Molecular and Computational Biology Program, Department of Biological Sciences
College of Letters Arts & Sciences
- Developmental Biology
- DNA Replication
- Gene Regulation/Transcription
- Cell Cycle
- Growth & Proliferation
Research OverviewChanges in gene expression during aging as a guide to aging mechanisms
We are using the laboratory fruit fly, Drosophila melanogaster, as a model to investigate basic mechanisms of aging. Our approach is to identify the changes in gene expression that occur during normal aging and use this information as a guide to investigating underlying aging mechanisms. We have used a variety of approaches, including traditional molecular biology techniques, transgenic reporter constructs, and micro-array analyses. The changes in gene expression during fly aging were found to be highly similar to the flys response to oxidative stress, and include increased expression of specific heat shock protein (hsp) genes, induction of purine biosynthesis genes, dramatic induction of innate immune response genes, and the down-regulation of genes in the mitochondrial electron transport chain (ETC); most or all of these changes appear to be conserved with mammalian aging.
We have investigated the induction of the Drosophila hsp genes hsp22 and hsp70, and have found that conserved heat shock response elements in the promoters of these genes are required for induction during aging, and that the time-course of induction is accelerated by mutations and environmental conditions that shorten life span and increase oxidative stress. Hsp22 exhibits one of the largest aging-related increases known for a eukaryotic protein (>150-fold). We hypothesize that many of the gene expression changes during fly aging result from a failure to maintain normally functional mitochondria and a consequent oxidative stress. This idea is consistent with results from yeast, C. elegans and mammals that point to a central role for the mitochondria in modulating life span and aging phenotypes.
Predictive biomarkers of aging & a novel video-tracking assay
Several genes that are induced during aging and by oxidative stress (including hsp22, hsp70, and the innate immune response gene Drosomycin) were analyzed using transgenic reporter constructs. Fusing the regulatory regions of the genes to GFP (or DsRED) produced transgenic reporters that could be quantified longitudinally during fly aging. To facilitate these analyses, we have collaborated with Simon Tavarés group to develop a video-based 3D tracking assay that allows fly movement, behavior, and transgenic reporter expression to be simultaneously quantified in multiple flies. Strikingly, the expression of these reporters was found to be predictive of remaining fly life span, and the hsp reporters were found to spike in expression in the hours preceding and overlapping the death of the animal. Hsps show promise as biomarkers of aging in C. elegans and humans as well. In the future we plan to use these reporters to investigate the mechanisms that cause different individual animals to have different life spans.
Identifying life span regulatory genes using conditional transgenic systems
To identify genes that directly regulate life span we have focused on the development and application of conditional transgenic systems. These include the FLP-out system based on yeast FLP recombinase (induced by a heat pulse), the Tet-on system (induced by doxycycline), and the Geneswitch system (induced by RU486/Mifepristone). Using these systems we have over-expressed a series of logical candidate genes in the adult fly, and find that the redox-regulatory enzymes Cu/Zn-superoxide dismutase and mitochondrial Mn-superoxide dismutase are both positive regulators of life span, and can yield life span increases ranging from 15-40%. Our data, as well as that from other labs demonstrates that the life span increases caused by SOD are dependent upon the flys genotype, gender, and specific diet/environment. Transcriptional profiling using micro-arrays suggests that MnSOD acts in young adult flies to inhibit the conserved insulin-like pathway, thereby causing increased life span. Finally, we are investigating the regulation of Drosophila life span by p53. We have found that p53 has developmental stage-specific and sex-specific effects on adult life span indicative of sexual antagonistic pleiotropy (Waskar et al 2009 Aging 1:903-936). Over-expression of wild-type p53 (isoform A) in adult flies can increase life span in a sex-specific and tissue-specific manner, and the foxo gene was found to act in males to create sexual dimorphism in the life span effects of p53 (Shen & Tower 2010 Exp Gerontol 45:97-105). These results suggest that both p53 and foxo exhibit sexual antagonistic pleiotropy for adult life span.
To screen for novel genes that might modulate aging we have developed a new way to make conditional mutations in Drosophila: A P element construct (called PdL) was generated with the doxycycline-inducible promoter directed out through the 3' end of the element. When mobilized to new positions in the genome, PdL creates conditional (doxycycline-dependent) gene over-expression mutations at high frequency. Using this method we have identified additional genes that can extend life span. For example, we have recently identified a gene called magu that when over-expressed in adult flies can increase life span, as well as increase late-age female fecundity and gonadal stem cell proliferation. The magu gene is related to the mammalian gene SMOC-2 that regulates angiogenesis by signaling through the integrin-linked kinase (ILK).
Taken together the data integrate well with current evolutionary theories of aging, and suggest a working model in which antagonistic pleiotropy of gene function between developmental stages and sexes causes a failure in nuclear-mitochondrial signaling and mitochondrial maintenance, thereby leading to oxidative stress and mortality (Tower 2006 Mech Ageing Dev 127:705-718). In the future we plan to investigate how the sex-determination pathway interacts with life span regulatory genes to affect mitochondrial maintenance, hsp expression and life span.
Chorion gene amplification
Chorion gene amplification provides an ideal model for the regulation of higher eukaryotic chromosomal DNA replication origins. To meet the demand for the rapid synthesis of chorion (eggshell) proteins, Drosophila ovarian follicle cells amplify the chromosomal loci containing the chorion gene clusters up to 60 fold. Amplification occurs by repeated firing of one or more origins located within each gene cluster. We have cloned and characterized two amplification trans-regulatory genes, k43 and chiffon, and found that they are related to origin regulatory proteins in yeasts, ORC2 and Dbf4, respectively. In the past the analysis of cis-sequence elements and their relationship to origins was hampered by severe chromosomal position effects: any given transgenic construct would amplify at only ~1/3 of chromosomal insertion sites, making comparison of constructs difficult. We found that flanking the ends of transgenic constructs with transcriptional insulator elements, called "Suppressor of Hairy-wing protein binding sites" (SHWBSs) protects the constructs from position effects. The data implicated chromatin structure in origin regulation, and provided a powerfully improved assay. Using such buffered constructs we have been able to analyze cis-sequence requirements and functions in detail. We have found that functionally distinct, sequence-specific replicator and origin elements are required for amplification. The replicator is not itself an origin, but is required in cis to activate nearby origin(s) in the locus. The data support and extend the replicator model for the organization of higher eukaryotic chromosomal replicons (Stillman, B. 1993 Replicator Renaissance. Nature 366:506).