10-甲醛四氫葉酸去氫酶 (10-formyltetrahydrofolate dehydrogenase; FDH)為參與葉酸單碳循環反應的酵素之一，主要是催化10-甲醛四氫葉酸轉變為四氫葉酸及二氧化碳的反應。研究發現在許多癌細胞株中10-甲醛四氫葉酸去氫酶基因表現有受到抑制的現象。此外過度表現10-甲醛四氫葉酸去氫酶則會造成癌細胞細胞週期終止以及細胞凋亡，顯示10-甲醛四氫葉酸去氫酶可能參與細胞增生與分化的過程，然而目前對於10-甲醛四氫葉酸去氫酶基因表現之調控機轉所知甚少。因此本研究欲藉由以斑馬魚做為動物模式，來探討參與在10-甲醛四氫葉酸去氫酶基因表現之順式調控因子與轉錄因子。之前我們實驗室已選殖出斑馬魚10-甲醛四氫葉酸去氫酶基因啟動子-124到+40的區域並證實其為具有啟動子活性之最小區域。而經由定點突變(site-directed mutagenesis)實驗結果，我們發現兩個可能的順式調控因子參與在斑馬魚10-甲醛四氫葉酸去氫酶基因啟動子之調控，並命名為site 1/2及site 4。因此我們進一步利用yeast one-hybrid screening，以重複序列的site 1/2及site 4作為bait進行篩選。結果得到五個可能的蛋白質coiled-coil domain containing protein 47 (CCDC47), prothymosin α (ProTα), sorcin, A3KPP3及hypoxia induced gene 1 (Hig-1)。經由暫時性表現共同轉染分析(transient expression cotransfection assay)，我們發現到ProTα, sorcin, A3KPP3及Hig-1可能於10-甲醛四氫葉酸去氫酶基因表現裡扮演轉錄活化子(transcriptional activators)的角色。我們同時利用DNA affinity precipitation assay (DAPA)的實驗，發現A3KPP3, sorcin, Hig-1, CCDC47以及Sp1會結合至10-甲醛四氫葉酸去氫酶基因啟動子-124到+40的區域上。總結以上之結果，我們認為A3KPP3, sorcin, Hig-1, CCDC47以及Sp1可能藉由結合到10-甲醛四氫葉酸去氫酶基因啟動子區域上，來進行啟動子活性與轉錄之調控。

10-formyltetrahydrofolate dehydrogenase (FDH; EC 1.5.1.6), one of the enzymes participating in the folate-mediated one carbon metabolism, catalyzes the NADP+-dependent conversion of 10-formyltetrahydrofolate (10-formyl-THF) to tetrahydrofolate (THF). It was reported that FDH was down-regulated in tumor tissues. In addition, over-expression of FDH induced cell cycle arrest and apoptosis in cancer cells32, suggesting the involvement of FDH in cell proliferation and differentiation. Currently, little is known about the regulatory mechanism of fdh gene expression. The aim of this study is to search for the cis-elements and transcription factors contributing to fdh expressional regulation using zebrafish as the animal model. Previously in our lab, a fragment (-124 to +40) in the fdh promoter region was found sufficient to maintain fdh promoter activity. Site-directed mutagenesis study revealed that two putative cis-elements, named site 1/2 and 4, that might participate in controlling zebrafish fdh expression. We used a yeast one-hybrid screening with the multimerized site 1/2 and 4 as bait to search for the potential transcriptional factors involved in fdh expressional regulation from zebrafish cDNA library. Five potential candidates are identified, including coiled-coil domain containing protein 47 (CCDC47), prothymosin α (ProTα), sorcin, A3KPP3 and hypoxia induced gene 1 (Hig-1), for bait 1/2. The further functional analysis revealed that ProTα, A3KPP3, sorcin, Hig-1 might act as transcriptional activators in fdh transcription. Furthermore, the DNA affinity precipitation assay (DAPA) results suggested that A3KPP3, sorcin, Hig-1, CCDC47 and Sp1 bound to the zebrafish fdh promoter region. These results indicated that A3KPP3, sorcin, Hig-1, CCDC47 and Sp1 can be considered as a transcriptional regulator of zebrafish fdh promoter.

1. Stanger, O. Physiology of folic acid in health and disease. Curr Drug Metab 3, 211-23 (2002).
2. Ifergan, I. & Assaraf, Y.G. Molecular mechanisms of adaptation to folate deficiency. Vitam Horm 79, 99-143 (2008).
3. Scott, J.M. Folate and vitamin B12. Proc Nutr Soc 58, 441-8 (1999).
4. Schweitzer, B.I., Dicker, A.P. & Bertino, J.R. Dihydrofolate reductase as a therapeutic target. FASEB J 4, 2441-52 (1990).
