The Role of MTF-1 as a Mediator of Ocular Toxicity

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 DESCRIPTION (provided by applicant): Eye pathologies, such as cataracts and macular degeneration, are the leading causes of impaired vision and blindness in the world. Although these pathologies form as a natural process of aging, there are several environmental and lifestyle factors that increase the risk of these diseases, such as cigarette smoking, exposure to light and ultraviolet radiation, oxidative stress, steroid treatment and stress-induced DNA damage. We propose to investigate the role of the metal-responsive transcription factor 1 (MTF-1) in the transcriptional regulation of a suite of genes involved in eye development that may also contribute to environmental stress- related eye pathologies. The main objectives of this proposal are to determine what role MTF-1 plays in normal eye development and whether or not inappropriate activation of MTF-1 signaling by cellular stress in the eye can lead to misexpression of genes that promote eye pathologies. Zebrafish embryos and adults will be used as our model organism and comparative experiments will be performed in a human primary lens epithelial cell line. Targeted gene expression assessments by in situ hybridization, real-time PCR and global transcriptomic profiling via RNA-seq will be used to determine the effects of MTF-1 activation or inhibition on the regulation of eye-specific genes. We have chosen the toxic metal cadmium as our model environmental risk factor because it is one of the best known activators of MTF-1 signaling and because it is believed to be a direct causative factor in the increased risk of cigarette smokers to cataractogenesis and macular degeneration. ChIP-Seq analysis complementary to the microarray expression profiling will be used to determine DNA binding by MTF- 1 as confirmation of a direct role in the regulation of genes responsible for eye development and stress-related pathologies. Additional promoter characterization via transient transfection assays and chromatin immunoprecipitation will be used for final confirmation of MTF-1 regulation of the eye-specific genes in zebrafish and humans. The results obtained from these experiments will yield public health significance by providing novel insight into mechanisms of action by which environmental stressors contribute to eye pathologies. In addition to the discovery of a potential new regulatory pathway involved in eye development with new insight into cataractogenesis and macular degeneration, successful completion of the proposed research will further expand the current use of zebrafish as a model organism for eye disease-related research.