Key Points
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C2H2 zinc fingers are the most common DNA-binding motif found in the human genome.
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The Zif268–DNA crystal structure shows how zinc fingers interact with DNA. The fingers act as modular units (each contacting three to four base pairs of DNA) and the structure reveals which residues should be changed to alter the specificity.
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Researchers have engineered zinc finger proteins (ZFPs) to bind a diverse set of DNA sequences, and thereby target specific locations in the human genome, such as the promoters of therapeutically relevant genes. Exquisite specificity can be obtained with proteins that have six fingers.
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ZFP transcription factors (ZFP TFs) are made by combining the ZFPs with domains that either activate or repress genes. ZFP TFs are used in drug discovery to regulate genes for target validation, high-throughput screening and human therapeutics.
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ZFP-mediated regulation of endogenous genes could make it possible to use genes in a drug discovery application that would otherwise require securing intellectual property rights for a corresponding complementary DNA sequence.
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Recently, ZFP TFs have been used to promote angiogenesis in a mouse ear model, and are now undergoing further preclinical testing.
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Combining ZFPs with novel functional domains makes it possible to target DNA for chromatin and DNA modification, DNA cleavage and for targeted integration of exogenous DNA.
Abstract
Zinc-finger proteins (ZFPs) that recognize novel DNA sequences are the basis of a powerful technology platform with many uses in drug discovery and therapeutics. These proteins have been used as the DNA-binding domains of novel transcription factors (ZFP TFs), which are useful for validating genes as drug targets and for engineering cell lines for small-molecule screening and protein production. Recently, they have also been used as a basis for novel human therapeutics. Most of our advances in the design and application of these ZFP TFs rely on our ability to engineer ZFPs that bind short stretches of DNA (typically 9–18 base pairs) located within the promoters of target genes. Here, we summarize the methods used to design these DNA-binding domains, explain how they are incorporated into novel transcription factors (and other useful molecules) and describe some key applications in drug discovery.
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Acknowledgements
The authors would like to express their indebtedness to all those who contributed to the critical reading of this manuscript, including Dr P. Gregory, D. E. Wolffe, Dr E. Rebar, Dr A. McNamara, Dr A. Reik, Dr F. Urnov, Dr M. Holmes and T. J. Cradick. The authors also apologize to those researchers whose many contributions were not included owing to space limitations.
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FURTHER INFORMATION
Encyclopedia of Life Sciences
Protein–nucleic acid interaction: major groove recognition determinants
Glossary
- NUCLEOSOME
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The basic structural subunit of chromatin, which consists of ∼200 base pairs of DNA and an octamer of histones (a family of small, highly conserved basic proteins).
- CHROMATIN
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The compact form that DNA is organized into in eukaryotic cells, which contains genomic DNA, histones and non-histone proteins.
- HISTONE ACETYLTRANSFERASES AND DEACETYLASES
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Enzymes that modify histones by adding and removing acetyl groups, a chemical modification that can affect chromatin structure.
- ANGIOGENESIS
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Growth of new blood vessels.
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Jamieson, A., Miller, J. & Pabo, C. Drug discovery with engineered zinc-finger proteins. Nat Rev Drug Discov 2, 361–368 (2003). https://doi.org/10.1038/nrd1087
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DOI: https://doi.org/10.1038/nrd1087
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