MTF-1 (metal response element-binding transcription factor )是一種具有啟動若干與抗氧化及抗金屬逆境相關基因的轉錄蛋白。目前有一些受到MTF-1調控的基因已被分析出。本篇文章試圖找出更多可能受MTF-1調控的下游基因。本研究是使用whole genome PCR的實驗方式，再將此技術稍加改良後，經由多次的篩選和放後，尋找出MTF-1的目標DNA。在此實驗中，挑選出了數十個DNA片段，其中DNA片段都含有MRE (metal response element)序列。再將這些片段利用in vitro和in vivo的實驗，如EMSA和報導基因的檢驗，驗證這些被篩選出來的DNA片段確實可以和MTF-1轉錄蛋白結合，再藉由比對於人類基因資料庫以協助找出MTF-1所調控的下游基因。由於本實驗中被篩選到的DNA片段離基因有一段距離，推測這些DNA片段或許扮演enhancer的角色，幫助或加強下游基因的轉錄。在本研究找出了一些基因，例如影響人類胚胎的肝臟細胞發育的ZNF268 (Zinc finger protein 268)，以及具有抗血管新生功能的BAI1 (brain-specific angiogenesis inhibitor 1)，都可能會受到MTF-1轉錄蛋白的調節。

The transcriptional activator metal-responsive transcription factor (MTF-1) exerts its function by activating down-stream genes to maintain heavy metal homeostasis and to respond to various oxidative stresses. A limited genes have been found recently that are regulated by MTF-1. In this study, we tried to employ whole genome PCR to search unidentifiied target genes of MTF-1. DNA fragment recovered after screening have been cloned, and sequenced. Several DNA fragments that contain MREs (metal response element) were obtained. Using EMSA (Electrophoretic Mobility Shift Assay) and reporter gene assay, the MTF-1 binding and transactivation activities of DNA fragment were examined. The DNA sequences were also searched from blast/NCBI human genome database to reveal the identity of the DNA. The sequences locate in the promoter region of several genes, such as zinc finger protein 268, brain-specific angiogenesis inhibitor 1. Since the sequences present far away from the identified genes, we speculate that the DNA sequence may serve as enhancer of the genes regulated by MTF-1.

Andrews, G.K. (2000) Regulation of metallothionein gene expression by oxidative stress and metal ions. Biochem Pharmacol, 59, 95-104.
Andrews, G.K. and Geiser, J. (1999) Expression of the mouse metallothionein-I and -II genes provides a reproductive advantage during maternal dietary zinc deficiency. J Nutr, 129, 1643-1648.
Andrews, G.K., Lee, D.K., Ravindra, R., Lichtlen, P., Sirito, M., Sawadogo, M. and Schaffner, W. (2001) The transcription factors MTF-1 and USF1 cooperate to regulate mouse metallothionein-I expression in response to the essential metal zinc in visceral endoderm cells during early development. Embo J, 20, 1114-1122.
Auf der Maur, A., Belser, T., Elgar, G., Georgiev, O. and Schaffner, W. (1999) Characterization of the transcription factor MTF-1 from the Japanese pufferfish (Fugu rubripes) reveals evolutionary conservation of heavy metal stress response. Biol Chem, 380, 175-185.
Auf der Maur, A., Belser, T., Wang, Y., Gunes, C., Lichtlen, P., Georgiev, O. and Schaffner, W. (2000) Characterization of the mouse gene for the heavy metal-responsive transcription factor MTF-1. Cell Stress Chaperones, 5, 196-206.
Bauman, J.W., Liu, J., Liu, Y.P. and Klaassen, C.D. (1991) Increase in metallothionein produced by chemicals that induce oxidative stress. Toxicol Appl Pharmacol, 110, 347-354.
Bittel, D.C., Smirnova, I.V. and Andrews, G.K. (2000) Functional heterogeneity in the zinc fingers of metalloregulatory protein metal response element-binding transcription factor-1. J Biol Chem, 275, 37194-37201.
Brugnera, E., Georgiev, O., Radtke, F., Heuchel, R., Baker, E., Sutherland, G.R. and Schaffner, W. (1994) Cloning, chromosomal mapping and characterization of the human metal-regulatory transcription factor MTF-1. Nucleic Acids Res, 22, 3167-3173.
Carter, A.D., Felber, B.K., Walling, M.J., Jubier, M.F., Schmidt, C.J. and Hamer, D.H. (1984) Duplicated heavy metal control sequences of the mouse metallothionein-I gene. Proc Natl Acad Sci U S A, 81, 7392-7396.
