Expression of EZH1-Polycomb Repressive Complex 2 and MALAT1 lncRNA and their Combined Role in Epigenetic Adaptive Response Thesis by Lamya Al Fuhaid In Partial Fulfillment of the Requirements For the Degree of Master of Science King Abdullah University of Science and Technology Thuwal, Kingdom of Saudi Arabia July, 2019 2 EXAMINATION COMMITTEE PAGE The thesis of Lamya Al Fuhaid is approved by the examination committee. Committee Chairperson: Prof. Valerio Orlando Committee Members: Prof. Stefan Arold, Prof. Salim Al Babili 3 COPYRIGHT © July, 2019 Lamya Al Fuhaid All Rights Reserved 4 ABSTRACT Expression of EZH1-Polycomb Repressive Complex 2 and MALAT1 lncRNA and their Combined Role in Epigenetic Adaptive Response Lamya Al Fuhaid Living cells maintain stable transcriptional programs while exhibiting plasticity that allows them to respond to environmental stimuli. The Polycomb repressive complex 2 (PRC2) is a key regulator of chromatin structure that maintains gene silencing through the methylation of histone H3 on lysine 27 (H3K27me), establishing chromatin-based memory. Two variants of PRC2 are present in mammalian cells, PRC2-EZH2 which is predominantly present in differentiating cells, and PRC2-EZH1 that predominates in post-mitotic tissues. PRC2-EZH1α/β pathway is involved in the response of muscle cells to oxidative stress. Atrophied muscle cells respond to oxidative stress by enabling the nuclear translocation of EED and its assembly with EZH1α and SUZ12. Here we prove that the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) long noncoding RNA (lncRNA) is required for the assembly of PRC2-EZH1 components. The absence of MALAT1 significantly decreased the association between EED and EZH1α proteins. Biochemical analysis shows that the presence of MALAT1 increases the enzymatic activity of PRC2-EZH1 in vitro. In addition, we show that the simultaneous expression of PRC2 core components is necessary for their solubility. The successful expression of PRC2 proteins enables the execution of several downstream experiments, which will further explain the nature of the interplay between MALAT1 and PRC2. 5 ACKNOWLEDGEMENTS I would like to thank my supervisor, Prof. Valerio Orlando for providing me with the opportunity to explore the field of epigenetics. I would also like to show great gratitude to Dr. Peng Liu, who was generous with his time and has put immense effort into training and assisting me. I extend my thanks to my committee members, Prof. Stefan Arold and Prof. Salim Al Babili, for their time and valuable input. Acknowledgment goes to my labmate, Dr. Nadine El Said, for her insightful data, and Dr. Muhammad Tehseen from the Laboratory of DNA Replication and Recombination, for providing SF9 insect cells and pFastBac vector. Most importantly, for his guidance and patience in answering all of my questions. Special thanks to my dear friend Mohammed Alomari, for his help in the in vitro transcription. Thank you for the support, the company, and for lending me autoclaved flasks when I was too lazy to autoclave my own. My best friend, Arwa Alghuneim, you have been with me through it all, and without you, it would have been dull. Thank you for your interest in my work and for helping me whenever I asked, or did not. Last but not least, my mom, from whom I got the love of knowledge and the pursuit of perfection, epigenetics I guess, I love you. 6 TABLE OF CONTENTS EXAMINATION COMMITTEE PAGE _________________________________ 2 COPYRIGHT _______________________________________________________ 3 ABSTRACT _________________________________________________________ 4 ACKNOWLEDGEMENTS ____________________________________________ 5 TABLE OF CONTENTS ______________________________________________ 6 LIST OF ABBREVIATIONS __________________________________________ 9 LIST OF SYMBOLS ________________________________________________ 12 LIST OF ILLUSTRATIONS __________________________________________ 13 LIST OF TABLES __________________________________________________ 17 CHAPTER 1: INTRODUCTION ______________________________________ 18 1.1. Polycomb Repressive Complex ____________________________________ 20 1.1.1. Classes of Polycomb repressive complex _________________________ 20 1.1.2. PRC2 Components __________________________________________ 21 1.1.2.1. EZH protein _____________________________________________ 21 1.1.2.2. EED protein _____________________________________________ 21 1.1.2.3. SUZ12 protein ____________________________________________ 22 1.1.2.4. RbAp46 and RbAp48 proteins _______________________________ 23 1.1.2.5. AEBP2 and JARID2 proteins ________________________________ 23 1.2. PRC2 in cell development and plasticity _____________________________ 23 1.2.1. PRC2 in cellular memory _____________________________________ 23 1.2.2. The interchange between PRC2-EZH2 and PRC2-EZH1 ____________ 24 1.2.3. PRC2 and adaptive response in postmitotic cells ___________________ 26 1.3. Interplay between PRC2 and MALAT1: _____________________________ 27 1.3.1. Non-coding RNA: ___________________________________________ 27 1.3.2. lncRNA and PRC2 function: __________________________________ 28 1.3.3. Interaction between MALAT1 and PRC2 ________________________ 30 1.3.3.1. MALAT1 and PRC2-EZH2 _________________________________ 30 7 1.3.4. Investigation of the involvement of MALAT1 in PRC2-EZH1 mediated plasticity 31 1.4. Scope of the thesis: Objectives, contributions, and model system _________ 34 CHAPTER 2: MATERIALS AND METHODS __________________________ 35 2.1. Mammalian cell culture __________________________________________ 35 2.2. Stable cell lines ________________________________________________ 35 2.3. Immunofluorescence ____________________________________________ 35 2.4. Cell treatments _________________________________________________ 36 2.5. RNA isolation and RT-qPCR ______________________________________ 36 2.6. Co-immunoprecipitation _________________________________________ 37 2.7. LC-MS analysis and MS data analysis ______________________________ 37 2.8. ChIP-seq ______________________________________________________ 39 2.9. Expression plasmids _____________________________________________ 39 2.10. RNA in vitro transcription ________________________________________ 40 2.11. Protein expression and purification in bacteria ________________________ 40 2.12. Protein expression in insect cells ___________________________________ 41 2.13. Western blotting ________________________________________________ 42 2.14. Histone methyltransferase activity __________________________________ 42 2.15. Statistical analysis ______________________________________________ 43 CHAPTER 3: RESULTS _____________________________________________ 44 3.1. generation of stable C2C12 cell line expressing tagged EZH1α __________ 44 3.2. Identification of suitable antisense oligos for MALAT1 loss of function experiments _________________________________________________________ 46 3.3. MALAT1 is required for the proper association of PRC2 core components in the nucleus _________________________________________________________ 47 8 3.4. MALAT1 is necessary for the binding of EZH1α and EED to muscle-specific promoters under oxidative stress ________________________________________ 50 3.5. Expression of PRC2 core components in E. coli _______________________ 51 3.6. PRC2 proteins are insoluble in E. coli except EED _____________________ 54 3.7. Expression of PRC2 core components in insect cells ___________________ 57 3.8. Simultaneous expression of the three core components of PRC2-EZH1 is necessary for their solubility ____________________________________________ 59 3.9. In vitro transcription of MALAT1 __________________________________ 60 3.10. MALAT1 positively affects the enzymatic activity of PRC2-EZH1 in vitro _ 61 CHAPTER 4: DISCUSSION __________________________________________ 63 CHAPTER 5: CONCLUSION _________________________________________ 68 APPENDIX A: VECTOR MAPS ______________________________________ 69 1. pOZ-FH-C-Puro vector ____________________________________________ 69 2. pJET1.2 vector ___________________________________________________ 70 3. pGEX-5X-1 vector _______________________________________________ 71 4. pFastBac HT C vector _____________________________________________ 72 APPENDIX B: PRIMER SEQUENCES ________________________________ 73 1. qPCR primers ___________________________________________________ 73 2. ChIP ___________________________________________________________ 73 3. Cloning primers __________________________________________________ 74 4. Sequencing primers: ______________________________________________ 74 APPENDIX C: ANTIBODIES _________________________________________ 76 1. Western blotting _________________________________________________ 76 REFERENCES _____________________________________________________ 77 9 LIST OF ABBREVIATIONS 3D Three dimensional ASO Antisense oligos ChIP Chromatin immunoprecipitation ChIP-qPCR Chromatin immunoprecipitation followed by real-time polymerase chain reaction CRPC Castration-resistant prostate cancer Cryo-EM Cryogenic electron microscopy DMEM Dulbecco's modified Eagle's medium DNA Deoxyribonucleic acid dSfmbt Scm-related gene containing four malignant brain tumor domains EED Embryonic ectoderm development ELISA Enzyme-linked immunosorbent assay EMSA Electrophoretic mobility shift assay EMT Epithelial to mesenchymal transmission ERK Extracellular signal-regulated kinase EZH1 Enhancer of zeste homolog 1 EZH1α-FH EZH1α fused with FLAg and HA tags EZH2 Enhancer of