The robust induction of metallothionein-I and II (MT-I and MT-II) genes by several heavy metals such as zinc and cadmium requires the specific transcription factor metal-responsive transcription factor 1 (MTF1). Chromium (VI), a major environmental carcinogen, not only failed to activate these genes but also inhibited their induction by Zn 2؉ or Cd 2؉ . The heavy metal-induced expression of another MTF1 target gene, zinc transporter 1 (ZnT-1), was also down-regulated by Cr 6؉ . By contrast, the expression of two MTF1-independent Cd 2؉ -inducible genes, heme oxygenase 1 (HO-1) that was augmented in presence Cr 6؉ , whereas the onset of apoptosis was significantly delayed in cells overexpressing MTF1.Chromium, a potential human mutagen and carcinogen (1, 2), exists in many different oxidation states in the environment, Cr 6ϩ and Cr 3ϩ being the most stable forms. In contrast to Cr 6ϩ , Cr 3ϩ is generally benign and is considered an essential nutrient required for normal sugar and fat metabolism (3, 4). Although the predominant natural source of Cr 3ϩ is present in the environment, the majority of Cr 6ϩ originates from industrial activities. Hexavalent chromium, a powerful oxidant, is actively internalized by the anion transporter located on the plasma membrane (5). Subsequently, Cr 6ϩ is reduced through the reactive Cr 5ϩ and Cr 4ϩ intermediates to trivalent chromium, (6, 7), which are highly impermeable to cell membrane.The molecular mechanism(s) for the Cr 6ϩ -mediated toxicity has not been fully elucidated. The chromium intermediates appear to interact directly with cellular constituents that lead to generation of reactive oxygen species (8, 9). Chromium treatment results in DNA strand breakage (10, 11) and DNA-protein cross-linking in vivo (12, 13). Reactive oxygen species generated during intracellular reduction of Cr 6ϩ affects almost every aspect of cellular function. Damage to cellular macromolecules and aberrations in gene expression ultimately lead to apoptosis or necrosis in the majority of the cells and uncontrolled cell proliferation in a few, causing cancer. Apoptosis induced by Cr 6ϩ treatment in lung epithelial cells involves both p53-dependent and p53-independent pathways (14, 15). Cr 6ϩ also activates a stress response protein (NF-B), which in turn activates anti-apoptotic proteins (16), thus protecting cells from apoptotic cell death.Our laboratory has been studying the molecular mechanisms of heavy metal-induced expression of metallothionein genes (17). Metallothionein I and II (MT-I 1 and MT-II) belong to a family of low molecular weight, cysteine-rich, and high metalcontaining (as metal-thiolate clusters) stress response proteins that protect cells not only from heavy metals but also from reactive oxygen species (18,19). Metallothioneins induced in response to heavy metals like zinc, cadmium, copper, mercury, gold, silver, cobalt, nickel, and bismuth act as scavengers of these toxic metals and help maintain zinc and copper homeostasis (17,20). It was of interest to examine the induction of ...

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Metallothioneins (MTs) are a group of cysteine-rich stress response proteins that scavenge reactive oxygen species and heavy metals. Recently, we have shown that MT-I promoter is methylated and suppressed in some solid and liquid tumors and can be robustly activated following treatment with inhibitors of DNA methyltransferase (DNMT) and histone deacetylase (HDAC). Here, we have analyzed MT-I chromatin structure in active, unmethylated (Hepa cells) and in repressed, methylated state (lymphosarcoma cells). Restriction enzyme accessibility assay showed that the MT-I promoter has an open conformation in unmethylated state as opposed to refractory chromatin structure in methylated state. Positioning of nucleosomal arrays on the methylated promoter further confirmed the closed chromatin structure of the methylated promoter. Chromatin immunoprecipitation (ChIP) assay demonstrated that the unmethylated promoter is associated with K9-acetyl, K4-methyl, and S10-phospho histone H3 whereas the methylated promoter is predominantly associated with K9-methyl H3. HP1alpha that recognizes K9-methyl H3 inhibited methylated MT-1 promoter activity whereas closely related HP1gamma repressed the promoter irrespective of its methylation status. Ubiquitously expressed DNA methyltransferase 1 (DNMT1) suppressed MT-I promoter activity irrespective of its methylation status that does not require its catalytic activity. The DNMT1-mediated repression of MT-I promoter was relieved by trichostatin A, an HDAC inhibitor. Among the methyl CpG binding proteins, MBD2 and MBD4 specifically associated with the methylated promoter and inhibited its activity. In contrast, MBD1 and MeCP2 interacted with both promoters and suppressed the promoter activity irrespective of its methylation status. These results demonstrate that the methylated and unmethylated MT-I promoter are differentially regulated by DNA methyltransferase and methyl-CpG binding proteins, and DNMT1 could suppress MT promoter by a transcriptional mechanism independent of its enzymatic function. These studies suggest that the components of epigenetic machinery differentially regulate methylated and unmethylated MT-I gene expression.

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