia induced cancer cell chromatin changes to identify key regulator for cancer 84 malignant progression 4.1 Summary 85 4.2 Introduction 85 4.3 Materials and methods 87 4.3.1 Reagents 87 4.3.2 Experimental methodology 87 4.3.2.1 A431 cell culture and hypoxia model 87 4.3.2.2 Chromatin isolation and digestion 88 4.3.2.3 In-gel Digestion and iTRAQ Labeling 89 4.3.2.6 Fractionation and LC-MS/MS analysis of iTRAQ labeled peptide 89 4.3.2.4 Mass Spectrometric Data Analysis and bioinformatics 89 4.3.2.5 Western blot analysis 89 4.3.2.6 Propidium iodide staining and flow cytrometry analysis 90 4.3.2.7 Preparation HP1BP3 knockdown cell phenotypes 90 4.3.2.8 Chromatin compaction measurement 90 4.3.2.9 Cell proliferation assay and clonogenic assay 90 4.3.2.10 Cell adhesion assay 90 4.3.2.11 Scratch-wound assay 91 4.3.2.12 Trans-well assay 91 4.3.2.13 Radiation-resistance and Chemo-resistance assay 92 4.3.2.14 Sphere formation assay 92 4.4 Result and Discussion 93 4.4.1 Mass spectrometric identification and quantification of the chromatin proteome 93 4.4.2 Chromatin organization and transcription regulation during hypoxia 96 4.4.3 HP1BP3 is a novel regulator of tumor biology during hypoxia 102 4.4.3.1 HP1BP3 and its functional role in tumor growth 104 4.4.3.2 Regulatory role of HP1BP3 in ECM adhesion and cell migration 106 4.4.3.3 Hypoxia and HP1BP1 in radio- and chemo-resistance 108 4.4.3.4 Effect hypoxia and HP1BP3 in cancer stem cell self-renewal of A431 cells 110 4.4.4 Conclusion 112 Chapter 5 114 5 Conclusion and future direction 114 5.1 Concluding remarks and future perspective 115 References 119 Appendix A 149 Appendix B 150 iv List of figures and tables List of figures Chapter 1 Figure 1.1: Levels of chromatin organization 2 Figure 1.2: Nuclease digestion of chromatin 5 Figure 1.3: Commonly used quantitative proteomic methods 6 Figure 1.4: Schematic representation of the isobaric tagging chemistry of iTRAQ 7 Figure 1.5: Schematic representation of the work flow of our iTRAQ based proteomic experiment 8 Figure 1.6: Schematic diagram of cell cycle 9 Figure 1.7: Different levels of cyclins and cyclin dependent kinase complexes at different stages of cell cycle. 10 Figure 1.8: Schematic diagram of cell cycle checkpoints. 11 Figure 1.9: Simplified schematic model of Rb pathway mediated regulatory control of G1 to S phase progression 13 Figure 1.10: Schematic diagram of hypoxia induced cancer progression 14 Figure 1.11: Schematic diagram represent the HIFs regulated mechanism of CSCs transformation.
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