Effect of Sirt1 and telomerase on stem cells
Effect of Sirt1 and telomerase on stem cells
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2008
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Coussens, Matthew J.
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Thesis (Ph.D.)--University of Hawaii at Manoa, 2008.
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 Sirt1. 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, Sirt1, a member of the sirtuin family of proteins, orthologous to the yeast Sir2, has important physiological roles in regulating glucose metabolism. We have now performed a detailed analysis of the molecular and functional effects of Sirt1 deficiency in the germ line of Sirt1 knock-out (-/-) mice. We find that Sirt1 deficiency markedly attenuates spermatogenesis, but not oogenesis. Microarray analysis in Sirt1 deficient testis revealed dysregulated expression of 85 genes, which were enriched (P<0.05) for genes involved in spermatogenesis and protein sumoylation. To assess the function of Sirt1 deficient germ cells, we compared the efficiency of generating embryos and offspring in in vitro fertilization (IVF) experiments using gametes from Sirt1 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 Sirt1-/- gametes. Using short hairpin RNAs, we found that inhibition of Sirt1 in immortalized human cells enhanced cell growth under normal and nutrient limiting conditions. Hematopoietic stem cells, which were shown to express Sirt1, showed increased growth capacity and decreased dependency on growth factors when the Sirt1 gene was deleted. These data support an important role for Sirt1 in spermatogenesis, including spermatogenic stem cells, as well as germ cell function, but also demonstrate that in certain cell lineages, Sirt1 can act as a growth suppressor.
Includes bibliographical references (leaves 117-132).
Also available by subscription via World Wide Web
132 leaves, bound 29 cm
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 Sirt1. 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, Sirt1, a member of the sirtuin family of proteins, orthologous to the yeast Sir2, has important physiological roles in regulating glucose metabolism. We have now performed a detailed analysis of the molecular and functional effects of Sirt1 deficiency in the germ line of Sirt1 knock-out (-/-) mice. We find that Sirt1 deficiency markedly attenuates spermatogenesis, but not oogenesis. Microarray analysis in Sirt1 deficient testis revealed dysregulated expression of 85 genes, which were enriched (P<0.05) for genes involved in spermatogenesis and protein sumoylation. To assess the function of Sirt1 deficient germ cells, we compared the efficiency of generating embryos and offspring in in vitro fertilization (IVF) experiments using gametes from Sirt1 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 Sirt1-/- gametes. Using short hairpin RNAs, we found that inhibition of Sirt1 in immortalized human cells enhanced cell growth under normal and nutrient limiting conditions. Hematopoietic stem cells, which were shown to express Sirt1, showed increased growth capacity and decreased dependency on growth factors when the Sirt1 gene was deleted. These data support an important role for Sirt1 in spermatogenesis, including spermatogenic stem cells, as well as germ cell function, but also demonstrate that in certain cell lineages, Sirt1 can act as a growth suppressor.
Includes bibliographical references (leaves 117-132).
Also available by subscription via World Wide Web
132 leaves, bound 29 cm
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Theses for the degree of Doctor of Philosophy (University of Hawaii at Manoa). Biomedical Sciences (Cell & Molecular Biology); no. 5097
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