5. Oleinik, N.V. & Krupenko, S.A. Ectopic expression of 10-formyltetrahydrofolate dehydrogenase in A549 cells induces G1 cell cycle arrest and apoptosis. Mol Cancer Res 1, 577-88 (2003).
6. Scott, J.M. & Weir, D.G. Folic acid, homocysteine and one-carbon metabolism: a review of the essential biochemistry. J Cardiovasc Risk 5, 223-7 (1998).
7. Stover, P.J. Physiology of folate and vitamin B12 in health and disease. Nutr Rev 62, S3-12; discussion S13 (2004).
8. Huang, R.F., Ho, Y.H., Lin, H.L., Wei, J.S. & Liu, T.Z. Folate deficiency induces a cell cycle-specific apoptosis in HepG2 cells. J Nutr 129, 25-31 (1999).
9. Kim, Y.I. et al. Effects of dietary folate on DNA strand breaks within mutation-prone exons of the p53 gene in rat colon. Gastroenterology 119, 151-61 (2000).
10. Taparia, S., Gelineau-van Waes, J., Rosenquist, T.H. & Finnell, R.H. Importance of folate-homocysteine homeostasis during early embryonic development. Clin Chem Lab Med 45, 1717-27 (2007).
11. Blom, H.J., Shaw, G.M., den Heijer, M. & Finnell, R.H. Neural tube defects and folate: case far from closed. Nat Rev Neurosci 7, 724-31 (2006).
12. Novakovic, P., Stempak, J.M., Sohn, K.J. & Kim, Y.I. Effects of folate deficiency on gene expression in the apoptosis and cancer pathways in colon cancer cells. Carcinogenesis 27, 916-24 (2006).
13. Krupenko, S.A. & Oleinik, N.V. 10-formyltetrahydrofolate dehydrogenase, one of the major folate enzymes, is down-regulated in tumor tissues and possesses suppressor effects on cancer cells. Cell Growth Differ 13, 227-36 (2002).
14. Min, H., Shane, B. & Stokstad, E.L. Identification of 10-formyltetrahydrofolate dehydrogenase-hydrolase as a major folate binding protein in liver cytosol. Biochim Biophys Acta 967, 348-53 (1988).
15. Kutzbach, C. & Stokstad, E.L. Partial purification of a 10-formyl-tetrahydrofolate: NADP oxidoreductase from mammalian liver. Biochem Biophys Res Commun 30, 111-7 (1968).
16. Rios-Orlandi, E.M., Zarkadas, C.G. & MacKenzie, R.E. Formyltetrahydrofolate dehydrogenase-hydrolase from pig liver: simultaneous assay of the activities. Biochim Biophys Acta 871, 24-35 (1986).
17. Cook, R.J., Lloyd, R.S. & Wagner, C. Isolation and characterization of cDNA clones for rat liver 10-formyltetrahydrofolate dehydrogenase. J Biol Chem 266, 4965-73 (1991).
18. Scrutton, M.C. & Beis, I. Inhibitory effects of histidine and their reversal. The roles of pyruvate carboxylase and N10-formyltetrahydrofolate dehydrogenase. Biochem J 177, 833-46 (1979).
19. Schirch, D., Villar, E., Maras, B., Barra, D. & Schirch, V. Domain structure and function of 10-formyltetrahydrofolate dehydrogenase. J Biol Chem 269, 24728-35 (1994).
20. Krupenko, S.A. FDH: an aldehyde dehydrogenase fusion enzyme in folate metabolism. Chem Biol Interact 178, 84-93 (2009).
21. Krupenko, S.A., Wagner, C. & Cook, R.J. Domain structure of rat 10-formyltetrahydrofolate dehydrogenase. Resolution of the amino-terminal domain as 10-formyltetrahydrofolate hydrolase. J Biol Chem 272, 10273-8 (1997).
22. Donato, H., Krupenko, N.I., Tsybovsky, Y. & Krupenko, S.A. 10-formyltetrahydrofolate dehydrogenase requires a 4'-phosphopantetheine prosthetic group for catalysis. J Biol Chem 282, 34159-66 (2007).
23. Krebs, H.A., Hems, R. & Tyler, B. The regulation of folate and methionine metabolism. Biochem J 158, 341-53 (1976).
24. Black, K.A., Eells, J.T., Noker, P.E., Hawtrey, C.A. & Tephly, T.R. Role of hepatic tetrahydrofolate in the species difference in methanol toxicity. Proc Natl Acad Sci U S A 82, 3854-8 (1985).
