Regulators Gene lin-11 và let-711: Điều chỉnh tỷ lệ lão hóa và tuổi thọ ở C. elegans

University of Kentucky UKnowledge University of Kentucky Doctoral Dissertations Graduate School 2011 GENE EXPRESSION REGULATORS lin-11 AND let-711, IN MODULATING THE RATE OF AGING AND LIFESPAN, IN C. Tseten Yeshi Jamling University of Kentucky, tseten.com Right click to open a feedback form in a new tab to let us know how this document benefits you. Recommended Citation Yeshi Jamling, Tseten, "GENE EXPRESSION REGULATORS lin-11 AND let-711, IN MODULATING THE RATE OF AGING AND LIFESPAN, IN C. University of Kentucky Doctoral Dissertations.edu/gradschool_diss/164 This Dissertation is brought to you for free and open access by the Graduate School at UKnowledge.

It has been accepted for inclusion in University of Kentucky Doctoral Dissertations by an authorized administrator of UKnowledge. For more information, please contact UKnowledge@lsv. ABSTRACT OF DISSERTATION Tseten Yeshi Jamling The Graduate School University of Kentucky 2011 GENE EXPRESSION REGULATORS lin-11 AND let-711, IN MODULATING THE RATE OF AGING AND LIFESPAN, IN C. ________________________________________________________________________ ABSTRACT OF DISSERTATION ________________________________________________________________________ A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Arts and Sciences at the University of Kentucky.

By Tseten Yeshi Jamling Lexington, Kentucky Director: Dr. Jim Lund, Assistant Professor of Biology Lexington, Kentucky 2011 Copyright  Tseten Yeshi Jamling 2011 ABSTRACT OF DISSERTATION GENE EXPRESSION REGULATORS lin-11 AND let-711, IN MODULATING THE RATE OF AGING AND LIFESPAN, IN C. lin-11 and let-711 are early-developmental gene expression regulators with no previously known roles in aging regulation. Yet, they show strong aging-correlated expression profiles (Lund, Tedesco et al.

lin-11 is strongly upregulated in very old worm populations, and let-711 is progressively downregulated in aging worm populations. Microarray studies were performed to identify their genome-wide targets, which were then subjected to further lifespan and genetic analysis to investigate their role in C. The results indicate that the target pools of both lin-11 and let-711 are enriched for aging genes, since a significant number of tested genes increased lifespan. This enrichment of aging genes in their target pools provides strong evidence that lin-11 and let-711 are indeed regulating the expression of aging genes in adult C.

The data suggests that increased lin-11 expression as well as reduced let-711 expression may be promoting longevity by downregulating the insulin/IGF-1 pathway. Decreasing let-711 may also be contributing to longevity by downregulating the germline signaling pathway.5 are four genes from the lin-11 target pool, whose knockdown produced increases inlifespan. These are unannotated genes, and the details of their roles in aging regulation are not known at this point. ins-3 expression was downregulated two-fold upon knockdown of lin-11, suggesting the possible involvement of lin-11 in regulation of the insulin/IGF-1 pathway.

An RNAi construct for ins-3 was not available and it is not known whether loss of ins-3 leads to lifespan extension. let-711 knockdown resulted in an almost four-fold reduction in pdk-1 expression. pdk-1 is an integral part of the insulin/IGF-1 pathway and its knockdown by RNAi extended lifespan. Four other genes from the let-711 target pool that increased lifespan, cdc-25.1, gna-2, meg-1 and ooc-3, all have germline specific functions.

The extensions in lifespan generated by these genes were completely dependent on DAF-16. Furthermore, for gna-2, meg-1 or ooc-3, they were independent of DAF-2. These results agree with previously established mechanisms for germline regulation of aging, suggesting the involvement of let-711 in regulating the germline-signaling pathway. elegans, aging, lin-11, let-711,germline signaling.

