Oleg Georgiev

The transcription factor MTF-1 is essential for basal and heavy metal-induced metallothionein gene expression.

We have described and cloned previously a factor (MTF‐1) that binds specifically to heavy metal‐responsive DNA sequence elements in the enhancer/promoter region of metallothionein genes. MTF‐1 is a protein of 72.5 kDa that contains six zinc fingers and multiple domains for transcriptional activation. Here we report the disruption of both alleles of the MTF‐1 gene in mouse embryonic stem cells by homologous recombination. The resulting null mutant cell line fails to produce detectable amounts of MTF‐1. Moreover, due to the loss of MTF‐1, the endogenous metallothionein I and II genes are silent, indicating that MTF‐1 is required for both their basal and zinc‐induced transcription. In addition to zinc, other heavy metals, including cadmium, copper, nickel and lead, also fail to activate metal‐responsive promoters in null mutant cells. However, cotransfection of an MTF‐1 expression vector and metal‐responsive reporter genes yields strong basal transcription that can be further boosted by zinc treatment of cells. These results demonstrate that MTF‐1 is essential for metallothionein gene regulation. Finally, we present evidence that MTF‐1 itself is a zinc sensor, which exhibits increased DNA binding activity upon zinc treatment.

Cloned transcription factor MTF-1 activates the mouse metallothionein I promoter.

Metallothioneins (MTs) are small cysteine‐rich proteins whose structure is conserved from fungi to man. MTs strongly bind heavy metals, notably zinc, copper and cadmium. Upon exposure of cells to heavy metal and other adverse treatments, MT gene transcription is strongly enhanced. Metal induction is mediated by several copies of a 15 bp consensus sequence (metal‐responsive element, MRE) present in the promoter region of MT genes. We and others have demonstrated the presence of an MRE‐binding factor in HeLa cell nuclear extracts. We found that this factor, termed MTF‐1 (MRE‐binding transcription factor) is inactivated/reactivated in vitro by zinc withdrawal/addition. Here we report that the amounts of MTF‐1‐DNA complexes are elevated several‐fold in zinc‐treated cells, as measured by bandshift assay. We have also cloned the cDNA of mouse MTF‐1, a 72.5 kDa protein. MTF‐1 contains six zinc fingers and separate transcriptional activation domains with high contents of acidic and proline residues. Ectopic expression of MTF‐1 in primate or rodent cells strongly enhances transcription of a reporter gene that is driven by four consensus MREd sites, or by the complete mouse MT‐I promoter, even at normal zinc levels.

Different activation domains stimulate transcription from remote ('enhancer') and proximal ('promoter') positions.

We reported previously that the lymphocyte‐derived octamer transcription factor 2A (Oct‐2A or OTF‐2A) activated both natural immunoglobulin promoters and synthetic promoters which contain the ‘octamer’ site, but was unable by itself to stimulate transcription from a remote enhancer position. Here we examine a larger set of transcription factors with respect to their proximal versus remote activation. Since a transcription factor may contain more than one activation domain, we have chosen to study the potential of individual activation domains in the context of fusion proteins that contain the DNA binding domain of GALA. We have identified at least two distinct functional classes of transcriptional activation domains. ‘Proximal’ activation domains, exemplified by glutamine‐rich domains of Oct‐1, Oct‐2A and Sp1, stimulate transcription only from a position close to the TATA box, usually in response to a remote enhancer. ‘General’ activation domains, derived from VP16, GAL4, p65 (NF‐chi B), TFE3, ITF‐1 and ITF‐2, can activate transcription from remote as well as proximal positions. These domains contain many acidic amino acids and/or other features such as clusters of serine and threonine. The proline‐rich activation domains of AP‐2 and CTF/NF1 may represent a third class with considerable promoter activity and low but significant enhancer activity. Furthermore, activation domains of both the acidic and glutamine‐rich types seem to have a modular structure, since duplicated subdomains can substitute for the entire domain.

Embryonic lethality and liver degeneration in mice lacking the metal-responsive transcriptional activator MTF-1.

We have shown previously that the heavy metal‐responsive transcriptional activator MTF‐1 regulates the basal and heavy metal‐induced expression of metallothioneins. To investigate the physiological function of MTF‐1, we generated null mutant mice by targeted gene disruption. Embryos lacking MTF‐1 die in utero at approximately day 14 of gestation. They show impaired development of hepatocytes and, at later stages, liver decay and generalized edema. MTF‐1−/− embryos fail to transcribe metallothionein I and II genes, and also show diminished transcripts of the gene which encodes the heavy‐chain subunit of the γ‐glutamylcysteine synthetase, a key enzyme for glutathione biosynthesis. Metallothionein and glutathione are involved in heavy metal homeostasis and detoxification processes, such as scavenging reactive oxygen intermediates. Accordingly, primary mouse embryo fibroblasts lacking MTF‐1 show increased susceptibility to the cytotoxic effects of cadmium or hydrogen peroxide. Thus, MTF‐1 may help to control metal homeostasis and probably cellular redox state, especially during liver development. We also note that the MTF‐1 null mutant phenotype bears some similarity to those of two other regulators of cellular stress response, namely c‐Jun and NF‐κB (p65/RelA).