25. Johlin, F.C., Fortman, C.S., Nghiem, D.D. & Tephly, T.R. Studies on the role of folic acid and folate-dependent enzymes in human methanol poisoning. Mol Pharmacol 31, 557-61 (1987).
26. Johlin, F.C., Swain, E., Smith, C. & Tephly, T.R. Studies on the mechanism of methanol poisoning: purification and comparison of rat and human liver 10-formyltetrahydrofolate dehydrogenase. Mol Pharmacol 35, 745-50 (1989).
27. Champion, K.M., Cook, R.J., Tollaksen, S.L. & Giometti, C.S. Identification of a heritable deficiency of the folate-dependent enzyme 10-formyltetrahydrofolate dehydrogenase in mice. Proc Natl Acad Sci U S A 91, 11338-42 (1994).
28. Cook, R.J. Disruption of histidine catabolism in NEUT2 mice. Arch Biochem Biophys 392, 226-32 (2001).
29. Martinasevic, M.K., Rios, G.R., Miller, M.W. & Tephly, T.R. Folate and folate-dependent enzymes associated with rat CNS development. Dev Neurosci 21, 29-35 (1999).
30. Neymeyer, V., Tephly, T.R. & Miller, M.W. Folate and 10-formyltetrahydrofolate dehydrogenase (FDH) expression in the central nervous system of the mature rat. Brain Res 766, 195-204 (1997).
31. Anthony, T.E. & Heintz, N. The folate metabolic enzyme ALDH1L1 is restricted to the midline of the early CNS, suggesting a role in human neural tube defects. J Comp Neurol 500, 368-83 (2007).
32. Oleinik, N.V., Krupenko, N.I., Priest, D.G. & Krupenko, S.A. Cancer cells activate p53 in response to 10-formyltetrahydrofolate dehydrogenase expression. Biochem J 391, 503-11 (2005).
33. Oleinik, N.V., Krupenko, N.I., Reuland, S.N. & Krupenko, S.A. Leucovorin-induced resistance against FDH growth suppressor effects occurs through DHFR up-regulation. Biochem Pharmacol 72, 256-66 (2006).
34. Oleinik, N.V., Krupenko, N.I. & Krupenko, S.A. Cooperation between JNK1 and JNK2 in activation of p53 apoptotic pathway. Oncogene 26, 7222-30 (2007).
35. Ghose, S., Oleinik, N.V., Krupenko, N.I. & Krupenko, S.A. 10-formyltetrahydrofolate dehydrogenase-induced c-Jun-NH2-kinase pathways diverge at the c-Jun-NH2-kinase substrate level in cells with different p53 status. Mol Cancer Res 7, 99-107 (2009).
36. Crabb, D.W., Matsumoto, M., Chang, D. & You, M. Overview of the role of alcohol dehydrogenase and aldehyde dehydrogenase and their variants in the genesis of alcohol-related pathology. Proc Nutr Soc 63, 49-63 (2004).
37. Blake, M.C., Jambou, R.C., Swick, A.G., Kahn, J.W. & Azizkhan, J.C. Transcriptional initiation is controlled by upstream GC-box interactions in a TATAA-less promoter. Mol Cell Biol 10, 6632-41 (1990).
38. Guimond, J., Devost, D., Brodeur, H., Mader, S. & Bhat, P.V. Characterization of the rat RALDH1 promoter. A functional CCAAT and octamer motif are critical for basal promoter activity. Biochim Biophys Acta 1579, 81-91 (2002).
39. Yanagawa, Y., Chen, J.C., Hsu, L.C. & Yoshida, A. The transcriptional regulation of human aldehyde dehydrogenase I gene. The structural and functional analysis of the promoter. J Biol Chem 270, 17521-7 (1995).
40. Feitsma, H. & Cuppen, E. Zebrafish as a cancer model. Mol Cancer Res 6, 685-94 (2008).
41. Chico, T.J., Ingham, P.W. & Crossman, D.C. Modeling cardiovascular disease in the zebrafish. Trends Cardiovasc Med 18, 150-5 (2008).
42. Kari, G., Rodeck, U. & Dicker, A.P. Zebrafish: an emerging model system for human disease and drug discovery. Clin Pharmacol Ther 82, 70-80 (2007).
43. Lieschke, G.J. & Currie, P.D. Animal models of human disease: zebrafish swim into view. Nat Rev Genet 8, 353-67 (2007).
44. McGrath, P. & Li, C.Q. Zebrafish: a predictive model for assessing drug-induced toxicity. Drug Discov Today 13, 394-401 (2008).