Tseten Yeshi Jamling 25 Febuary 2011 GENE EXPRESSION REGULATORS lin-11 AND let-711, IN MODULATING THE RATE OF AGING AND LIFESPAN, IN C. By Tseten Yeshi Jamling Jim Lund Director of Dissertation Rebecca Kellum Co-director of Dissertation Brian Rymond Director of Graduate Studies 25 Febuary 2011 RULES FOR THE USE OF DISSERTATIONS Unpublished dissertations submitted for the Doctor’s degree and deposited in the University of Kentucky Library are as a rule open for inspections, but are able to be used only with due regard to the rights of the authors.Bibliographiccal references may be noted, but quatations or summaries of parts may be published only with the permission of the author, and with the usual scholarly acknowledgements. Extensive copying or publication of the dissertation in whole or in part also requires the consent of the Dean of the Graduate School of the University of Kentucky. A Library that borrows this dissertation for use by its patrons is expected to secure the signature of each user.

NAMEDATE ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ _______________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ DISSERTATION Tseten Yeshi Jamling The Graduate School University of Kentucky 2011 GENE EXPRESSION REGULATORS lin-11 AND let-711, IN MODULATING THE RATE OF AGING AND LIFESPAN, IN C. ________________________________________________________________________ DISSERTATION ________________________________________________________________________ A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Arts and Sciences at the University of Kentucky. By Tseten Yeshi Jamling Lexington, Kentucky Director: Dr. Jim Lund, Assistant Professor of Biology Lexington, Kentucky 2011 Copyright  Tseten Yeshi Jamling 2011 FOR MY SON, TO BE BORN JUNE 29th 2011, MY WIFE, PHUNTSOK DOLMA, AND MY PARENTS, BUMO TSERING AND TSEWANG YESHI JAMLING.

THIS ACHEIVEMENT IS AS MUCH YOURS AS IT IS MINE. ACKNOWLEDGMENTS The followitng dissertation would never have been, were it not for the contributions of numerous people in numerous different ways.I begin by thanking my advisor, Dr. Jim Lund, for introducing me to the fascinating field of aging biology and C. elegans genetics, for his patience and guidancethoughout my graduate career.

I thank my committee members, Drs. Rebecca Kellum, Peter Mirabito and Brett Spear, for valuable insight and feedback towards my improvement as a scientist. I would also like to thank all my lab members, Scott Frasure, Dustin Perry, David Keesling, Daipayan Banerjee, Lin Xue, Suchita Desai and especially George Chaffins (late) for being comrades through the trials and tribulations of graduate school. George, you are always in my thoughts and prayers.

I have had a wonderful graduate school experience and would like to thank all graduate students, staff and faculty of the Biology department, for contributing towards my amazing experience. I thank Beverly Taulbee for her help with all the administrative details, and to Dr. John Seabolt for his genuine care and concern. The completion of my doctoral degree would not have been possible also, without the continuous support of my loved ones.

My beautiful wife, Phuntsok Dolma, never ceases to amaze me with her unwavering conviction in my capabilities, even when I am in doubt myself. Your love and support has been the single greatest contribution towards my completion of graduate school. Likewise, my parents BumoTsering and Tsewang Yeshi, along with my sisters Tenzin Nangkyi and Lhakpa Dolma, have provided incredible support and understanding throughout this entire process iii Table of Contents ACKNOWLEDGMENTS. iii LIST OF TABLES.

vi LIST OF FIGURES. vii CHAPTER 1: Background and Significance .1 Cellular pathways that regulate aging .3 The DAF-2 Insulin/IGF-1 Pathway. 3 Role of the reproductive system in aging regulation. 5 Other pathways that regulate aging.

8 Gene Expression Regulators lin-11 and let-711 .12 Expression profiles of lin-11 and let-711 are aging co-regulated. 12 lin-11, an early-developmental transcription factor. 13 let-711, an early-developmental gene expression regulator .21 CHAPTER 2: Characterization of early developmental transcription factors lin-11 and let-711 as aging regulators.24 Results and Discussion .26 Individual knockdown of lin-11 and let-711 in adult worms shortens lifespan. 26 Genome-wide target identification of lin-11 and let-711 in adult worms utilizing microarray analysis.

28 lin-11 knockdown increased expression of an insulin-related peptide. 29 lin-11 target pool is enriched for aging genes. 30 let-711 interaction with the Insulin-like signaling pathway. 32 let-711 target pool is enriched for aging genes that may be contributing to lifespan extension by downregulating germline signaling.

