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Functional Analysis of Trf1 Phosporylation in Telomere FUNCTIONAL ANALYSIS OF TRF1 PHOSPORYLATION IN TELOMERE MAINTENANCE FUNCTIONAL ANALYSIS OF THE ROLE OF TRF1 PHOSPHORYLATION ON THREONINE 271 AND THREONINE 371 IN TELOMERE MAINTENANCE By ANGUS HO, B.Sc (Hon) A Thesis Submitted to the School of Graduate Studies in Partial Fulfillment of the Requirements for the Degree Master of Science McMaster University © Copyright by Angus Ho, September 2016 McMaster University MASTER OF SCIENCE (2016) Hamilton, Ontario (Biology) TITLE: Functional Analysis of the Role of TRF1 Phosphorylation on threonine 271 and threonine 371 in Telomere Maintenance AUTHOR: Angus Ho, B.Sc (Hon) SUPERVISOR: Dr. Xu-Dong Zhu NUMBER OF PAGES: cxcvii; 197 (excluding publication) ii ABSTRACT TRF1, telomeric-repeat binding factor 1, is a component of the six-subunit protein complex, referred to as shelterin, which is essential for not only regulating telomere length maintenance but also protecting mammalian telomeres from being recognized as damaged DNA. TRF1 acts as a negative mediator of telomerase-dependent telomere elongation in telomerase-expressing cells, whereas it promotes alternative lengthening of telomeres (ALT) activity by regulating ALT features including the production of extrachromosomal telomere-repeat (ECTR) DNA such as C-circles, and ALT-associated promyelocytic leukemia bodies, or APBs. The activity of TRF1 is tightly regulated by post-translational modification such as phosphorylation. This thesis sets out to investigate the function of TRF1 phosphorylation on threonine-271 (T271) and threonine-371 in telomere maintenance. The results presented in this thesis demonstrate that TRF1 phosphorylation on T271 positively regulates the association of TRF1 to telomeric DNA in telomerase- expressing cells. In ALT cells, TRF1 phosphorylation on both T271 and T371 is shown to be important for the formation of APBs. Furthermore, the work presented here suggests that transcription-associated DNA damage mediates the association of phosphorylated (pT371)TRF1 with APBs. iii ACKNOWLEDGEMENTS I entered the Zhu lab in September of 2013 as an undergraduate thesis student and have since then been motivated to strive to become an effective researcher. Dr. Xu-Dong Zhu has been a very effective and motivating supervisor throughout my undergraduate and graduate studies. Her critical evaluations and feedbacks have been tough but, at the same time, very rewarding. Without her constant support throughout my graduate work, I would not have been able to succeed in my project. To this end, I am grateful and appreciative of her guidance in my growth as a scientist. I would also like to extend my gratitude to Dr. Jon Draper and Dr. Bhagwati Gupta for their assistance as my supervisory committee members, both of whom have provided with great advice and criticisms in helping me to succeed and grow as a scientist. Moreover, members of the Zhu lab have also been an integral part of my graduate life at McMaster University. I would like to give a HUGE thank you to Nicole L. Batenburg, who has been a constant support in helping me out with all kinds of lab techniques. She was a great mentor and a great friend. I would like to give special thanks to past member, Florence R. Wilson, who has also mentored me when I first entered the lab. Who would have known we ended up becoming great friends and co-authors on my first publication! I would also like to thank past member, Kyle. R. Lindsay, who has also provided me with constant moral support when things aren’t going well. Whether it is in the lab, coffee breaks in the hallway, or at the pub, memories with these unique individuals will be cherished. I wish them all the best and success in their current and future endeavors. iv Finally, there are two very important individuals whom I must thank: Bosco and Cherly, my parents, for their unconditional support and love throughout my life. v TABLE OF CONTENTS ABSTRACT ...................................................................................................................... iii ACKNOWLEDGEMENT ................................................................................................ iv TABLE OF CONTENTS ................................................................................................. vi LIST OF FIGURES .......................................................................................................... xi LIST OF ABBREVIATIONS ........................................................................................ xiii CHAPTER 1 INTRODUCTION.............................................................................................................. 1 1.1 Telomeres and the Maintenance of Telomere Length .................................................... 1 1.1.1 Telomere structure and the end-replication problem in human cells ..................1 1.1.2 The shelterin complex and telomeres ..................................................................4 1.2 The Role of Shelterin subunits in Telomere Length Maintenance ................................ 8 1.2.1 Telomere Repeat Binding Factor 1 (TRF1).........................................................8 1.2.2 The role of TRF1 in telomere length maintenance ............................................11 1.2.3 Telomere Repeat Binding Factor 2 (TRF2).......................................................12 1.2.4 The role of TRF2 in telomere length maintenance ............................................13 1.2.5 TERF1-interacting nuclear factor 2 (TIN2) ......................................................14 1.2.6 The role of TIN2 in telomere length maintenance ............................................15 1.2.7 Telomeric repeat-binding factor 2-interacting protein 1 (Rap1) .......................16 1.2.8 The role of Rap1 in telomere length maintenance.............................................17 1.2.9 Protection of Telomeres 1 (POT1) and TINT1/PTOP1/PIP1 (TPP1) ...............18 1.2.10 The role of POT1 and TPP1 in telomere length maintenance .........................20 1.3 Telomeres and the DNA Damage Response ................................................................... 24 1.3.1 Telomeres and the DNA Damage Response .....................................................24 1.3.2 The DNA double-strand break repair pathways ................................................28 vi 1.4 Alternative Lengthening of Telomeres (ALT) ............................................................... 30 1.4.1 Telomere maintenance in telomerase-negative cells .........................................30 1.4.2 Molecular hallmarks of ALT Cells....................................................................34 1.4.3 Chromatin landscape of telomeres is important for ALT activity .....................41 1.4.4 Dysfunctional chromatin promotes ALT activity..............................................43 1.5 TRF1 Functions and Post-translational Modifications ................................................ 45 1.5.1 The role of TRF1 in genome integrity ...............................................................45 1.5.2 Phosphorylation of TRF1 ..................................................................................46 1.5.3 The role of TRF1 in ALT activity .....................................................................51 1.6 OBJECTIVES AND SIGNIFICANCE ........................................................................... 52 CHAPTER 2 MATERIALS AND METHODS ........................................................................................... 55 2.1 Plasmids and Antibodies ......................................................................................55 2.2 Cell Culture and Stable Cell Lines Generation ....................................................56 2.3 Inhibitor Treatments .............................................................................................57 2.4 Cycloheximide Chase Analysis of TRF1 protein stability ...................................58 2.5 Cell synchronization of ALT cells .......................................................................58 2.6 Protein Extracts and Differential Salt Extraction of Chromatin ...........................58 2.7 Western Blotting (Immunoblotting) .....................................................................60 2.8 Immunofluorescence (IF) microscopy .................................................................61 2.9 Quantifying cells positive for APBs using IF microscopy ...................................61 2.10 Genomic DNA Isolation and Digestion .............................................................62 2.11 Ethanol precipitation of DNA ............................................................................63 2.12 C-circle Amplification Assay .............................................................................64 vii 2.13 Northern analysis of TERRA .............................................................................64 2.14 Chromatin Immunoprecipitation (ChIP) ............................................................66 2.15 Radioactive probe preparation for detecting TERRA or telomeric DNA sequences ....................................................................................................................70 2.16 Radioactive probe preparation for detecting C-circles .......................................71 2.17 Radioactive probe preparation for GAPDH and Alu sequences ........................71 2.18 Image analysis of IF staining of anti-(pT271)TRF1 antibody............................72 2.19 Statistical Analysis .............................................................................................73
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