IDENTIFICATION OF TELOMERE LENGTH REGULATORS BY POOLED LIBRARY SCREENING by Steven Wang A dissertation submitted to The Johns Hopkins University in conformity with the requirements of the degree of Doctor of Philosophy Baltimore, Maryland January 2017 © 2017 Steven Wang All Rights Reserved Abstract Telomeres protect chromosome ends from damage (d'Adda di Fagagna et al., 2003). In the presence of telomerase, telomere length is maintained at an established equilibrium (Greider, 1996). Alterations to this equilibrium cause short telomeres, which manifest as degenerative disease, or long telomeres, which can facilitate initiation and growth of cancer (Stanley and Armanios, 2015). Altering the activity of telomere length regulators has therapeutic application in short and long telomere syndromes (Stanley and Armanios, 2015). We tested whether elongation of short telomeres, in bone marrow stem and progenitor cells, was sufficient to enhance engraftment. We also identified and characterized new regulators of telomere length using a pooled screening approach. We transplanted short telomere or wildtype mouse hematopoietic stem and progenitor cells, with telomerase or GFP lentivirus, into lethally irradiated hosts. Short telomere mice, transduced with telomerase, had greater survival and engraftment, compared to short telomere mice transduced with GFP. We found that this effect was not due to changes in mean telomere length, but a reduction in critically short telomeres. This suggests that elongation of the shortest telomeres is sufficient to ameliorate degenerative phenotypes. We also developed a pooled genetic screen to identify new telomere length regulators. We transduced cells with an shRNA or sgRNA library against human kinases, then isolated short telomere cells by fluorescently labeling telomeres and cell sorting. Enrichment of shRNA or sgRNA inserts in the short telomere fraction were determined using bowtie2 (Langmead and Salzberg, 2012) and MAGeCK analysis (Li et al., 2014). ii We identified BRD4 as a strong positive regulator of telomere length. We also identified CK1, and the MEK/ERK pathway as more moderate positive regulators of length. Inhibition of BRD4, with 4 different BRD4 inhibitors, was sufficient to completely block telomere elongation by telomerase over expression, in a drug dependent manner. BRD4 has pleiotropic roles and is currently being investigated in cancer and inflammatory disease (Devaiah et al., 2016b). We believe shortening telomeres, is one mechanism by which BRD4 inhibition slows cancer proliferation. Telomere length should also be considered when using BRD4 inhibitors for inflammatory disease, particularly in the context of short telomere syndromes. Thesis Advisor: Carol W. Greider Ph.D. Thesis Reader: Heng Zhu Ph.D. iii Acknowledgements I feel very fortunate to have been a member of the Greider lab, and am thankful to many people for making my time here an enriching experience. First, I’d like to thank Carol for her mentorship and support. Carol’s passion for science has created a lab environment where it is a real joy to discuss science, and I’ve benefitted tremendously from it. I would also like to thank her for her support of my career and professional development. She is truly someone who recognizes the importance of education, as much as research. I’d also like to thank Carla and Margaret for all their help. Many people say they couldn’t have done it without them, but this is probably literally true in my case. Thank you Carla for entertaining all my late night conversations, and Margaret for always bringing a positive perspective when things go wrong. I’ve had the privilege of being surrounded by many talented and fun graduate students. Thanks to Stella for being a fantastic friend/mentor/life consultant since day one of my rotation. I’d also like to thank Alex for always being down for an excursion for free beer or food. I am indebted to many alumni, including Jon Alder, Sofia Rehermann and Chris Viggiani, who welcomed me to the lab and from whom I learned a lot. I am grateful to my committee, Heng, Steve, DJ and Jef for helpful discussion and feedback throughout the years. I’d also like to thank Hao Zhang at the Public Health Cell Sorting Facility and John Weger at UC Riverside for many long and helpful discussions about cell sorting and sequencing. Lastly, I’d like to thank all my friends and family from Toronto, college and Baltimore. Thanks for all your support through the years and always making me laugh. iv Table of Contents Abstract ii Acknowledgements iv Table of Contents v List of Tables ix List of Figures x Chapter 1 Introduction 1 1.1 Telomeres and telomerase 