Using the technique of genomic footprinting, we demonstrate cadmium-inducible protection from dimethyl sulfate (DMS) modification of guanine residues in vivo in five metal-responsive elements (MREs) Studies on eucaryotic gene promoters indicate that they consist of extended regions of DNA containing different functional elements. The metallothionein (MT) genes provide a good example of eucaryotic promoter architecture. MT genes specify the synthesis of low-molecular-weight metal-binding proteins. They are transcriptionally regulated by the metal ions cadmium and zinc (11), glucocorticoid hormones (18), interferon (14), interleukin-1 (22), and tumor promoters (2). The metal ion regulation of MTs is conferred by a short sequence element called the metal-responsive element (MRE [21]) or TGC box (31, 34), which functions as a metal ion-dependent enhancer. Studies with the MT promoter by using deletion mutation analysis (21, 31) or the introduction of synthetic DNA sequence elements (35) indicate that the presence of repeated copies of the MRE confers metal ion regulation of transcription. MREs also confer metal responsiveness on heterologous promoters normally not inducible by metal ions (21,30,35). It has recently been reported that a nuclear factor(s) binds to the mouse MT-1 promoter in the presence of metal ions in vitro (31). No evidence has yet been presented which demonstrates the binding of a transcription factor to MREs in vivo. In addition, DNase hypersensitivity analysis does not show a metal ion-inducible hypersensitive site overlapping the rat MT-1 MREs (1, 37). We chose the methodology of genomic sequencing (8, 9) with the chemical reagent dimethyl sulfate (DMS) on intact cells to observe changes in the reactivity of the N-7 position of guanine residues in the promoter of the MT-1 gene in vivo after treatment of the cells with cadmium ions. Transfection experiments with portions of the MT-1 promoter fused to a reporter gene were used to correlate the functional roles of these sequences with the observed changes in their DMS reactivity. We identify a trans-acting factor(s) which binds to MRE sequences only after metal ion * Corresponding author.induction. We are able to follow the kinetics of binding or dissociation of this factor in vivo after cells are exposed to cadmium ions or upon removal of this inducer from culture medium. Additional evidence suggests that the basal level of MT gene expression is regulated by another transcription factor, possibly Spl. MATERIALS AND METHODSCell lines. Fao cells, a rat hepatoma cell line (10), were maintained in alpha medium (Irvine Scientific) supplemented with 10% fetal calf serum (Gemini Bio-Products). CdR cells are Fao cells selected for growth in 50 ,uM CdCl2. This selection causes a five-to sixfold amplification of the MT genes (S. J. Taplitz and H. R. Herschman, manuscript in preparation). Stocks of CdR cells were maintained by growth and passage in medium containing 50 ,uM CdCl2.Preparation of DNA and genomic footprint analysis. CdR cells were grown for 1...

Genomic footprinting studies in vivo and experiments using synthetic metal regulatory elements (MREs) in vitro suggest protein binding to the MREs of the mouse and rat metallothionein I (MT-I) genes. Using gel-retardation assays of promoter fragments, we observe a cadmium-dependent binding factor for the rat MT-I promoter in rat hepatoma cells. This factor is present in extracts from both uninduced and cadmium-induced cells, but requires the presence of cadmium to bind to the promoter. The formation of a cadmium-dependent complex is competed by an oligonucleotide containing two MREs. This competition is lost when when one of the MREs is mutated, indicating a requirement for at least two MREs for binding of this factor. The cadmium-dependent factor dissociates more rapidly from the MT-I promoter than does a factor that binds to a consensus Sp1 site present on the same DNA fragment. UV crosslinking analysis using nuclear extracts from cadmium induced cells, in the presence of an oligonucleotide probe containing both 5-bromodeoxyuridine and 32P-deoxycytidine, identifies a 39 kDalton protein associated with the metal inducible complex.

We examined in the H4IIE rat hepatoma cell line the relationship between butyrate-induced changes in the nuclease sensitivity of chromatin and changes in transcriptional activity of specific genes. The butyrate-inducible metallothionein I (MT-I) gene underwent a dramatic increase in DNase I sensitivity after 3 h of butyrate treatment. However, genes not transcribed in H4IIE cells underwent the same changes in DNase I sensitivity. Thus, butyrate-induced increases in DNase I sensitivity are not sufficient for the transcriptional activation of a gene. Butyrate treatment has also been reported to alter the sensitivity of sequences to micrococcal nuclease (MNase) in a manner reflecting their tissue-specific expression. Butyrate exposure caused increased digestion of the MT-I gene by MNase. However, butyrate-induced MNase sensitivity also occurred for genes which are neither transcribed in untreated cells nor butyrate inducible. Moreover, cadmium, a potent transcriptional activator of the MT-I gene, does not alter the sensitivity of the MT-I gene to MNase. Thus, the butyrate-induced alterations in MNase sensitivity are neither sufficient for, necessary for, nor indicative of transcriptional activation.

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