45. Sun, S.N. et al. Effect of dihydrofolate reductase gene knock-down on the expression of heart and neural crest derivatives expressed transcript 2 in zebrafish cardiac development. Chin Med J (Engl) 120, 1166-71 (2007).
46. Denvir, M.A., Tucker, C.S. & Mullins, J.J. Systolic and diastolic ventricular function in zebrafish embryos: influence of norepenephrine, MS-222 and temperature. BMC Biotechnol 8, 21 (2008).
47. Kao, T.T. et al. Characterization and comparative studies of zebrafish and human recombinant dihydrofolate reductases--inhibition by folic acid and polyphenols. Drug Metab Dispos 36, 508-16 (2008).
48. Chang, W.N., Tsai, J.N., Chen, B.H. & Fu, T.F. Cloning, expression, purification, and characterization of zebrafish cytosolic serine hydroxymethyltransferase. Protein Expr Purif 46, 212-20 (2006).
49. Chang, W.N., Tsai, J.N., Chen, B.H., Huang, H.S. & Fu, T.F. Serine hydroxymethyltransferase isoforms are differentially inhibited by leucovorin: characterization and comparison of recombinant zebrafish serine hydroxymethyltransferases. Drug Metab Dispos 35, 2127-37 (2007).
50. Kao, T.T., Chang, W.N., Wu, H.L., Shi, G.Y. & Fu, T.F. Recombinant zebrafish {gamma}-glutamyl hydrolase exhibits properties and catalytic activities comparable with those of mammalian enzyme. Drug Metab Dispos 37, 302-9 (2009).
51. Lassen, N. et al. Molecular cloning, baculovirus expression, and tissue distribution of the zebrafish aldehyde dehydrogenase 2. Drug Metab Dispos 33, 649-56 (2005).
52. Westerfield, M. The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio rerio) (2000).
53. Miranda, C.L., Collodi, P., Zhao, X., Barnes, D.W. & Buhler, D.R. Regulation of cytochrome P450 expression in a novel liver cell line from zebrafish (Brachydanio rerio). Arch Biochem Biophys 305, 320-7 (1993).
54. Mirhendi H., D.K., Rezaei A., Jalalizand N., Hosseinpur L.,Khodadadi H. Colony-PCR Is a rapid and sensitive method for DNA amplification in yeasts. Iranian J Publ Health 36, 40-44 (2007).
55. Evstafieva, A.G. et al. Overproduction in Escherichia coli, purification and properties of human prothymosin alpha. Eur J Biochem 231, 639-43 (1995).
56. Yi, S., Brickenden, A. & Choy, W.Y. A new protocol for high-yield purification of recombinant human prothymosin alpha expressed in Escherichia coli for NMR studies. Protein Expr Purif 57, 1-8 (2008).
57. Kalthoff, C. A novel strategy for the purification of recombinantly expressed unstructured protein domains. J Chromatogr B Analyt Technol Biomed Life Sci 786, 247-54 (2003).
58. Karetsou, Z. et al. Prothymosin alpha modulates the interaction of histone H1 with chromatin. Nucleic Acids Res 26, 3111-8 (1998).
59. Papamarcaki, T. & Tsolas, O. Prothymosin alpha binds to histone H1 in vitro. FEBS Lett 345, 71-5 (1994).
60. Sukhacheva, E.A. et al. Sensing prothymosin alpha origin, mutations and conformation with monoclonal antibodies. J Immunol Methods 266, 185-96 (2002).
61. Karey, K.P. & Sirbasku, D.A. Glutaraldehyde fixation increases retention of low molecular weight proteins (growth factors) transferred to nylon membranes for western blot analysis. Anal Biochem 178, 255-9 (1989).
62. Takeya, K., Loutzenhiser, K., Shiraishi, M., Loutzenhiser, R. & Walsh, M.P. A highly sensitive technique to measure myosin regulatory light chain phosphorylation: the first quantification in renal arterioles. Am J Physiol Renal Physiol 294, F1487-92 (2008).
63. Gast, K. et al. Prothymosin alpha: a biologically active protein with random coil conformation. Biochemistry 34, 13211-8 (1995).
64. Wang, N., Ahmed, S. & Haqqi, T.M. Genomic structure and functional characterization of the promoter region of human IkappaB kinase-related kinase IKKi/IKKvarepsilon gene. Gene 353, 118-33 (2005).
65. Azizkhan, J.C., Jensen, D.E., Pierce, A.J. & Wade, M. Transcription from TATA-less promoters: dihydrofolate reductase as a model. Crit Rev Eukaryot Gene Expr 3, 229-54 (1993).
