Intellectual merit: DNA damage is important and costly enough that organisms devote scores of genes to its repair, but little is known about how individuals in natural populations vary in their underlying mutation rates, or in their ability to repair those mutations. Furthermore, little is understood about the identity of the genetic determinants of this variation, and whether these genes differ from those found in studies on laboratory organisms. This gap in knowledge seriously limits the ability to understand the mechanisms that maintain genome integrity, and to understand natural variation in mutation-dependent phenomena such as senescence. The long-term goal of this project is to understand the genetic and environmental factors that affect genome stability in nature. The central hypothesis of this project is that individuals vary in somatic mutation rate, and that this variation is caused by differences in gene sequences and gene expression. To test the central hypothesis, this project examines variation in somatic mutation rate in 40 inbred lines of Drosophila melanogaster derived from a natural population in North Carolina. Genome-wide expression and sequence data are available for these lines. The study is made possible by the recent creation of a transgenic model to measure somatic mutation rates in vivo in the fruit fly with a lacZ reporter gene. Aim 1 will involve experiments to test the hypothesis that genetic variation exists for somatic mutation rate by placing a lacZ reporter gene into the 40 inbred lines and measuring line-, sex- and tissue-specific variation for somatic mutation. This hypothesis is strongly supported by preliminary data showing heritable variation for somatic mutation rate. In Aim 2, genetic and regulatory factors associated with somatic mutation rate will be investigated. Genome-wide association studies of single nucleotide polymorphisms will be used to identify genes associated with somatic mutation rate. Expression data will be analyzed to search for networks of co-regulated genes whose expression levels correlate with somatic mutation rate. Aim 3 will determine whether genotypes with naturally high somatic mutation rate show relatively large increases in mutation frequency when exposed to paraquat, a herbicide that has been shown previously to increase the frequency of mutations in Drosophila. This project uses a creative combination of quantitative genetics, molecular genetics, and high-throughput genomics. In its focus on natural genetic variation, this work is expected to lead to the discovery of new genes that influence evolutionarily relevant variation in somatic mutation rates and to create a novel paradigm for understanding the genetic basis of somatic mutation rate. This new framework should provide better understanding of the evolution of genome stability and genome structure.
Broader impacts: This proposal provides the first direct study of natural variation for somatic mutation rate, and should lead to the identification of novel genes associated with somatic mutation rate. All novel strains developed and all expression data collected during the course of this project will be made freely available to the research community. The project will provide continued hands-on research training for high school and undergraduate students, including students from underrepresented populations. Trainees will be given the opportunity to publish scientific articles and to attend national scientific meetings with the investigator.
Intellectual Merit: The overarching goals of this project were to determine the heritability of somatic mutation rate (SMR) and to characterize the genetic and environmental factors that shape SMR. To address these questions, we developed a reporter system, whereby a bacterial gene (lacZ, or β-galactosidase) was inserted permanently into the fly genome. When flies reach adulthood, we excise the gene from the fly (many thousands of copies per fly), and put the gene into a bacterium that lacks the gene (lac- bacteria). If the gene is still functioning, the bacteria will revert to the lac+ state. Thus, the frequency with which flies fail to revert provides a measure of the frequency of mutations that knock out gene function. Over the course of three years, we were able to obtain repeatable results showing that there is, in fact, genetic variation for SMR, with some genotypes showing much higher rates than others (Figure 1). To our knowledge, this is the first demonstration of heritable variation for somatic mutation rate in a genetically variable population. Our results from multiple studies provide the strongest evidence to date in support of our central hypothesis—namely, that genetic differences among individuals account for inter-individual variation in somatic mutation rate. Having established this important finding, our primary goal has been to determine the specific genetic factors that shape somatic mutation rate. Unfortunately, until very recently, the available methods have proved too slow and cumbersome for the scale at which we had hoped to actually map genes that affect SMR. Work was further slowed by a viral infection in the lab that eliminated many stocks. Fortunately, success is now on the horizon. In the third year of the grant, the Principal Investigator relocated to the University of Washington, and initiated a new and exciting collaboration with Dr. Jason Bielas (Fred Hutchinson Cancer Research Center, Seattle WA) that has led to a dramatic increase in our ability to screen SMR in a high-throughput manner. Dr. Bielas’ method, called Random Mutation Capture (RMC, Bielas and Loeb 2005, Vermulst et al. 2008a), allows us to measure the rate at which specific restriction enzyme sites in the mitochondrial genome experience spontaneous mutations. Importantly, our results from the RMC method suggest an average per-base mitochondrial mutation rate of approximately 1 per 100,000 base pairs, similar to recent measures of fly mitochondrial DNA somatic mutation rate carried out using whole genome sequencing(Itsara et al. 2014). Studies in mice have identified a mitochondrial-specific gene (DNA polymerase gamma, or pol-γ) which, when knocked out, leads to a ~100-fold increase in mtDNA mutation frequency compared to wild-type (Vermulst et al. 2008b). We obtained tissue from the fly equivalent of the pol-γ mutant. With the loss of this key gene that is required for the integrity of replication of the mitochondrial genome, we found an average 21-fold increase (21.8 ± 5.5, mean ± s.e.) in mitochondrial somatic mutation rate, demonstrating that the Drosophila pol-γ gene has effects similar to the mammalian version, and that we can measure mtDNA mutation frequency with sufficient accuracy to differentiate wild-type and mutant lines (Figure 2). We are now in the process of collecting samples from the complete library of sequenced Drosophila Genome Reference Panel (DGRP, Mackay et al. 2012) flies to measure genetic variation in mtDNA mutation rate. We anticipate that within the coming months, we will have sufficient estimates to allow us to map the genetic basis of mitochondrial mutation rate for the first time and with a high level of accuracy. Broader Impacts: Throughout the course of this project, the PI has actively involved undergraduates in the research program. Trainees have had the opportunity to learn key concepts in fruit fly genetics, as well as in the methods used to study aging in Drosophila. Minority UGA undergraduates supervised during this time included Chiemeka Ugochukwo, Amirah Pittman and Kathryn Woods. At the University of Washington, the PI has recruited a new undergraduate, Mr. Nick Force, to work on this project. Mr. Force is a veteran of the United States Navy (Petty Officer First Class), attending UW on the GI Bill. Literature Cited Bielas, J. H. and L. A. Loeb. 2005. Quantification of random genomic mutations. Nat Methods 2:285-290. Itsara, L. S., S. R. Kennedy, E. J. Fox, S. Yu, J. J. Hewitt, M. Sanchez-Contreras, F. Cardozo-Pelaez, and L. J. Pallanck. 2014. Oxidative stress is not a major contributor to somatic mitochondrial DNA mutations. PLoS Genet 10:e1003974. Mackay, T. F., et al. 2012. The Drosophila melanogaster Genetic Reference Panel. Nature 482:173-178. Vermulst, M., J. H. Bielas, and L. A. Loeb. 2008a. Quantification of random mutations in the mitochondrial genome. Methods 46:263-268. Vermulst, M., et al.. 2008b. DNA deletions and clonal mutations drive premature aging in mitochondrial mutator mice. Nat Genet 40:392-394.
