Identification of multiple metal regulatory elements in mouse metallothionein-I promoter by assaying synthetic sequences

Stuart, Gary W.; Searle, Peter F.; Palmiter, Richard D.

doi:10.1038/317828a0

Cited by 347 publications

(253 citation statements)

References 17 publications

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“…As up to five tandem copies of MRE have been used previously for optimal hypoxic induction, 8,37,38 we have yet to evaluate the consequences of three to five copies of MRE or EBS. Therefore, there is still room for further improvement of the E-M-H construct.…”

Section: Discussionmentioning

confidence: 99%

A novel chimeric promoter that is highly responsive to hypoxia and metals

et al. 2006

Gene Ther

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To develop a potent hypoxia-inducible promoter, we evaluated the usefulness of chimeric combinations of the (Egr-1)-binding site (EBS) from the Egr-1 gene, the metal-response element (MRE) from the metallothionein gene, and the hypoxia-response element (HRE) from the phosphoglycerate kinase 1 gene. In transient transfection assays, combining three copies of HRE (3 Â HRE) with either EBS or MRE significantly increased hypoxia responsiveness. When a three-enhancer combination was tested, the EBS-MRE-3 Â HRE (E-M-H) gave a hypoxia induction ratio of 69. The expression induced from E-M-H-pGL3 was 2.4-fold higher than that induced from H-pGL3 and even surpassed the expression from a human cytomegalovirus promoter-driven vector. The high inducibility of E-M-H was confirmed by validation studies in different cells and by expressing other cDNAs. Gel shift assays together with functional overexpression studies suggested that increased levels of hypoxiainducible factor 1a, metal transcription factor-1 and Egr-1 may be associated with the high inducibility of the E-M-H chimeric promoter. E-M-H was also induced by hypoxia mimetics such as Co 2+ and deferoxamine (DFX) and by hydrogen peroxide. Gene expression from the E-M-H was reversible as shown by the reduced expression of the transgene upon removal of inducers such as hypoxia and DFX. In vivo evaluation of the E-M-H in ischemic muscle revealed that erythropoietin secretion and luciferase and LacZ expression were significantly higher in the E-M-H group than in a control or H group. With its high induction capacity and versatile means of modulation, this novel chimeric promoter should find wide application in the treatment of ischemic diseases and cancer.

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Section: Discussionmentioning

confidence: 99%

A novel chimeric promoter that is highly responsive to hypoxia and metals

et al. 2006

Gene Ther

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“…MTs are small, cysteine-rich proteins with the ability to complex heavy metals via their sulfhydryl groups. The enhancer-promoter regions of the genes encoding mammalian metallothioneins contain multiple characteristic DNA sequence motifs (TGCRCNC), so-called metal response elements (MREs) which are essential for metalinduced transcription (Stuart et al, 1984(Stuart et al, , 1985.…”

Section: Introductionmentioning

confidence: 99%

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

Balamurugan

Egli²,

Selvaraj³

et al. 2004

Biological Chemistry

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The zinc finger transcription factor MTF-1 (metal-responsive transcription factor-1) is conserved from insects to vertebrates. Its major role in both organisms is to control the transcription of genes involved in the homeostasis and detoxification of heavy metal ions such as Cu 2q , Zn 2q and Cd 2q . In mammals, MTF-1 serves at least two additional roles. First, targeted disruption of the MTF-1 gene results in death at embryonic day 14 due to liver degeneration, revealing a stage-specific developmental role. Second, under hypoxic-anoxic stress, MTF-1 helps to activate the transcription of the gene placental growth factor (PlGF), an angiogenic protein. Recently we characterized dMTF-1, the Drosophila homolog of mammalian MTF-1. Here we present a series of studies to compare the metal response in mammals and insects, which reveal common features but also differences. A human MTF-1 transgene can restore to a large extent metal tolerance to flies lacking their own MTF-1 gene, both at low and high copper concentrations. Likewise, Drosophila MTF-1 can substitute for human MTF-1 in mammalian cell culture, although both the basal and the metal-induced transcript levels are lower. Finally, a clear difference was revealed in the response to mercury, a highly toxic heavy metal: metallothionein-type promoters respond poorly, if at all, to Hg 2q in mammalian cells but strongly in Drosophila, and this response is completely dependent on dMTF-1.