Activity of Metal-Responsive Transcription Factor 1 by Toxic Heavy Metals and H2O2 In Vitro Is Modulated by Metallothionein

ABSTRACT Metallothioneins are small, cysteine-rich proteins that avidly bind heavy metals such as zinc, copper, and cadmium to reduce their concentration to a physiological or nontoxic level. Metallothionein gene transcription is induced by several stimuli, notably heavy metal load and oxidative stress. Transcriptional induction of metallothionein genes is mediated by the metal-responsive transcription factor 1 (MTF-1), an essential zinc finger protein that binds to specific DNA motifs termed metal-response elements. In cell-free DNA binding reactions with nuclear extracts, MTF-1 requires elevated zinc concentrations for efficient DNA binding but paradoxically is inactivated by other in vivo inducers such as cadmium, copper, and hydrogen peroxide. Here we have developed a cell-free, MTF-1-dependent transcription system which accurately reproduces the activation of metallothionein gene promoters not only by zinc but also by these other inducers. We found that while transcriptional induction by zinc can be achieved by elevated zinc concentration alone, induction by cadmium, copper, or H2O2 additionally requires the presence of zinc-saturated metallothionein. This is explained by the preferential binding of cadmium or copper to metallothionein or its oxidation by H2O2; the concomitant release of zinc in turn leads to the activation of transcription factor MTF-1. Conversely, thionein, the metal-free form of metallothionein, inhibits activation of MTF-1. The release of zinc from cellular components, including metallothioneins, and the sequestration of zinc by newly produced apometallothionein might be a basic mechanism to regulate MTF-1 activity upon cellular stress.

The Drosophila homolog of mammalian zinc finger factor MTF-1 activates transcription in response to heavy metals.

ABSTRACT Metallothioneins (MTs) are short, cysteine-rich proteins for heavy metal homeostasis and detoxification; they bind a variety of heavy metals and also act as radical scavengers. Transcription of mammalian MT genes is activated by heavy metal load via the metal-responsive transcription factor 1 (MTF-1), an essential zinc finger protein whose elimination in mice leads to embryonic lethality due to liver decay. Here we characterize the Drosophila homolog of vertebrate MTF-1 (dMTF-1), a 791-amino-acid protein which is most similar to its mammalian counterpart in the DNA-binding zinc finger region. Like mammalian MTF-1, dMTF-1 binds to conserved metal-responsive promoter elements (MREs) and requires zinc for DNA binding, yet some aspects of heavy metal regulation have also been subject to divergent evolution between Drosophila and mammals. dMTF-1, unlike mammalian MTF-1, is resistant to low pH (6 to 6.5). Furthermore, mammalian MT genes are activated best by zinc and cadmium, whereas in Drosophila cells, cadmium and copper are more potent inducers than zinc. The latter species difference is most likely due to aspects of heavy metal metabolism other than MTF-1, since in transfected mammalian cells, dMTF-1 responds to zinc like mammalian MTF-1. Heavy metal induction of bothDrosophila MTs is abolished by double-stranded RNA interference: small amounts of cotransfected double-stranded RNA ofdMTF-1 but not of unrelated control RNA inhibit the response to both the endogenous dMTF-1 and transfected dMTF-1. These data underline an important role for dMTF-1 in MT gene regulation and thus heavy metal homeostasis.

Two major branches of anti-cadmium defense in the mouse: MTF-1/metallothioneins and glutathione

