The DNA Binding Activity of Metal Response Element-binding Transcription Factor-1 Is Activated in vivo and in Vitro by Zinc, but Not by Other Transition Metals

Abstract

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...

Role of oxidative stress in the induction of metallothionein-2A and heme oxygenase-1 gene expression by the antineoplastic agent gallium nitrate in human lymphoma cells

“…However, it is unlikely that the increase in MTF-1-MRE binding in cells exposed to gallium nitrate is directly facilitated by gallium. Bittel et al have shown that zinc, but not a variety of other metals, can activate MTF-1-MRE binding in vitro [33]. Based on those studies, we considered the possibility that MTF-1 activation in gallium-treated cells involved changes in cellular zinc homeostasis.…”

“…It is well known that an increase in cellular zinc content may upregulate metallothionein gene expression by increasing the binding of MTF-1, a zinc finger transcription factor, to MREs present on the promoter region of the metallothionein gene [33,34]. To determine whether gallium produced changes in cellular zinc homeostasis, cells exposed to zinc sulfate or gallium nitrate were allowed to incorporate Zinquin, a zinc-specific fluorophore that fluoresces upon binding to zinc, and then examined by fluorescence microscopy.…”

Metal-responsive transcription factor (MTF-1) and heavy metal stress response in Drosophila and mammalian cells: a functional comparison

“…We have recently identified and characterized dMTF-1, the MTF-1 homolog of the fruit fly Drosophila (Zhang et al, 2001;Egli et al, 2003). In both mammals and Drosophila, MTF-1 requires zinc and its function is compromised by direct exposure to other heavy metals (Bittel et al, 1998;Giedroc et al, 2001;Zhang et al, 2001). Accordingly, in a cell-free transcription system, MTF-1 activation by cadmium and copper (and H 2 O 2 ) was shown to occur indirectly, via release of zinc from metallothionein (for convenience, the designations Zn or zinc, Cu or copper, Cd or cadmium and Hg or mercury are also used here to denote Zn 2q , Cu 2q , Cd 2q and Hg 2q , respectively.)…”

A molecular technique for detecting the liberation of intracellular zinc in cultured neurons

“…Zinc, but not other heavy metals, increases the binding activity of MTF-1 to MRE, which is present in multiple copies in the promoter region of metal-lothionein genes (Bittel et al, 1998;Koizumi et al, 1992Koizumi et al, , 1999Zhang et al, 2003). As such, MTF-1 serves as a selective intracellular Zn 2+ sensor to activate MT gene expression (Giedroc et al, 2001;Smirnova et al, 2000;Zhang et al, 2003).…”