UNrVERSrTY OF HAWAr'r LIBRARY EFFECT OF SIRT! AND TELOMERASE ON STEM CELLS A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAW AI'I IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOMEDICAL SCIENCES (CELL AND MOLECULAR BIOLOGy) AUGUST 2008 By Matthew 1. Cousseus Dissertation Committee: Richard Allsopp, Chairperson Marla Berry Scott Lozanoff Pratibha Nerurkar David Duffy We Certify that we have read this dissertation and that, in our opinion, it is satisfactory is scope and quality as a dissertation for the degree of Doctor of Philosophy in Biomedical Sciences (Cell and Molecular Biology). Dissertation Committee: Copyright Information SIRT! AND TELOMERASE AFFECT STEM CELL SURVIVAL AND FUNCTION by Coussens et aI. Copyright 2006 by Society for the Study of Reproduction. Reproduced with permission of Society for Study of Reproduction in the format of Dissertation via Copyright Clearance Center. SIRT! DEFICIENCY A'ITENUATES SPERMATOGENESIS AND GERM CELL FUNCTION by Coussens et aI. Copyright: © 2008 Coussens et aI. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which pennits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. SIRT! ACTS AS A NUfRIENT-SENSITIVE GROWTH SUPPRESSOR AND ITS LOSS IS ASSOCIATED WITH INCREASED AMPK AND TELOMERASE ACTIVITY by NaraJa et aI. Copyright 2008 by Molecular Biology of the Cell. Reproduced with permission of Molecular Biology of the Cell in the format of Dissertation via Copyright Clearance Center. ABSTRACT Two genes that have established roles in the regulation of cell survival are telomerase reverse transcriptase (Tert) and the nicotinamide adenine dinucleotide­ dependent protein deactylase Sirtl. However, the relative importance of these genes to the survival of stem cells, and the regulation of expression of these genes in stem cells, has yet to be thoroughly established. We now show that telomerase suppression in quiescent male primordial germ cells (PGCs) from murine embryos is accompanied by a decrease in expression of TERT. Telomerase activity was detected in quiescent PGCs from transgenic embryos, that constitutively express TERT, demonstrating that re-activation of TERT expression is sufficient to restore telomerase activity in these cells and implying that TERT expression is an important mechanism of telomerase regulation in PGCs. These results also demonstrate that TERT per se does not affect proliferation or development of PGCs in mammals, Sirtl, a member of the sirtuin family of proteins, orthologous to the yeast SirZ, has important physiological roles in regulating glucose metabolism. We have now performed a detailed analysis of the molecular and functional effects of Sirtl deficiency in the germ line of Sirtl knock-out (-1-) mice. We fmd that Sirtl deficiency markedly attenuates spermatogenesis, but not oogenesis. Microarray analysis in Sirtl deficient testis revealed dysregulated expression of 85 genes, which were enriched (p<O.05) for genes involved in spermatogenesis and protein sumoylation. To assess the function of Sirtl deficient germ cells, we compared the efficiency of generating embryos and offspring in in vitro fertilization (IVF) experiments using gametes from Sirtl deficient mice. While viable animals were derived in all experiments, the efficiency of producing both 2-cell zygotes and viable offspring was diminished when IVF was performed with Sirtl-/- gametes. Using short hairpin RNAs, we found that inhibition of Sirtl in immortalized human cells enhanced cell growth under normal and nutrient limiting conditions. Hematopoietic stem cells, which were shown to express Sirtl, showed increased growth capacity and decreased dependency on growth factors when the Sin1 gene was deleted. These data support an important role for Sirtl in spermatogenesis, including spermatogenic stem cells, as well as germ cell function, but also demonstrate that in certain cell lineages, Sirtl can act as a growth suppressor. ii Table of Contents Abstract................................................................................................................................ i List of Tables...................................................................................................................... v List of Figures ................................................................................................................... vi Chapter 1. Introduction...................................................................................................... 1 Chapter 2. Regulation and Effects of Modulation of Telomerase Reverse Transcriptase Expression in Primordial Germ Cells During Development............................................ 19 Abstract. ................................................................................................................ 20 Introduction........................................................................................................... 21 Materials and Methods.......................................................................................... 24 Mice .......................................................................................................... 24 CAG-TERT Transgenic Construct Assembly.......................................... 24 Transgenesis .............................................................................................. 25 Fluorescence-Activated Cell-Sorting (FACS) Analysis and Purification of PGCs......................................................................................................... 25 Analysis of Telomerase Activity .............................................................. 26 Reverse Transcription-PCR...................................................................... 26 Quantitative RT-PCR ................................................................................ 27 Cell-Cycle Analysis .................................................................................. 28 Results ................................................................................................................... 29 Analysis of Telomerase Activity and TERT Expression in Purified PGCs......................................................................................................... 29 Expression of TERT is Sufficient to Restore Telomerase Activity in Quiescent PGCs........................................................................................ 30 Analysis of the Effect of Modulation of TERT Expression on PGC Numbers and Cell-Cycle Status................................................................ 31 Discussion............................................................................................................. 33 Acknowledgements ............................................................................................... 37 Chapter 3. Sirtl Deficiency Attenuates Spermatogenesis and Germ Cell Function......... 43 Abstract................................................................................................................. 44 Introduction. .......................................................................................................... 46 Materials and Methods .......................................................................................... 49 Mice.......................................................................................................... 49 Sperm Count. ............................................................................................ 49 Analysis of Primitive Spermatogenic Stem Cells ..................................... 50 Histology.................................................................................. 50 In Vitro Fertilization. ........................................................................ 50 Embryo Transfer....................................................................................... 51 Comet Assay..... ..... ......... ............ ... ............... ... ......... .................. ... ........... 51 Apoptosis Analysis ................................................................................... 52 TRAP Assay............................................................................................. 52 RNA Isolation and Labeling for Array Analysis...................................... 52 cDNA Hybridization and Data Analysis................................................... 53 III Quantitave RT-PCR .................................................................................. 54 Results ................................................................................................................... 55 Sirtl Deficiency Abrogates Spermatogenesis........................................... 55 Sirtl Deficiency Affects Male Gametogenesis Owing Development...... 56 Sirtl Deficiency Induces Elevated DNA Damage in Male Germ Cells... 57 Sirtl Deficiency Affects Expression of Genes Involved in Spermatogenesis....................................................................................... 57 Discussion............................................................................................................. 59 Acknowledgments................................................................................................. 63 Author Contributions............................................................................................ 63 Chapter 4. Sirtl Acts as a Nutrient-sensitive