Contribution

1 More information about MTF-1

Metal response transcription factor-1 (MTF-1, also called MRE-binding transcription factor-1) is a multi-functional transcriptional regulator that participates in cellular adaptation to various stress conditions, primarily exposed to heavy metals, but also to hypoxia or oxidative stress. MTF-1 is evolutionarily conserved from insects to humans and is the main activator of metallothionein genes, which encode small proteins rich in cysteine that can remove toxic heavy metals and free radicals. In the early stage of the project, we obtained a lot of information about MTF-1 through literature review. We would like to share this information here.

Most animals have mechanisms to deal with heavy metal stress. These species contain small, cysteine-rich proteins, called metallothioneins (MTs), to bind heavy metals for redistribution and/or detoxification (DeMoo 2010; Kägi 1987,1991; Simpkins 2000). Vertebrates contain four types of MTs, of which MT-I and MT-II are stress induced and expressed in all organs, while MT-III and MT-IV are cell type specific and respond only moderately to heavy metal load (Palmiter 1992; Uchida 1991). Thereinto, the ones related to our project are MT-I and MT-II.

The stress-induced MT-I and MT-II genes contain in their promotors multiple sequence copies called metal-responsive element (MRE) (Searle 1987; Stuart 1985). The transcription factor that binds to these MREs and confers metal inducibility are called MTF-1 (for MRE-binding transcription factor 1 or metal-responsive transcription factor 1) (Heuchel 1994; Radtke 1993; Westin 1988). MTF-1 is essential for heavy metal response and for embryonic development (Günes 1998). MTF-1, which also contributes to the activation of genes other than MTs, in resting cells localizes to the cytoplasm and enters the nucleus under stress conditions (Saydam 2001; Smirnova 2000). Zinc induction works by direct metal binding to the MTF-1 zinc fingers (Bittel 2000; Chen 1999; Dalton 1997; Heuchel 1994). Even though other metals, such as cadmium and copper, also activate transcription via MTF-1, activation must be indirect since these metals cannot replace zinc in zinc finger binding (6, 25). MTF-1 also mediates response to oxidative stress and hypoxia (Dalton 1996; Günes 1998; Murphy 1999; Yoo 1999).

The MTF-1 we used is marked as dMTF, which is the Drosophila homolog of vertebrate MTF-1. dMTF 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 (Zhang er al., 2001).

The above is some information we have obtained about MTF-1.We hope that this information will be helpful for iGEMers in future in synthetic biology, especially in research related to biological responses to heavy metals.

2 Vector construction of pUAST-MTF-1, pMRE-Hid and pMRE-GFP

(1) Construction of pUAST-MTF-1 plasmid

We took a commercialized recombinant plasmid pUAST as template, and used restrictive endonuclease (BglII and XhoI) digestion to obtain a linearized pUAST vector. MTF-1 gene fragment was amplified from the cDNA of wildtype Drosophila melanogaster by PCR. DNA electrophoresis confirmed the length of the PCR product (2376bp). MTF-1 gene fragment was ligated with the pUAST linearized vector by T4 ligase and vector pUAST-MTF-1 was obtained later.

(2) Construction of pMRE-Hid and pMRE-GFP plasmids

We also reformed the pUAST plasmid into pMRE plasmid by restrictive endonuclease (Pst1) digestion to obtain a linearized pUAST vector and then substitute UAS sequence for MRE sequence. MRE was obtained by DNA synthesis and T4 ligase was used to combine the linearized vector into complete plasmid to get the pMRE plasmid.

Start from pMRE as template, we used restrictive endonuclease (NotI and XbaI) digestion to obtain a linearized pMRE vector. Hid gene fragment was amplified from the cDNA of wildtype Drosophila melanogaster by PCR. GFP gene fragment was amplified from the plasmid of pUAST-GFP by PCR. Hid and GFP gene fragments were ligated with pMRE linearized vector by T4 ligase to obtained pMRE-Hid plasmid and pMRE-GFP plasmid , respectively.

Figure 1 Schematic representations of pUAST-MTF-1, pMRE-Hid and pMRE-GFP plasmids

Figure 2 Genetic circuits of pUAST-MTF-1, pMRE-Hid, and pMRE-GFP plasmids

3 Grasp the demand for heavy metal monitoring in remote and backward areas

For the concern of heavy metal pollution, the public is most concerned about the environment and whether the heavy metal content of products will endanger health. Therefore, people's demand for monitoring/detection of heavy metal pollution is also clear. According to Mr. Zhou and Ms. Li, environmental experts in Guangxi and Chongqing, two relatively remote and backward province in China, there are roughly these points: comprehensive monitoring, regular detection, fast detection speed and accurate detection results to minimize pollution risks.

