Abstract
Zinc finger (ZF) proteins belonging to the Cys2–His2 class provide a simple and versatile framework to design novel artificial transcription factors (ATFs) targeted to the desired genes. Our work is based on ZF ATFs engineered to up-regulate the expression level of the dystrophin-related gene utrophin in Duchenne muscular dystrophy (DMD). In particular, on the basis of the “recognition code” that defines specific rules between zinc finger primary structure and potential DNA-binding sites we engineered and selected a new family of artificial transcription factors, whose DNA-binding domain consists in a three zinc finger peptide called “Jazz.” Jazz protein binds specifically the 9 bp DNA sequence (5′-GCT-GCT-GCG-3′) present in the promoter region of both the human and mouse utrophin gene. We generated a transgenic mouse expressing Jazz protein fused to the strong transcriptional activation domain VP16 and under the control of the muscle specific promoter of the myosin light chain gene. Vp16-Jazz mice display a strong up-regulation of the utrophin at both mRNA and protein levels. To our knowledge, this represents the first example of a transgenic mouse expressing an artificial gene coding for a zinc finger-based transcription factor.
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References
Corbi, N., Libri, V., Onori, A., and Passananti, C. (2004) Synthetic zinc finger peptides: old and novel applications. Biochem Cell Biol. 82, 428–436.
Blancafort, P., Segal, D.J., and Barbas, C.F., III. (2004) Designing transcription factor architectures for drug discovery. Mol Pharmacol 66, 1361–1371.
Choo, Y. and Klug, A. (1997) Physical basis of a protein-DNA recognition code. Curr Opin Struct Biol. 7, 117–125.
Pabo, C.O., Peisach, E., and Grant, R.A. (2001) Design and selection of novel Cys2His2 zinc finger proteins. Annu Rev Biochem. 70, 313–340.
Dreier, B., Segal, D.J., and Barbas, C.F., III. (2000) Insights into the molecular recognition of the 5′-GNN-3′ family of DNA sequences by zinc finger domains. J Mol Biol. 303, 489–502.
Dreier, B., Beerli, R.R., Segal, D.J., Flippin, J.D., and Barbas, C.F., III. (2001) Development of zinc finger domains for recognition of the 5′-ANN-3′ family of DNA sequences and their use in the construction of artificial transcription factors. J Biol Chem. 276, 29466–29478.
Dreier, B., Fuller, R.P., Segal, D.J., Lund, C.V., Blancafort, P., Huber, A., Koksch, B., and Barbas, C.F., III. (2005) Development of zinc finger domains for recognition of the 5′-CNN-3′ family DNA sequences and their use in the construction of artificial transcription factors. J Biol Chem. 280, 35588–35597.
Papworth, M., Kolasinska, P., and Minczuk, M. (2006) Designer zinc-finger proteins and their applications. Gene. 366, 27–38.
Beltran, A., Liu, Y., Parikh, S., Temple, B., and Blancafort, P. (2006) Interrogating genomes with combinatorial artificial transcription factor libraries: asking zinc finger questions. Assay Drug Dev Technol. 4, 317–331.
Blancafort, P. and Beltran, A.S. (2008) Rational design, selection and specificity of artificial transcription factors (ATFs): the influence of chromatin in target gene regulation. Comb Chem High Throughput Screen. 11(2), 146–158.
Papworth, M., Moore, M., Isalan, M., Minczuk, M., Choo, Y., and Klug, A. (2003) Inhibition of herpes simplex virus 1 gene expression by designer zinc-finger transcription factors. Proc Natl Acad Sci USA. 100, 1621–1626.
Reynolds, L., Ullman, C., Moore, M., Isalan, M., West, M.J., Clapham, P., Klug, A., and Choo, Y. (2003) Repression of the HIV-1 5′ LTR promoter and inhibition of HIV-1 replication by using engineered zinc-finger transcription factors. Proc Natl Acad Sci USA. 100, 1615–1620.
Beltran, A., Parikh, S., Liu, Y., Cuevas, B.D., Johnson, G.L., Futscher, B.W., and Blancafort, P. (2007) Re-activation of a dormant tumor suppressor gene maspin by designed transcription factors. Oncogene. 26(19), 2791–2798.
