The overall long-term objective of these studies is to apply molecular, biochemical and transgenic approaches to study environmental health-related issues. Current studies focus on the mechanisms by which the toxic metal Cd, and the essential metal Zn regulate gene expression. Cd is a widespread environmental toxin that poses an increasing threat to public health. It is a common industrial pollutant that is also present in cigarette smoke. Cd poisoning causes damage to many major organ systems, leading to Itai-Itai disease, retardation of growth, sterility and cancer. In contrast, Zn is an essential metal, which is required for the activity of hundreds of proteins, but is toxic when in high concentration. Specifically, our studies concentrate on metal-response element-binding transcription factor-1 (MTF-1), which coordinates the Cd- and Zn-induced transcription of several protective genes (e.g., the metal chelator metallothionein, the metal exporter zinc-transporter-1, and gamma-glutamylcysteine synthetase heavy chain, the rate limiting step in glutathione synthesis). MTF-1 functions as a cellular metal-sensor, but the molecular mechanisms by which it senses different metals are not well understood. Our studies suggest that Cd and Zn utilize overlapping yet distinct mechanisms for activation of gene expression using MTF-1. Remarkably, the mouse MTF-1 gene is essential and homozygous knockout embryos die at midgestation, thus underscoring the importance of this transcription factor. Whether MTF-1 is an essential gene in other species remains to be determined, as does the function of this protein during development. The six zinc-finger domain of this factor is highly conserved from insects to mammals, which indicates a conserved function. Therefore, the specific aims of this proposal are to: 1) Identify proteins which interact with MTF-1 specifically during Cd versus Zn induction, and examine their roles in MTF-1 -regulation of gene expression; 2) Determine the roles of histone acetylation in the MTF-1 activation of gene expression; and 3) Develop the Zebrafish as a model system to reveal essential functions of MTF-1 during embryonic development. Proteins which interact with MTF-1 will be studied using Superose-6 HPLC, immunoprecipitation, binding-site chromatography and mass spectrometry of proteolytic peptides. Roles of histone acetylation will be examined using chromatin immunoprecipitation, co-transfection with HAT expression vectors, detection of specific HAT proteins in the MTF-l complex, and analysis of MTF-l functions in yeast with mutations in HAT genes. Functions of MTF-l during development will be examined in Zebrafish using morpholino antisense oligonucleotides, whole mount in situ hybridization and morphometric analyses of developing embryos.
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