We examined the DNA binding activity of mouse and human MTF-1 in whole cell extracts from cells cultured in medium containing zinc or cadmium and from untreated cells after the in vitro addition of zinc or cadmium, as well as using recombinant MTF-1 transcribed and translated in vitro and treated with various transition metals. Incubation of human (HeLa) or mouse (Hepa) cells in medium containing cadmium (5-15 M) did not lead to a significant increase (<2-fold) in the amount of MTF-1 DNA binding activity, whereas zinc (100 M) led to a 6 -15-fold increase within 1 h. MTF-1 binding activity was low, but detectable, in control whole cell extracts and was increased (>10-fold) after the in vitro addition of zinc (30 M) and incubation at 37°C for 15 min. In contrast, addition of cadmium (6 or 60 M) did not activate MTF-1 binding activity. Recombinant mouse and human MTF-1 were also dependent on exogenous zinc for DNA binding activity. Cadmium did not facilitate activation of recombinant MTF-1, but instead inhibited the activation of the recombinant protein by zinc. Interestingly, glutathione (1 mM) protected recombinant MTF-1 from inactivation by cadmium, and allowed for activation by zinc. It was also noted that zinc-activated recombinant MTF-1 was protected from cadmium only when bound to DNA. These results suggest that cadmium interacts with the zinc fingers of MTF-1 and forms an inactive complex. Of the several transition metals (zinc, cadmium, nickel, silver, copper, and cobalt) examined, only zinc facilitated activation of the DNA binding activity of recombinant MTF-1. These data suggest that transition metals, other than zinc, that activate MT gene expression may do so by mechanisms independent of an increase in the DNA binding activity of MTF-1. Metallothioneins (MT)1 constitute a conserved family of cysteine-rich heavy metal-binding proteins (1). In the mouse, MT-I and MT-II display a wide tissue distribution and have been demonstrated to participate in detoxification of transition metals such as cadmium (2, 3), zinc homeostasis (4), and protection against oxidative stress (5). MT-I and MT-II gene transcription is induced dramatically by heavy metals (especially zinc and cadmium) (6). Metal response elements (MRE) are essential for this induction, and these elements are present in multiple copies in the proximal promoters of MT genes. MREs were initially shown to mediate transcriptional response of MT genes to zinc and cadmium (7-9), and more recently to oxidative stress (10, 11).A protein that binds specifically to MREs and that transactivates MT gene expression has been cloned from mouse and human, and is termed MTF-1 (MRE-binding transcription factor-1) (12, 13). MTF-1 is a zinc finger transcription factor in the Cys 2 His 2 family. The DNA binding activity of MTF-1 is reversibly regulated by zinc interactions with the zinc finger domain (14). In contrast with some zinc finger proteins, including zinc finger transcription factors, which can bind zinc with picomolar to nanomolar disassociation constants (15...

No abstract

In this study, the contributions of the two metal-responsive elements (MREs) of the rainbow trout (Salmo gairdnerii) metallothionein (tMT)-B gene promoter (-137 to +5) were analyzed. The effect of MRE mutations on the basal and zinc-induced activities of tMT-B promoter-reporter gene fusions were determined by transfection of a rainbow trout hepatoma (RTH-149) cell line. Together, MREa and MREb cooperate to elicit a significant response to zinc but exhibit differential basal and metal-induced activity. The MREa sequence (-62 to -51) is important for basal promoter activity and can function independently, whereas the more distal MREb (-89 to -100) mainly contributes to metal induction through cooperative interactions with MREa. The degree of basal character of the MREs is partially determined by nucleotide differences at the flexible position N of the MRE consensus TGC(G/A)CNC. In mouse L and HepG2 cells, MREa activity is conserved, but the contributions of the MREb region differ, including reduced cooperativity with MREa. There are also differences in the apparent molecular masses of the rainbow trout and mammalian nuclear factors that bind to the tMT-B promoter and MREa sequence.

No abstract

We have analyzed the human (h) metallothionein (MT)-IG proximal promoter region (؊174 to ؉5) using a TATA box mutation (TATCA) and four trinucleotide mutants of the proximal MREa. Transient transfection of HepG2 cells was complemented by in vitro transcription with rat liver nuclear extracts. In both systems, mutations of the TATA box and conserved core of metal responsive element (MRE)a were detrimental to hMT-IG promoter activity suggesting that both elements make significant contributions to hMT-IG transcription. Although MRE binding factors were active in vitro, further metal activation of MT promoter activity was accomplished only by in vivo metal treatment rather than addition of zinc in vitro. Southwestern blotting identified nuclear proteins in rat liver and HepG2 cells which physically interact with MREa in a zinc-dependent manner and could be responsible for MREa function in each system. In addition, the functional effects of the TATCA mutation correlate with altered physical interaction with TATA box-binding protein as observed using DNase I protection. Metallothioneins (MTs)1 are low molecular mass (6 -7 kDa), cysteine-rich, metal-binding proteins which are thought to be important for trace metal homeostasis, as with zinc and copper, and detoxification, as with cadmium (1). Consistent with these putative functions, MTs are transcriptionally induced by these metals, mediated by multiple copies of metal-responsive elements (MREs) in the MT gene 5Ј-regulatory regions (2). MREs are 12-bp sequences which contain a heptanucleotide core sequence TGC(A/G)CNC surrounded by less conserved flanking nucleotides (2).We are interested in the regulation of the human (h)MT genes. This gene family consists of a minimum of 12 members which include both non-functional and processed pseudogenes as well as seven functional genes (3-5). The single MT-II isoform gene hMT-IIA is responsive to a variety of inducers including cytokines, growth factors, UV light, and glucocorticoids, and its regulatory region is complicated by numerous cis-acting elements which mediate the effects of such inducers (2). However, the promoters of the five hMT-I isoform genes are comparatively simple (3, 6) only containing MREs and GC boxes, which are possible binding sites for the SP1 transcription factor (3). Because of this simplicity, the hMT-I promoters are excellent candidates to study metal-regulated gene expression without complication by other inducible non-MRE elements as are found in hMT-IIA. Although the hMT-I promoters display remarkable homology of sequence and MRE organization (3), these genes exhibit cell type-specific patterns of expression as well as distinct levels of basal and metal-induced transcription within one cell type (3, 4).Previous studies of MRE contributions to transcription have employed synthetic MRE sequences fused to minimal promoters (7-9). However, we wished to examine MRE contributions to transcription regulation in the context of native MT promoter organization and sequences. For this purpose, we have chosen...

No abstract