66. Ramsay, R.G. & Gonda, T.J. MYB function in normal and cancer cells. Nat Rev Cancer 8, 523-34 (2008).
67. Driever, W. & Rangini, Z. Characterization of a cell line derived from zebrafish (Brachydanio rerio) embryos. In Vitro Cell Dev Biol Anim 29A, 749-54 (1993).
68. Ghosh, C., Zhou, Y.L. & Collodi, P. Derivation and characterization of a zebrafish liver cell line. Cell Biol Toxicol 10, 167-76 (1994).
69. He, S. et al. Genetic and transcriptome characterization of model zebrafish cell lines. Zebrafish 3, 441-53 (2006).
70. Wei, Z., Angerer, R.C. & Angerer, L.M. Identification of a new sea urchin ets protein, SpEts4, by yeast one-hybrid screening with the hatching enzyme promoter. Mol Cell Biol 19, 1271-8 (1999).
71. Uversky, V.N. et al. Zn(2+)-mediated structure formation and compaction of the "natively unfolded" human prothymosin alpha. Biochem Biophys Res Commun 267, 663-8 (2000).
72. Segade, F. & Gomez-Marquez, J. Prothymosin alpha. Int J Biochem Cell Biol 31, 1243-8 (1999).
73. Donizetti, A. et al. Differential expression of duplicated genes for prothymosin alpha during zebrafish development. Dev Dyn 237, 1112-8 (2008).
74. Gomez-Marquez, J. Function of prothymosin alpha in chromatin decondensation and expression of thymosin beta-4 linked to angiogenesis and synaptic plasticity. Ann N Y Acad Sci 1112, 201-9 (2007).
75. Karapetian, R.N. et al. Nuclear oncoprotein prothymosin alpha is a partner of Keap1: implications for expression of oxidative stress-protecting genes. Mol Cell Biol 25, 1089-99 (2005).
76. Mosoian, A. et al. Novel function of prothymosin alpha as a potent inhibitor of human immunodeficiency virus type 1 gene expression in primary macrophages. J Virol 80, 9200-6 (2006).
77. Karetsou, Z., Kretsovali, A., Murphy, C., Tsolas, O. & Papamarcaki, T. Prothymosin alpha interacts with the CREB-binding protein and potentiates transcription. EMBO Rep 3, 361-6 (2002).
78. Marfatia, K.A., Harreman, M.T., Fanara, P., Vertino, P.M. & Corbett, A.H. Identification and characterization of the human MOG1 gene. Gene 266, 45-56 (2001).
79. Hamada, H., Okochi, E., Oh-hara, T. & Tsuruo, T. Purification of the Mr 22,000 calcium-binding protein (sorcin) associated with multidrug resistance and its detection with monoclonal antibodies. Cancer Res 48, 3173-8 (1988).
80. Xie, X., Dwyer, M.D., Swenson, L., Parker, M.H. & Botfield, M.C. Crystal structure of calcium-free human sorcin: a member of the penta-EF-hand protein family. Protein Sci 10, 2419-25 (2001).
81. Matsumoto, T. et al. Sorcin interacts with sarcoplasmic reticulum Ca(2+)-ATPase and modulates excitation-contraction coupling in the heart. Basic Res Cardiol 100, 250-62 (2005).
82. Denko, N. et al. Epigenetic regulation of gene expression in cervical cancer cells by the tumor microenvironment. Clin Cancer Res 6, 480-7 (2000).
83. Jin, K. et al. cDNA microarray analysis of changes in gene expression induced by neuronal hypoxia in vitro. Neurochem Res 27, 1105-12 (2002).
84. Salnikow, K. et al. The involvement of hypoxia-inducible transcription factor-1-dependent pathway in nickel carcinogenesis. Cancer Res 63, 3524-30 (2003).
85. Bedo, G., Vargas, M., Ferreiro, M.J., Chalar, C. & Agrati, D. Characterization of hypoxia induced gene 1: expression during rat central nervous system maturation and evidence of antisense RNA expression. Int J Dev Biol 49, 431-6 (2005).
86. Looft, C. et al. Isolation and assignment of the UDP-glucose pyrophosphorylase gene (UGP2) to porcine chromosome 3q21-->q22 by FISH and by analysis of somatic cell and radiation hybrid panels. Cytogenet Cell Genet 89, 154-5 (2000).
87. Wierstra, I. Sp1: emerging roles--beyond constitutive activation of TATA-less housekeeping genes. Biochem Biophys Res Commun 372, 1-13 (2008).