REGULATION AND FUNCTION OF METALLOTHIONEIN GENES

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.