33 lin-11 and let-711 coregulated genes .2, a common target in both microarray experiments, extends lifespan .36 lin-11 regulates aging genes in C. 36 let-711 is an aging regulator in C. 36 Materials and Methods. 38 CHAPTER 3: let-711 Regulates Aging Through the Germline Signaling Pathway .58 iv Regulation of lifespan by the germline.

58 The germline regulates lifespan by inhibiting DAF-16 activity, independent of the daf- 2/insulin-like signaling pathway. 59 let-711 regulated genes that influence germline survival and proliferation. 60 Results and Discussion .64 daf-16 mutation suppresses lifespan extensions produced by RNAi of germline-related let- 711-regulated genes. 64 meg-1, ooc-3 and gna-2 regulate lifespan independently of daf-2.

65 Lifespan extension by cdc-25.1 is dependent on daf-2 .67 let-711 regulates lifespan through the germline signaling pathway. 67 let-711 regulates aging through daf-2 dependent check-point signaling mechanisms. 67 Model for let-711 regulation of lifespan in adult C. 68 Materials and Methods .69 Strains and maintenance.

69 CHAPTER 4: Conclusions and Future Directions. 77 lin-11 regulation of aging. 77 let-711 regulation of aging. 88 v LIST OF TABLES Table 2.

Seven genes showed significant changes in expression in both lin-11 and let- 711 microarray experiments. Sixteen genes were differentially expressed by at least two-fold when lin-11 is knocked down…………………………………. Sixty genes showed at least two-fold change in expression in response to let- 711 RNAi………………………………………………………………………54 Table 3. Summary of lifespan assays……….……85 vi LIST OF FIGURES Figure 1.

The Insulin/IGF-1 signaling pathway……. The early embryonic lineage and summary of the asymmetric distribution of various cell-fate determinants in 4-cell embryos……. Development of the reproductive system in C. Effects of germline and whole gonad ablation on C.

A genetic working model for the effects of the reproductive system on Aging.lin-11 RNAi and let-711 RNAi effects on lifespan………………………. M vs A plots show good agreement among replicate GeneChips…. Venn Diagram summarizing the results of the microarray analyses………. Expression profiles of the genes that were differentially expressed in the microarray experiments….2 RNAi extended lifespan……………….

RNAi of pdk-1 extended lifespan………………….grsp-4 RNAi and meg-1 RNAi extended lifespan…………………………. RNAi of cdc-25.1, ooc-3 or gna-2 individually extended lifespan. The daf-16 gene is required for the longevity induced bycdc-25.1, meg-1, ooc-3 or gna-2RNAi…. Loss of daf-2 supressed the lifespan extension of cdc-25.

Lifespan extensionsdue to knockdown of meg-1, ooc-3 and gna-2were independent of daf-2……………………………………………………………83 Figure 3. A model for let-711 regulation of lifespan in adult C.84 vii CHAPTER 1: Background and Significance Introduction Aging and death are something we are very familiar with, and accept as universal biological phenomena that cannot be avoided. Nonetheless, human life expectancy has increased by decades due to modern medicine and technology over the last century. Infant mortality rates have declined, cures for deadly diseases have been discovered, and our cars and buildings have become safer, allowing us to live longer.

As such, the elderly population is growing at a rapid rate and diseases associated with aging, including Alzheimer’s disease and cancer, are gaining notoriety. The US Department of Health and Human Services estimates that 20% of Americans, more than 70 million individuals, will be over the age of 65, by the year 2030. With ever-greater numbers living to grow old, there is strong interest in understanding the aging process at a fundamental level. But what do we really know about the biology of aging? The increased lifespan expectancy of the modern man is due to a reduction in risks or external factors and not due to an intimate understanding of the biology of aging.

All organisms age, and the similar vulnerability to deleterious effects of aging across phyla is remarkable, hinting at a conserved mechanism that regulates the aging process. Aging exponentially increases the mortality rate, producing a characteristic population survival curve observed in aging populations of a wide range of animals with lifespans ranging from days to years. Physiological changes such as organ function decline, loss of muscle mass, and weakened stress resistance are common aging phenotypes. The similarities are even more remarkable at the cellular level.

Accumulation of DNA damage, lipofuscin deposits, and accumulation of misfolded and damaged proteins are common cellular changes observed with aging in all living organisms.