REGULATION AND FUNCTION OF METALLOTHIONEIN GENES

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Metallothionein (MTs) are small, Cys-rich metal-binding proteins that are ubiquitous among eukaryotes. MTs are thought to subserve metal homeostasis and detoxification and recently, have been implicated in stress responses, embryogenesis/development, Cd-induced cancer, cis-DDP resistance and the regulation of transcription factor activities. However, incisive tests of MT functions, based on the ablation or overexpression of MTs in specific tissues, have not been performed. Likewise, current knowledge of the molecular mechanisms that govern metal-inducible, cell-specific and/or developmentally-modulated expression/transcription of MTs in multicellular eukaryotes is limited. We will study the regulations and function of MT genes in the nematode Caenorhabdities elegans (C. elegans). Protein immunocytochemistry and in situ mRNA hybridization will be used to determine temporal, cellular and subcellular patterns of expression for 2 iso-MTs, designated CeMT1 and CeMT2, during successive developmental stages. In vivo transcriptional activities of the promoters of the MT genes (mtl-1 and mtl-2) will be monitored in individual cells by creating and characterizing transgenic C. elegans that express reporter genes driven by tl promoters. DNA flanking the 5' ends of the mtl genes will be modified by mutagenesis in order to identify and characterize metal regulatory elements (MREs) and DNA sequences that control the cellular specificity and developmental timing of transcription. The function of each cis element will be determined in the physiological environment of intact (transgenic) animals. The occurrence of cadmium/zinc-activated MRE binding proteins (MRE-BP) will be established in gel shift and footprinting assays. The MRE-BP will be (a) purified to homogeneity via conventual procedures and affinity chromatography, (b) characterized biochemically (c) tested in an in vitro transcription system and (d) partially sequenced. cDNA clones encoding MRE-BPs will be obtained by conventional and expression screening of cDNA libraries of complementation in a yeast system. Finally CeMTs will be laminated, overexpressed or ectopically expressed in specific cells and tissues of transgenic C. elegans. We will determine whether CeMTs are essential for normal development, a stress response, broad spectrum detoxification and the intracellular distribution of zinc. New concepts derived from the proposed studies will be applicable to Cd-induced carcinogenesis because of the conserved nature of both MT gene regulation by metals/stress and MT functions. Analysis of C. elegans' response to the environmental carcinogen cadmium may suggest mechanistic causes and therapies for (a) metal-promoted malignancies and (b) tumors that become cross-resistant to cis-DDP and alkylating agents.