Metal-responsive transcription factor 1 (MTF-1) regulates expression of its target genes in response to various stress conditions, notably heavy metal load, via binding to metal response elements (MREs) in the respective enhancer/promoter regions. Furthermore, it serves a vital function in embryonic liver development. However, targeted deletion of Mtf1 in the liver after birth is no longer lethal. For this study, Mtf1 conditional knockout mice and control littermates were both mock- or cadmium-treated and liver-specific transcription was analyzed. Besides the well-characterized metallothionein genes, several new MTF-1 target genes with MRE motifs in the promoter region emerged. MTF-1 is required for the basal expression of selenoprotein W, muscle 1 gene (Sepw1) that encodes a glutathione-binding and putative antioxidant protein, supporting a role of MTF-1 in the oxidative stress response. Furthermore, MTF-1 mediates the cadmium-induced expression of N-myc downstream regulated gene 1 (Ndrg1), which is induced by several stress conditions and is overexpressed in many cancers. MTF-1 is also involved in the cadmium response of cysteine- and glycine-rich protein 1 gene (Csrp1), which is implicated in cytoskeletal organization. In contrast, MTF-1 represses the basal expression of Slc39a10, a putative zinc transporter. In a pathway independent of MTF-1, cadmium also induced the transcription of genes involved in the synthesis and regeneration of glutathione, a cadmium-binding antioxidant. These data provide strong evidence for two major branches of cellular anti-cadmium defense, one via MTF-1 and its target genes, notably metallothioneins, the other via glutathione, with an apparent overlap in selenoprotein W.

The B cell coactivator Bob1 shows DNA sequence-dependent complex formation with Oct-1/Oct-2 factors, leading to differential promoter activation.

We have shown previously that both octamer binding transcription factors, namely the ubiquitous Oct‐1 and the B cell‐specific Oct‐2A protein, can be enhanced in transcriptional activity by their association with the B cell‐specific coactivator protein Bob1, also called OBF‐1 or OCA‐B. Here we study the structural requirements for ternary complex formation of DNA‐Oct‐Bob1 and coactivation function of Bob1. In analogy to DNA‐bound transcription factors, Bob1 has a modular structure that includes an interaction domain (amino acids 1–65) and a C‐terminal domain (amino acids 65–256), both important for transcriptional activation. A mutational analysis has resolved a region of seven amino acids (amino acids 26–32) in the N‐terminus of Bob1 that are important for contacting the DNA binding POU domain of Oct‐1 or Oct‐2. In contrast to the viral coactivator VP16 (vmw65), which interacts with Oct‐1 via the POU homeosubdomain, Bob1 association with Oct factors requires residues located in the POU‐specific subdomain. Because the same residues are also involved in DNA recognition, we surmised that this association would affect the DNA binding specificity of the Oct‐Bob1 complex compared with free Oct factors. While Oct‐1 or Oct‐2 bind to a large variety of octamer sequences, Bob1 ternary complex formation is indeed highly selective and occurs only in a subset of these sequences, leading to the differential coactivation of octamer‐containing promoters. The results uncover a new level in selectivity that furthers our understanding in the regulation of cell type‐specific gene expression.

A recent evolutionary change affects a regulatory element in the human FOXP2 gene

The FOXP2 gene is required for normal development of speech and language. By isolating and sequencing FOXP2 genomic DNA fragments from a 49,000-year-old Iberian Neandertal and 50 present-day humans, we have identified substitutions in the gene shared by all or nearly all present-day humans but absent or polymorphic in Neandertals. One such substitution is localized in intron 8 and affects a binding site for the transcription factor POU3F2, which is highly conserved among vertebrates. We find that the derived allele of this site is less efficient than the ancestral allele in activating transcription from a reporter construct. The derived allele also binds less POU3F2 dimers than POU3F2 monomers compared with the ancestral allele. Because the substitution in the POU3F2 binding site is likely to alter the regulation of FOXP2 expression, and because it is localized in a region of the gene associated with a previously described signal of positive selection, it is a plausible candidate for having caused a recent selective sweep in the FOXP2 gene.

Knockout of ‘metal-responsive transcription factor’ MTF-1 in Drosophila by homologous recombination reveals its central role in heavy metal homeostasis

‘Metal‐responsive transcription factor‐1’ (MTF‐1), a zinc finger protein, is conserved from mammals to insects. In the mouse, it activates metallothionein genes and other target genes in response to several cell stress conditions, notably heavy metal load. The knockout of MTF‐1 in the mouse has an embryonic lethal phenotype accompanied by liver degeneration. Here we describe the targeted disruption of the MTF‐1 gene in Drosophila by homologous recombination. Unlike the situation in the mouse, knockout of MTF‐1 in Drosophila is not lethal. Flies survive well under laboratory conditions but are sensitive to elevated concentrations of copper, cadmium and zinc. Basal and metal‐induced expression of Drosophila metallothionein genes MtnA (Mtn) and MtnB (Mto), and of two new metallothionein genes described here, MtnC and MtnD, is abolished in MTF‐1 mutants. Unexpectedly, MTF‐1 mutant larvae are sensitive not only to copper load but also to copper depletion. In MTF‐1 mutants, copper depletion prevents metamorphosis and dramatically extends larval development/lifespan from normally 4–5 days to as many as 32 days, possibly reflecting the effects of impaired oxygen metabolism. These findings expand the roles of MTF‐1 in the control of heavy metal homeostasis.