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“…In animals, MT genes are regulated transcriptionally by metal-regulatory elements (MREs), which are present in multiple copies (Stuart et al, 1985). MREs consist of a highly conserved heptanucleotide core (5#-TGCRCNC-3#) and less conserved flanking nucleotides (Searle et al, 1987).…”

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confidence: 99%

Characterization of a Novel Plant Promoter Specifically Induced by Heavy Metal and Identification of the Promoter Regions Conferring Heavy Metal Responsiveness

Zhang

Chai

2006

Plant Physiology

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The bean (Phaseolus vulgaris) stress-related gene number 2 (PvSR2) gene responds to heavy metals but not to other forms of environmental stresses. To elucidate its heavy metal-regulatory mechanism at the transcriptional level, we isolated and characterized the promoter region (21623/148) of PvSR2. Deletions from the 5# end revealed that a sequence between 2222 and 2147 relative to the transcriptional start site was sufficient for heavy metal-specific induction of the promoter region of PvSR2. Detailed analysis of this 76-bp fragment indicated that heavy metal-responsive elements were localized in two regions (2222/2188 and 2187/2147), each of which could separately confer heavy metal-responsive expression on the b-glucuronidase gene in the context of a minimal cauliflower mosaic virus 35S promoter. Region I (2222/2188) contains a motif (metalregulatory element-like sequence) similar to the consensus metal-regulatory element of the animal metallothionein gene, and mutation of this motif eliminated the heavy metal-inducible function of region I. Region II (2187/2147) had no similarity to previously identified cis-acting elements involved in heavy metal induction, suggesting the presence of a novel heavy metalresponsive element. Transformed tobacco (Nicotiana tabacum) seedlings expressing b-glucuronidase under control of the PvSR2 promoter region (2687/148) showed heavy metal-specific responsive activity that depended on the type and concentration of the heavy metal and the type of organ. These findings further our understanding of the regulation of PvSR2 expression and provide a new heavy-metal-inducible promoter system in transgenic plants.In plants, a number of heavy metals are essential for normal growth as cofactors and as structural and catalytic components of proteins and enzymes. These micronutrients, as well as nonessential heavy metals such as cadmium, are toxic at high levels. Plants have evolved a suite of mechanisms that control and respond to the uptake and accumulation of both essential and nonessential heavy metals. These mechanisms include the chelation and sequestration of heavy metals by particular ligands including small peptides, organic acids, and amino acids, which bind free metal ions. They contribute to metal detoxification by buffering cytosolic metal ions (Clemens, 2001). The two best-characterized heavy metal-binding ligands in plant cells are the phytochelatins (PCs) and metallothioneins (MTs; for review, see Cobbett and Goldsborough, 2002). MTs are Cys-rich polypeptides encoded by a family of genes. In contrast, PCs are a family of enzymatically synthesized Cys-rich peptides. The induction of PC synthesis is based on the posttranscriptional activation of preformed PC synthase and is not under transcriptional control.Several heavy metal-inducible genes have been reported in plants (Hagen et al., 1988;Lescure et al., 1991;Berna and Bernier, 1999), but surprisingly, little is known about the transcriptional regulation of gene expression in response to heavy metals. In particular, the cis-...

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Identification of multiple metal regulatory elements in mouse metallothionein-I promoter by assaying synthetic sequences

Cited by 347 publications

References 17 publications

A novel chimeric promoter that is highly responsive to hypoxia and metals

A novel chimeric promoter that is highly responsive to hypoxia and metals

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

Characterization of a Novel Plant Promoter Specifically Induced by Heavy Metal and Identification of the Promoter Regions Conferring Heavy Metal Responsiveness

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