Rebar, E.J., Huang, Y., Hickey, R., Nath, A.K., Meoli, D., Nath, S., Chen, B., Xu, L., Liang, Y., Jamieson, A.C., Zhang, L., Spratt, S.K., Case, C.C., Wolffe, A., and Giordano, F.J. (2002) Induction of angiogenesis in a mouse model using engineered transcription factors. Nat Med 8, 1427–1432.
Lu, Y., Tian, C., Danialou, G., Gilbert, R., Petrof, B.J., Karpati, G., and Nalbantoglu, J. (2008) Targeting artificial transcription factors to the Utrophin A promoter: effects on dystrophic pathology and muscle function. J Biol Chem. 283(50), 34720–34727.
Corbi, N., Libri, V., Fanciulli, M., Tinsley, J.M., Davies, K.E., and Passananti, C. (2000) The artificial zinc finger coding gene “Jazz” binds the utrophin promoter and activates transcription. Gene Ther. 7, 1076–1083.
Onori, A., Desantis, A., Buontempo, S., Di Certo, M.G., Fanciulli, M., Salvatori, L., Passananti, C., and Corbi, N. (2007) The artificial 4-zinc-finger Bagly binds human utrophin promoter A at the endogenous chromosomal site and activates transcription. Biochem Cell Biol. 85(3), 358–365.
Mattei, E., Corbi, N., Di Certo, M.G., Strimpakos, G., Severini, C., Onori, A., Desantis, A., Libri, V., Buontempo, S., Floridi, A., Fanciulli, M., Baban, D., Davies, K.E., and Passananti, C. (2007) Utrophin up-regulation by an artificial transcription factor in transgenic mice. PLoS ONE. 22(1), e774.
Desantis, A., Onori, A., Di Certo, M.G., Mattei, E., Fanciulli, M., Passananti, C., and Corbi, N. (2009) Novel activation domain derived from Che-1 cofactor coupled with the artificial protein Jazz drives utrophin upregulation. Neuromuscular Disorders. 19(2), 158–162.
Tinsley, J.M., Potter, A.C., Phelps, S.R., Fisher, R., Trickett, J.I., and Davies, K.E. (1996) Amelioration of the dystrophic phenotype of mdx mice using a truncated utrophin transgene. Nature. 384, 349–353.
Tinsley, J., Deconinck, N., Fisher, R., Kahn, D., Phelps, S., Gillis, J.M., and Davies, K.E. (1998) Expression of full-length utrophin prevents muscular dystrophy in mdx mice. Nat Med. 4, 1441–1444.
Miura, P. and Jasmin, B.J. (2006) Utrophin upregulation for treating Duchenne or Becker muscular dystrophy: how close are we? Trends Mol Med. 3, 122–129.
Dennis, C.L., Tinsley, J.M., Deconinck, A.E., and Davies, K.E. (1996) Molecular and functional analysis of the utrophin promoter. Nucleic Acids Res. 24, 1646–1652.
Musarò, A., McCullagh, K., Paul, A., Houghton, L., Dobrowolny, G., Molinaro, M., Barton, E.R., Sweeney, H.L., and Rosenthal, N. (2001) Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle. Nat Genet. 27(2), 195–200.
Nagy, A., Gertsenstein, M., Vintersten, K., and Behringer, R. (2003) Manipulating the Mouse Embryo: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press, New York.
Laemmli, U.K. (1970) Cleavege of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227, 680–685.
Acknowledgments
This work was supported by: Telethon-Italy (Grant # GGP07177), Health Ministery RF-INP-2007-653996, and Paul Blümel Stiftung für medizinische Forschung. M.G. Di Certo is a recipient of a fellowship supported by Regione Lazio/Filas fundings for “Sviluppo della Ricerca sul Cervello.”.
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Passananti, C., Corbi, N., Onori, A., Certo, M.G.D., Mattei, E. (2010). Transgenic Mice Expressing an Artificial Zinc Finger Regulator Targeting an Endogenous Gene. In: Mackay, J., Segal, D. (eds) Engineered Zinc Finger Proteins. Methods in Molecular Biology, vol 649. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-753-2_11
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DOI: https://doi.org/10.1007/978-1-60761-753-2_11
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