However, the experts added that due to differences in geographical environment and economic development level, there are still problems such as insufficient funds, relatively low technical level of personnel and inconvenient transportation for monitoring/detection heavy metal pollution in remote and backward areas. On the one hand, the social and economic backwardness makes the local environmental monitoring/detection departments lack sufficient funds to purchase conventional experimental equipment and introduce and maintain high-tech personnel; On the other hand, for conventional detection technologies, samples often need to be sent to the laboratory for testing, and the backward local traffic conditions seriously limit the convenience of sampling and sample delivery by technicians.

Therefore, in the demand for heavy metal pollution monitoring/detection in remote and backward areas, in addition to the above conventional needs, the low cost, low technical requirements and high portability of instruments and equipment are particularly important.

4 Reference:

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Chen, X., M. Chu, and D. P. Giedroc. 1999. MRE-Binding transcription factor-1: weak zinc-binding finger domains 5 and 6 modulate the structure, affinity, and specificity of the metal-response element complex. Biochemistry 38:12915–12925.

Dalton, T. P., D. Bittel, and G. K. Andrews. 1997. Reversible activation of mouse metal response element-binding transcription factor 1 DNA binding involves zinc interaction with the zinc finger domain. Mol. Cell. Biol. 17: 2781–2789.

Dalton, T. P., Q. Li, D. Bittel, L. Liang, and G. K. Andrews. 1996. Oxidative stress activates metal-responsive transcription factor-1 binding activity. Occupancy in vivo of metal response elements in the metallothionein-I gene promoter. J. Biol. Chem. 271:26233–26241.

DeMoor, J. M., and D. J. Koropatnick. 2000. Metals and cellular signaling in mammalian cells. Cell. Mol. Biol. (Noisy-le-Grand) 46:367–381.

Günes, C., R. Heuchel, O. Georgiev, K.-H. Müller, P. Lichtlen, H. Blüthmann, S. Marino, A. Aguzzi, and W. Schaffner. 1998. Embryonic lethality and liver degeneration in mice lacking the metal-responsive transcriptional activator MTF-1. EMBO J. 17:2846–2854.

Heuchel, R., F. Radtke, O. Georgiev, G. Stark, M. Aguet, and W. Schaffner. 1994. The transcription factor MTF-1 is essential for basal and heavy metalinduced metallothionein gene expression. EMBO J. 13:2870–2875.

Heuchel, R., F. Radtke, O. Georgiev, G. Stark, M. Aguet, and W. Schaffner. 1994. The transcription factor MTF-1 is essential for basal and heavy metalinduced metallothionein gene expression. EMBO J. 13:2870–2875.

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Palmiter, R. D., S. D. Findley, T. E. Whitmore, and D. M. Durnam. 1992. MT-III, a brain-specific member of the metallothionein gene family. Proc.Natl. Acad. Sci. USA 89:6333–6337.

Radtke, F., R. Heuchel, O. Georgiev, M. Hergersberg, M. Gariglio, Z. Dembic, and W. Schaffner. 1993. Cloned transcription factor MTF-1 activates the mouse metallothionein I promoter. EMBO J. 12:1355–1362.

Saydam, N., O. Georgiev, M. Y. Nakano, U. Greber, and W. Schaffner. 2001 Nucleo-cytoplasmic trafficking of metal-responsive transcription factor MTF-1 is regulated by diverse stress signals. J. Biol. Chem., in press.

Searle, P. F., G. W. Stuart, and R. D. Palmiter. 1987. Metal regulatoryelements of the mouse metallothionein-I gene. Exper. Suppl. 52:407–414.

Simpkins, C. O. 2000. Metallothionein in human disease. Cell. Mol. Biol.(Noisy-le-Grand) 46:465–488.

Smirnova, I. V., D. C. Bittel, R. Ravindra, H. Jiang, and G. K. Andrews. 2000. Zinc and cadmium can promote rapid nuclear translocation of metal response element-binding transcription factor-1. J. Biol. Chem. 275:9377–9384.

Stuart, G. W., P. F. Searle, and R. D. Palmiter. 1985. Identification of multiple metal regulatory elements in mouse metallothionein-I promoter by assaying synthetic sequences. Nature 317:828–831

Uchida, Y., K. Takio, K. Titani, Y. Ihara, and M. Tomonaga. 1991. The growth inhibitory factor that is deficient in the Alzheimer’s disease brain is a 68 amino acid metallothionein-like protein. Neuron 7:337–347.

Westin, G., and W. Schaffner. 1988. A zinc-responsive factor interacts with a metal-regulated enhancer element (MRE) of the mouse metallothionein-I gene. EMBO J. 7:3763–3770.

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