Chen, X., Agarwal, A. and Giedroc, D.P. (1998) Structural and functional heterogeneity among the zinc fingers of human MRE-binding transcription factor-1. Biochemistry, 37, 11152-11161.
Chen, X., Chu, M. and Giedroc, D.P. (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., Fu, K., Palmiter, R.D. and Andrews, G.K. (1996) Transgenic mice that overexpress metallothionein-I resist dietary zinc deficiency. J Nutr, 126, 825-833.
Dalton, T.P., Li, Q., Bittel, D., Liang, L. and Andrews, G.K. (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.
Datta, P.K. and Jacob, S.T. (1997) Activation of the metallothionein-I gene promoter in response to cadmium and USF in vitro. Biochem Biophys Res Commun, 230, 159-163.
Duda, D.G., Sunamura, M., Lozonschi, L., Yokoyama, T., Yatsuoka, T., Motoi, F., Horii, A., Tani, K., Asano, S., Nakamura, Y. and Matsuno, S. (2002) Overexpression of the p53-inducible brain-specific angiogenesis inhibitor 1 suppresses efficiently tumour angiogenesis. Br J Cancer, 86, 490-496.
Durnam, D.M. and Palmiter, R.D. (1981) Transcriptional regulation of the mouse metallothionein-I gene by heavy metals. J Biol Chem, 256, 5712-5716.
Guerrerio, A.L. and Berg, J.M. (2004) Metal ion affinities of the zinc finger domains of the metal responsive element-binding transcription factor-1 (MTF1). Biochemistry, 43, 5437-5444.
Gunes, C., Heuchel, R., Georgiev, O., Muller, K.H., Lichtlen, P., Bluthmann, H., Marino, S., Aguzzi, A. and Schaffner, W. (1998) Embryonic lethality and liver degeneration in mice lacking the metal-responsive transcriptional activator MTF-1. Embo J, 17, 2846-2854.
Heuchel, R., Radtke, F., Georgiev, O., Stark, G., Aguet, M. and Schaffner, W. (1994) The transcription factor MTF-1 is essential for basal and heavy metal-induced metallothionein gene expression. Embo J, 13, 2870-2875.
Kaur, B., Brat, D.J., Calkins, C.C. and Van Meir, E.G. (2003) Brain angiogenesis inhibitor 1 is differentially expressed in normal brain and glioblastoma independently of p53 expression. Am J Pathol, 162, 19-27.
Kelly, E.J., Quaife, C.J., Froelick, G.J. and Palmiter, R.D. (1996) Metallothionein I and II protect against zinc deficiency and zinc toxicity in mice. J Nutr, 126, 1782-1790.
Kinzler, K.W. and Vogelstein, B. (1989) Whole genome PCR: application to the identification of sequences bound by gene regulatory proteins. Nucleic Acids Res, 17, 3645-3653.
LaRochelle, O., Gagne, V., Charron, J., Soh, J.W. and Seguin, C. (2001) Phosphorylation is involved in the activation of metal-regulatory transcription factor 1 in response to metal ions. J Biol Chem, 276, 41879-41888.
Lichtlen, P., Georgiev, O., Schaffner, W., Aguzzi, A. and Brandner, S. (1999) The heavy metal-responsive transcription factor-1 (MTF-1) is not required for neural differentiation. Biol Chem, 380, 711-715.
Lichtlen, P., Wang, Y., Belser, T., Georgiev, O., Certa, U., Sack, R. and Schaffner, W. (2001) Target gene search for the metal-responsive transcription factor MTF-1. Nucleic Acids Res, 29, 1514-1523.
Masters, B.A., Kelly, E.J., Quaife, C.J., Brinster, R.L. and Palmiter, R.D. (1994) Targeted disruption of metallothionein I and II genes increases sensitivity to cadmium. Proc Natl Acad Sci U S A, 91, 584-588.
Michalska, A.E. and Choo, K.H. (1993) Targeting and germ-line transmission of a null mutation at the metallothionein I and II loci in mouse. Proc Natl Acad Sci U S A, 90, 8088-8092.
Mori, K., Kanemura, Y., Fujikawa, H., Nakano, A., Ikemoto, H., Ozaki, I., Matsumoto, T., Tamura, K., Yokota, M. and Arita, N. (2002) Brain-specific angiogenesis inhibitor 1 (BAI1) is expressed in human cerebral neuronal cells. Neurosci Res, 43, 69-74.
Mueller, P.R., Salser, S.J. and Wold, B. (1988) Constitutive and metal-inducible protein:DNA interactions at the mouse metallothionein I promoter examined by in vivo and in vitro footprinting. Genes Dev, 2, 412-427.