1 1.2 Telomere length homeostasis 2 1.3 Telomere disease 3 1.4 Role of kinases in telomere length regulation 4 1.5 Measuring telomere length 5 1.6 Screens for telomere length regulators 6 Chapter 2 Restoration of telomerase in mTR-/- mice enhances hematopoietic stem and progenitor cell engraftment 9 2.1 Introduction 9 v 2.2 Results and Discussion 11 2.2.1 Bone marrow transplant to test the effect of lentiviral telomerase restoration on hematopoietic stem and progenitor cell engraftment 12 2.2.2 Effect of telomerase restoration on hematopoietic stem and progenitor cell engraftment 13 2.2.3 Effect of telomerase restoration on telomere length 15 2.2.4 Generation of novel lentiviral constructs to restore telomerase in vivo 18 2.3 Materials and Methods 20 2.3.1 Transgenic Mice 20 2.3.2 Lentivirus production and transduction 21 2.3.3 Bone marrow harvest and collecting lineage depleted cells 22 2.3.4 Bone Marrow Transplant 23 2.3.5 Peripheral blood collection and flow cytometry 24 2.3.6 Cell Sorting 25 2.3.7 Quantitative Real-Time PCR 25 2.3.8 Telomere flow-FISH 26 2.3.9 Telomere Quantitative FISH 27 2.3.10 Telomerase Repeat Amplification Protocol 28 2.3.11 Telomere Restriction Fragment Southern Blot 30 vi Chapter 3 Identifying novel kinase regulators of telomere length by pooled screening approach 48 3.1 Introduction 48 3.2 Results and Discussion 49 3.2.1 Identifying cell line for pooled telomere length screening 49 3.2.2 Validating shRNA screening approach 52 3.2.3 Pooled shRNA screening 53 3.2.4 Analysis of sequencing results from pooled shRNA screen 55 3.2.5 Pooled CRISPR Screening approach 58 3.2.2 Analysis of sequencing results from pooled CRISPR screen 60 3.2.3 Summary 61 3.3 Materials and Methods 62 3.3.1 shRNA knockdown 62 3.3.2 qPCR Analysis 62 3.3.3 Telomere flow-FISH analysis 62 3.3.4 Pooled shRNA kinase library 63 3.3.5 Pooled CRISPR kinase library 63 3.3.6 Generating constitutive Cas9 expressing cell line 63 3.3.7 Western Blotting 64 3.3.8 Transduction and tissue culture of transduced cells 65 3.3.9 Telomere flow-FISH cell sorting 65 3.3.10 FACS of cells by telomere length 66 vii 3.3.11 Illumina sample preparation and sequencing 67 3.3.12 shRNA screen sequencing analysis 68 3.3.13 CRISPR screen sequencing analysis 68 Chapter 4 Chemical inhibition of BRD4, CK1 and MEK/ERK block telomere elongation 81 4.1 Introduction 81 4.2 Results and Discussion 83 4.2.1 Telomeres are rapidly elongated by telomerase overexpression 83 4.2.2 BRD4 inhibition blocks telomere elongation 85 4.2.3 CK1 inhibition blocks telomere elongation 86 4.2.3 MEK1/2 and ERK1/2 inhibition blocks telomere elongation 87 4.2.3 Summary 89 4.3 Materials and Methods 90 4.4.1 Small molecule inhibitors 90 4.4.2 Virus Production and Titering 91 4.4.3 Inhibition of SVA mediated telomere elongation 92 4.4.4 Telomere Southern Blot 92 References 102 Curriculum Vitae 115 viii List of Tables Table 2.1 Primer list 46 Table 2.2 Mouse mTR and mTERT lentiviral constructs 47 Table 3.1 HeLa shRNA screen top 20 hits 77 Table 3.2 293FT shRNA screen top 20 hits 78 Table 3.3 CRISPR screen top 20 hits 79 Table 3.3 Primer List 80 ix List of Figures Figure 2.1 Experimental design to test effect of mTR expression on hematopoietic stem and progenitor cell engraftment 30 Figure 2.2 Kaplan-Meier survival curve for primary and secondary transplant 32 Figure 2.3 Donor engraftment in primary transplant 34 Figure 2.4 Donor engraftment in secondary transplant 36 Figure 2.5 mTR expression and telomere length in mTR or FUGW transduced WT or G4 cells 38 Figure 2.6 Signal free ends and P/Q ratio of GFP+ sorted splenocytes 40 Figure 2.7 Expression of mTR and mTERT compared to mTR alone 42 Figure 2.8 LVT3b lentiviral telomerase construct elongates telomeres and generates bright GFP 44 Figure 3.1 Selecting cell line to perform pooled telomere length screen 69 Figure 3.2 Knockdown of hTERT or POT1 shortens, or elongates telomeres respectively, in 293FT cells 71 Figure 3.3 Optimizations for fluorescence activated cell sorting and flow-FISH 73 Figure 3.4 POT1 and hTERT shRNA inserts are recovered in the expected fractions upon sorting long and short telomere cells 75 Figure 4.1 Inhibition of BRD4 with small molecules blocks SVA mediated telomere elongation 94 x Figure 4.2 Inhibition of BRD4 by JQ1 blocks telomere elongation in a dose dependent manner 96 Figure 4.3 Inhibition of CK1 by D4476 inhibits telomere elongation in a dose dependent manner 98 Figure 4.4 Chemical inhibition of ERK1/2 or MEK1/2 partially blocks telomere elongation 100 xi CHAPTER 1. INTRODUCTION Chapter 1. Introduction 1.1 Telomeres and telomerase Eukaryotic cells organize their DNA as linear chromosomes.