METALLOTHIONEIN FUNCTION AND GENE REGULATION

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An understanding of the role of metallothionein (MT) in the normal processes of gene expression and cellular differentiation is essential for evaluating its involvement in carcinogenesis. Our approach to such a role is to address the regulation of the MT gene and the function of the MT protein as interrelated processes, since both are influenced by the binding and homeostasis of metal ions. The Strongylocentrotus purpuratus metallothionein gene, SpMTA, is an appropriate model for testing these relationships, principally because its expression is restricted to specific cell lineages (the aboral ectoderm) of the sea urchin embryo. To take advantages of this membership in a cell-type specific set of genes, diverse aspects of SpMTA function and SpMTA gene regulation will be studied, including the possibility that the SPMTA protein may be involved in regulating the expression of other genes in the set, as well as in influencing cellular differentiation. The effect of increasing MT expression will be tested by monitoring the expression of these genes in embryos whose intracellular levels of MT have been elevated through transgenic expression. For this, SpMTA mRNA or SpMTA cDNA, the latter under the regulation of either a globally expressed or aboral ectoderm cell-type specific promoter, will be microinjected into eggs for expression in developing embryos. Effects on cellular differentiation will be monitored morphologically as well as by the levels of the marker mRNAs. A second and complementary aim of this project will be to analyze the components and mechanisms of metal-induced transcription of the SpMTA gene. Nuclei from normal embryos and embryos chronically exposed to An(II) ions (known to have their SpMTA genes transcribed at extraordinarily high levels) will be used as sources of transcription factors. The metal-response factors (MRFs) will be subsequently cloned. An in vitro transcription system will also be generated from these nuclei, in order to analyze (a) the functions of protein factors, especially of cloned MRFs, and (b) the involvement of specific DNA elements in the regulation of SpMTA gene transcription. The functional properties of MRFs and other metal-associated factors in SpMTA gene transcription, and the deduced structural properties of cloned MRFs, will be compared with the properties of MT and metal-containing transcription factors, especially to understand the dynamics of metal regulation, in the interrelated processes of MT gene regulation and MT functionality. The results of these studies will provide a basic conceptual framework for evaluation of the potential role of human MT in cancer, and for development of future hypotheses and approaches to diagnosis and therapies.