MOLECULAR MECHANISMS OF METALLOTHIONEIN INDUCTION

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DESCRIPTION (Adapted from the applicant's abstract): This laboratory has demonstrated that rat fibroblast cells that overexpress the large subunit of the protein Ku (Ku-80) repress metallothionein (MT) induction by heavy toxic metals, whereas the parental cells (Rat 1) or cells that overexpress the small Ku subunit (Ku-70) of both subunits as a heterodimer (Ku-7080) conintue to induce MT. Further study revealed that this repression is caused by hypermethylation of MT promoter, and probably by the induction of a diffusible repressor (R) that can function independent of the chromatin. The present proposal is aimed at elucidation of the mechanisms responsible for hypermethylation of MT-1 promoter and the mechanisms by with the methyl C binding protein (MeCP) and R mediate the rpression of Mt-1 gene expression. Bisulfite genomic sequencing showed that unlike Rat-1 cells, all 21 CpG dinucleotides in MT-1 immediate promoter are methylated in Ku-80 cells. This work will identify the nature of the methyl C-binding protein (MeCP) (activated or induced) by UV crosslinking and Southwestern blotting analysis using methylated DNA as probe, and Western blot analysis. If the MeCP activated or induced in Ku-80 cells is distinct from the two characterized MeCPs, it will be purified and cloned, the repressor domain identified, the effect of the recombinant MeCP on transcription from methylated MT promoter determined, and the mechanism by which it inhibits transcription will be elucidated. Next, the investigators will explore the signal transduction mechanisms by which overexpression of Ku subunit enhances methylation of MT-1 promoter, particularly with regard to enhancement of DNA methyltransferase activity and the probably role of H ras in mediating the increase in this enzyme activity. R will be characterized in addition to MeCP, and attempts will be made to elucidate the mechanisms by which it represses MT-1 transcription. Furthermore, tumor cells that do not expresss MT and exhibit higher levels of P80 compared to p70 will be investigated. MT is known to protect cells from damage against toxic metals and agents that generate free oxygen radicals and cause oxidative stress. Since the basal level of MT is negliglible in most tissues, its dramatic induction (as much as 50-100 fold, depending upon the inducer and the cell type) appears to be crucial for the survival of cells against the harmful effects of these agents. It is hoped that the present study will help design ways to augment MT induction by the inactivation of the signal transduction reactions that lead to hypermethylation of the MT promoter. Because MT induction is repressed in many cancer cells due to hypermethylation, the re-expresssion of MT may be one effective means to arrest malignant growth of these cells.