Suppression of metallothionein gene expression in a rat hepatoma because of promoter-specific DNA methylation

Metallothionein I can be induced in response to a variety of agents that in clude heavy metals and oxidative stress, On the contrary, its induction was suppressed in some lymphoid-derived cancer cells. The mechanism of this re pression has not been elucidated. Here, we show silencing of MT I gene in a solid transplanted rat tumor as a result of promoter methylation at all th e 21 CpG dinucleotides that span the region from -225 bp to +1 bp. By contr ast, none of these CpG dinucleotides were methylated in the livers from the rats bearing the tumor, which was consistent with the efficient induction of the gene in this tissue by zinc sulfate. Genomic footprinting revealed l ack of access of the transcriptional activators to the respective cis-actin g elements of the methylated MT-I promoter in the hepatoma. The absence of footprinting was not due to inactivation of the metal regulatory transcript ion factor MTF-1, because it was highly active in the hepatoma. Treatment o f the hepatoma bearing rats with 5-azacytidine, a demethylating agent, indu ced basal as well as heavy metal-activated MT-I gene expression in the hepa toma, implying that methylation was indeed responsible for silencing the ge ne. Bisulfite genomic sequencing showed significant (>90%) demethylation of CPG dinucleotides spanning MT-I promoter in the hepatoma following treatme nt with 5-AzaC, The hypermethylation of MT-I promoter was probably caused b y significantly higher (as much as 7-fold) level of DNA methyl transferase activity as well as enhanced expression of its gene in the hepatoma relativ e to the host liver. These data elucidated for the first time the molecular mechanism for the silencing of a highly inducible gene in a solid tumor tr ansplanted in an animal, as compared with the robust induction in the corre sponding parental tissue and have discussed the probable reasons for the su ppression of this gene in some tumors.