CEGIB Pilot Grant Awards 2010
University of Louisville
CENTER FOR ENVIRONMENTAL GENOMICS AND INTEGRATIVE BIOLOGY
2010 Pilot Project Grants
Comprehensive Identification of LINE1 Retrotransposons in an Individual's Genome - Dr. Ted Kalbfleisch
Long Interspersed Nuclear Element 1 (LINE-1), an active retrotransposon found pervasively within the human genome, has been demonstrated to be of clinical significance, and is known to be regulated by pollutants and other elements found commonly in the environment. In this work, we propose the development of a molecular assay utilizing both sequence capture, and Next Generation Sequencing technologies that is capable of measuring the comprehensive genetic profile for an individual with respect to LINE-1 insertions. Previous work in our lab, and that of others has demonstrated that there is heterogeneity with respect to the number and position of copies of the LINE-1 element. The assay that will be developed in this work will result in an enriched sequence dataset that contains the 5′ splice junction of all sequence elements that suggest full length LINE-1 insertion. These insertions may serve as targets for further laboratory research necessary to fully characterize the LINE-1 insertion as intact, capable of retrotransposition, or otherwise capable of imparting regulatory function. This work provides a robust, accurate, inexpensive assay that will provide a comprehensive profile of the LINE-1 elements within an individual’s genome. This will help define the intersection between genetic and epigenetic events in response to environmental insults. As such, it has huge implications for environmental diseases involving deficits of cellular growth and differentiation.
Toxic Air Pollutants and Endothelial Progenitor Cells - Dr. Timothy O'Toole
Several epidemiological and experimental studies suggest that exposure to fine particulate air pollution (particulate matter: PM) is associated with a dysfunctional endothelium and consequently, cardiovascular pathologies such as atherosclerosis angina, myocardial infarction, and stroke. Acute and chronic damage to the endothelium is repaired by circulating pluripotent cells or the endothelial progenitor cells (EPCs) which promote angiogenesis and wound healing. Several previous studies show that the presence of cardiovascular risk-factors including physical inactivity, hypertension, diabetes, hyperlipidemia and exposure to tobacco smoke, decreases the number and proliferative capacity of EPCs or their ability to differentiate into functional blood vessels. Thus we hypothesize that that low-grade systemic inflammation induced by exposure to toxic air pollutants prevents vascular regeneration by similarly decreasing EPC mobilization and/or their reparative potential. This hypothesis will be addressed in two Aims. In the first we will quantify the levels of antigenically-defined EPC populations in peripheral blood and bone marrow of mice exposed to concentrated ambient PM2.5, identify the time course and recovery of these effects, and determine whether the effects of pollutants are unique to EPCs, or whether mesenchymal (MSC) and hematopoietic (HSC) stem cells are affected as well. In the second Aim we will attempt to identify interventions which reverse the effects of pollutants on EPC mobilization including approaches to resolve low-grade inflammation and integrin-mediated adhesion in the bone marrow. Successful completion of this project will provide novel insights into the mechanisms by which exposure to air pollutants impairs endothelial function and contributes to cardiovascular disease. Demonstration of the efficacy of our approaches to limit inflammation and adhesion in mobilizing EPCs might suggest avenues for developing novel therapies for promoting angiogenesis and wound healing.
Molecular mechanisms of stalled DNA replication fork recognition - Dr. W. Glenn McGregor
Recent advances implicate error-prone DNA polymerases in the generation of virtually all mutations induced by environmental carcinogens in higher eukaryotic cells. These data have supported the promise of cancer chemoprevention based on the selective modulation of the activity of these proteins, based on the assumption that reducing the mutant frequency will reduce the incidence of cancer. However, carcinogenesis studies using newly-developed mouse models in which one or another of these polymerases is deficient have yielded unexpected results. Specifically, the deficiency of one such polymerase, termed DNA polymerase iota (pol iota), results in greatly decreased mutation frequencies induced by ultraviolet (UV) radiation in an endogenous reporter gene. These results would predict a protective effect against the carcinogenic effects of UV. However, pol iota-deficient animals exhibit a highly accelerated development of aggressive skin cancer. This unexpected result is not consistent with the somatic mutation hypothesis of carcinogenesis, and highlights the fact that there are critical gaps in our knowledge of the cellular function of this universe of polymerases. This application proposes to confirm and extend our recent gene and microRNA expression data that strongly implicate pol iota in UV-induced cell cycle arrest and apoptotic pathways. This proposal addresses the overall hypothesis that pol iota modulates genomic responses to UV that are distinct from its activity as an error-prone polymerase. To examine this, we propose a pilot project that will consist of two Specific Aims. In Aim 1, we propose to examine the effect of pol iota deficiency on UV-induced changes in gene expression, damage-induced cell cycle checkpoint protein modification,and markers of proliferation in intact skin of newly-derived cancer-prone murine models. In Aim 2, we will examine the effect of pol iota deficiency in UV carcinogenesis studies that employ these novel murine models. This proposal will fill critical gaps in our knowledge of how cancer is initiated by the most ubiquitous environmental carcinogen. The ultimate goal of this research is to understand how carcinogens cause cancer in order to design strategies to prevent the disease.
Exosomal microRNA in the detection and diagnosis of renal disease - Dr. Michael L. Merchant
The incidence of chronic kidney disease has seen a worldwide increase; some of which is suggested to result of chronic or acute exposure to environmental pollutants. More specifically, chronic kidney diseases such as nephrotic syndrome have been causally linked to environmental factors such as metals and hydrocarbon exposure. These factors may exaggerate the systemic, pro-inflammatory state which occur in susceptible individuals and contribute to the development of a profibrotic state. In the case of diabetes and diabetic kidney disease, the cytotoxic diabetic milieu (hyperglycemia and proteinuria) has been demonstrated to up-regulate specific profibrotic microRNA expression. These microRNAs contribute to increased expression of growth factors and fibrotic proteins which drive the development of end-stage renal disease. We hypothesize that similar conditions found in other proteinuric diseases also contribute to the upregulation of specific microRNA expression in the kidney. Preliminary data suggest that urinary microparticles known as exosomes contain unique profiles of proteins and microRNA. We propose to conduct experiments to determine the utility of urinary exosomal microRNAs to diagnose renal disease.