Nishimori, H., Shiratsuchi, T., Urano, T., Kimura, Y., Kiyono, K., Tatsumi, K., Yoshida, S., Ono, M., Kuwano, M., Nakamura, Y. and Tokino, T. (1997) A novel brain-specific p53-target gene, BAI1, containing thrombospondin type 1 repeats inhibits experimental angiogenesis. Oncogene, 15, 2145-2150.
Otsuka, F., Iwamatsu, A., Suzuki, K., Ohsawa, M., Hamer, D.H. and Koizumi, S. (1994) Purification and characterization of a protein that binds to metal responsive elements of the human metallothionein IIA gene. J Biol Chem, 269, 23700-23707.
Palmiter, R.D. (1994) Regulation of metallothionein genes by heavy metals appears to be mediated by a zinc-sensitive inhibitor that interacts with a constitutively active transcription factor, MTF-1. Proc Natl Acad Sci U S A, 91, 1219-1223.
Palmiter, R.D., Sandgren, E.P., Koeller, D.M. and Brinster, R.L. (1993) Distal regulatory elements from the mouse metallothionein locus stimulate gene expression in transgenic mice. Mol Cell Biol, 13, 5266-5275.
Radtke, F., Heuchel, R., Georgiev, O., Hergersberg, M., Gariglio, M., Dembic, Z. and Schaffner, W. (1993) Cloned transcription factor MTF-1 activates the mouse metallothionein I promoter. Embo J, 12, 1355-1362.
Saydam, N., Adams, T.K., Steiner, F., Schaffner, W. and Freedman, J.H. (2002) Regulation of metallothionein transcription by the metal-responsive transcription factor MTF-1: identification of signal transduction cascades that control metal-inducible transcription. J Biol Chem, 277, 20438-20445.
Saydam, N., Georgiev, O., Nakano, M.Y., Greber, U.F. and Schaffner, W. (2001) Nucleo-cytoplasmic trafficking of metal-regulatory transcription factor 1 is regulated by diverse stress signals. J Biol Chem, 276, 25487-25495.
Searle, P.F. (1990) Zinc dependent binding of a liver nuclear factor to metal response element MRE-a of the mouse metallothionein-I gene and variant sequences. Nucleic Acids Res, 18, 4683-4690.
Serfling, E., Lubbe, A., Dorsch-Hasler, K. and Schaffner, W. (1985) Metal-dependent SV40 viruses containing inducible enhancers from the upstream region of metallothionein genes. Embo J, 4, 3851-3859.
Smirnova, I.V., Bittel, D.C., Ravindra, R., Jiang, H. and Andrews, G.K. (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., Searle, P.F., Chen, H.Y., Brinster, R.L. and Palmiter, R.D. (1984) A 12-base-pair DNA motif that is repeated several times in metallothionein gene promoters confers metal regulation to a heterologous gene. Proc Natl Acad Sci U S A, 81, 7318-7322.
Sun, Y., Shao, H., Li, Z., Liu, J., Gao, L., Peng, X., Meng, Y. and Li, W. (2004) ZNF268, a novel kruppel-like zinc finger protein, is implicated in early human liver development. Int J Mol Med, 14, 971-975.
Tolsma, S.S., Volpert, O.V., Good, D.J., Frazier, W.A., Polverini, P.J. and Bouck, N. (1993) Peptides derived from two separate domains of the matrix protein thrombospondin-1 have anti-angiogenic activity. J Cell Biol, 122, 497-511.
Wang, Y., Lorenzi, I., Georgiev, O. and Schaffner, W. (2004) Metal-responsive transcription factor-1 (MTF-1) selects different types of metal response elements at low vs. high zinc concentration. Biol Chem, 385, 623-632.
Westin, G. and Schaffner, W. (1988) A zinc-responsive factor interacts with a metal-regulated enhancer element (MRE) of the mouse metallothionein-I gene. Embo J, 7, 3763-3770.
Wimmer, U., Wang, Y., Georgiev, O. and Schaffner, W. (2005) Two major branches of anti-cadmium defense in the mouse: MTF-1/metallothioneins and glutathione. Nucleic Acids Res, 33, 5715-5727.
Yu, C.W., Chen, J.H. and Lin, L.Y. (1997) Metal-induced metallothionein gene expression can be inactivated by protein kinase C inhibitor. FEBS Lett, 420, 69-73.
Zhang, B., Egli, D., Georgiev, O. and Schaffner, W. (2001) The Drosophila homolog of mammalian zinc finger factor MTF-1 activates transcription in response to heavy metals. Mol Cell Biol, 21, 4505-4514.
石致遠 (2004) Isolation and characterization of binding site for transsription factor FOXP2.January 清華大學生命科學　碩士論文