A Noncanonical Function of the Telomerase RNA Component in Human Embryonic Stem Cells
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Washington University in St. Louis Washington University Open Scholarship Arts & Sciences Electronic Theses and Dissertations Arts & Sciences Winter 12-15-2019 A Noncanonical Function of the Telomerase RNA Component in Human Embryonic Stem Cells Kirsten Ann Brenner Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/art_sci_etds Part of the Cell Biology Commons, Genetics Commons, and the Molecular Biology Commons Recommended Citation Brenner, Kirsten Ann, "A Noncanonical Function of the Telomerase RNA Component in Human Embryonic Stem Cells" (2019). Arts & Sciences Electronic Theses and Dissertations. 1990. https://openscholarship.wustl.edu/art_sci_etds/1990 This Dissertation is brought to you for free and open access by the Arts & Sciences at Washington University Open Scholarship. It has been accepted for inclusion in Arts & Sciences Electronic Theses and Dissertations by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS Division of Biology and Biomedical Sciences Molecular Genetics and Genomics Dissertation Examination Committee: Luis Batista, Chair Sheila Stewart, Co-Chair Sergei Djuranovic Todd Fehniger James Skeath Christopher Sturgeon A Noncanonical Function of the Telomerase RNA Component in Human Embryonic Stem Cells by Kirsten Ann Brenner A dissertation presented to The Graduate School of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy December 2019 St. Louis, Missouri © 2019, Kirsten Ann Brenner Table of Contents List of Figures............................................................................................................................v List of Tables ........................................................................................................................... vi Acknowledgments .................................................................................................................. vii Abstract of the Dissertation ......................................................................................................x Chapter 1: Introduction and Rationale ....................................................................................1 Telomeres ................................................................................................................................2 The Telomerase Canon ............................................................................................................6 Noncanonical Functions of Telomerase ................................................................................. 13 Rationale…. .......................................................................................................................... 18 Chapter 2: TERC suppresses apoptosis in human embryonic stem cells independent of other telomerase components ................................................................................................ 23 Introduction ........................................................................................................................... 24 Methods ................................................................................................................................ 25 Results .................................................................................................................................. 30 Discussion…. ........................................................................................................................ 36 Figures and Tables…. ............................................................................................................ 41 Chapter 3: Investigating the mechanism behind the iTERC_KO phenotype ....................... 56 Introduction ........................................................................................................................... 57 Methods ................................................................................................................................ 59 Results .................................................................................................................................. 62 Discussion…. ........................................................................................................................ 66 Figures and Tables…. ............................................................................................................ 71 ii Chapter 4: Understanding phenotypic differences between conditional and constitutive TERC knock-out human embryonic stem cells...................................................................... 81 Introduction ........................................................................................................................... 82 Methods ................................................................................................................................ 83 Results .................................................................................................................................. 85 Discussion…. ........................................................................................................................ 90 Figures and Tables…. ............................................................................................................ 97 Chapter 5: Conclusions and Future Directions.................................................................... 107 Conclusions ......................................................................................................................... 108 Remaining Questions and Future Directions ........................................................................ 110 Figures and Tables ............................................................................................................... 118 References.............................................................................................................................. 119 iii List of Figures Figure 1.1: Telomere structure…………………………………………………………………...21 Figure 1.2: Telomerase RNP……………………………………………………………………..22 Figure 2.1: Generation of conditional TERC knock-out human embryonic stem cells (iTERC_KO)……………………………………………………………………………………..41 Figure 2.2: DOX-controlled TERC expression in iTERC_KO hESCs………………………….42 Figure 2.3: TERC ablation induces cell death in iTERC_KO hESCs…………………………...43 Figure 2.4: TERC ablation triggers p53-mediated apoptosis…………………………………….44 Figure 2.5: DDR activation in iTERC_KO hESCs……………………………………………....45 Figure 2.6: iTERC_KO cells do not exhibit signs of telomere dysfunction……………………..46 Figure 2.7: Characterization of iTERT_KO hESCs……………………………………………..47 Figure 2.8: iTERT_KO does not recapitulate the iTERC_KO phenotype………………………48 Figure 2.9: Characterization of TERC ΔscaRNA in U2OS cells………………………………..49 Figure 2.10: The scaRNA domain is dispensable in iTERC_KO hESCs………………………..50 Figure 2.11: Low levels of TERC are sufficient to prevent apoptosis in iTERC_KO…………..51 Figure 2.12: TERC ΔscaRNA prevents apoptosis in iTERC_KO without restoring telomere maintenance…………………………………………………………………………………...…52 Figure 2.13: Differentiation of iTERC_KO hESCs into fibroblast-like cells……………………53 Figure 2.14: TERC is not essential in differentiated iTERC_KO cells………………………….54 Figure 3.1: Principal Component Analysis of RNAseq Results…………………………………71 Figure 3.2: The proposed hTERP ORF…………………………………………………………..72 Figure 3.3: hTERP is not required to prevent apoptosis in iTERC_KO hESCs…………………73 Figure 3.4: TERC mutations targeting mitochondrial trafficking and RNA processing………...74 Figure 3.5: TERC nucleotides 1-63 are required to prevent apoptosis in iTERC_KO hESCs…..75 Figure 3.6: TERC53 is sufficient to prevent apoptosis in iTERC_KO hESCs…………………..76 iv Figure 4.1: Generation of constitutive TERC knock-out hESCs (TERC_KO hESCs)………….97 Figure 4.2: TERC_KO hESCs display reduced viability………………………………………...98 Figure 4.3: Transgenic TERC rescues TERC_KO hESCs……………………………………....99 Figure 4.4: Average telomere lengths in TERC_KO hESCs do not appear critically short……100 Figure 4.5: Characterization of telomerase-deficient and telomerase-dead TERC mutations in U2OS cells……………………………………………………………………………………...101 Figure 4.6: Telomerase-deficient TERC_305g>a rescues TERC_KO hESCs………………....102 Figure 4.7: Low telomerase activity is sufficient to prevent telomere dysfunction in hESCs….103 Figure 4.8: Principal Component Analysis of RNAseq results………………………………...104 Figure 4.9: Identified DEGs in different TERC expression models do not coincide…………..105 Figure 5.1: Proposed model of apoptosis suppression by TERC53………...…………………..118 v List of Tables Table 2.1: List of oligonucleotides used for qRT-PCR and CRISPR/Cas9 gene-editing.….…....55 Table 3.1: RNAseq analysis conditions…………………………………………………...……..77 Table 3.2: Differentially expressed gene quantification following TERC or TERT ablation…...78 Table 3.3: KEGG pathway analysis of RNAseq results…………………………………………79 Table 3.4: Summary of iTERC_KO rescue experiments………………………………………...80 Table 4.1: Summary of results from tested telomerase-dead and telomerase-deficient engineered hESC cell lines……………………………………………………………...........…………..…106 vi Acknowledgements This work was supported by the Hematology Training Grant (T32HL007088) and the National Science Foundation Graduate Research Fellowship Program (DGE-1143954). I want to thank the Batista Lab for their support during