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

Abstract

In previous studies we have developed Cys 2 -His 2 zinc finger domains that specifically recognized each of the 16 5-GNN-3 DNA target sequences and could be used to assemble six-finger proteins that bind 18-base pair DNA sequences (Beerli, R. R., Dreier, B., and Barbas, C. F., III (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 1495-1500). Such proteins provide the basis for the construction of artificial transcription factors to study gene/function relationships in the post-genomic era. Central to the universal application of this approach is the development of zinc finger domains that specifically recognize each of the 64 possible DNA triplets. Here we describe the construction of a novel phage display library that enables the selection of zinc finger domains recognizing the 5-ANN-3 family of DNA sequences. Library selections provided domains that in most cases showed binding specificity for the 3-base pair target site that they were selected to bind. These zinc finger domains were used to construct 6-finger proteins that specifically bound their 18-base pair target site with affinities in the pM to low nM range. When fused to regulatory domains, these proteins containing various numbers of 5-ANN-3 domains were capable of specific transcriptional regulation of a reporter gene and the endogenous human ERBB-2 and ERBB-3 genes. These results suggest that modular DNA recognition by zinc finger domains is not limited to the 5-GNN-3 family of DNA sequences and can be extended to the 5-ANN-3 family. The domains characterized in this work provide for the rapid construction of artificial transcription factors, thereby greatly increasing the number of sequences and genes that can be targeted by DNA-binding proteins built from pre-defined zinc finger domains.The study of protein-DNA interactions is central to our understanding of the regulation of genes and the flow of genetic information characteristic of life. One practical application of the development of a protein-DNA recognition system is the construction of artificial transcription factors that might be used to purposefully regulate gene expression. We have demonstrated that gene expression can be specifically altered through the use of designed polydactyl zinc finger transcription factors that bind 18 base pairs (bp) 1 of DNA sequence. Because of their extended DNA recognition site, these proteins have the potential to be genome-specific transcriptional regulators (1, 2). Targeting of only 9 bp of sequence can also result in gene regulation wherein chromatin structure provides for an additional level of specificity (3, 4). Because a universal system for gene regulation would provide many new opportunities in basic and applied biology and medicine, the development of such a system is of considerable interest.Two key features have made Cys 2 -His 2 zinc finger domains the most promising DNA recognition motifs for the construction of artificial transcription factors, modular structure, and modular recognition. Each domain consists of ϳ30 amino acids and folds into a ␤␤␣ ...

Drug-inducible and simultaneous regulation of endogenous genes by single-chain nuclear receptor-based zinc-finger transcription factor gene switches

“…By taking comprehensive approaches and using phage display selection strategies, we and others have successfully prepared ZF domains that target almost all GNN, ANN and CNN DNA triplets. [3][4][5][6][7] Typically, designer TF ZF s are composed of up to six ZF domains; a six-finger ZF binds to 18 bp of DNA; this allows recognition of a unique sequence within the human genome. 8 When combined with repressor or activator domains, ZF proteins can be used to regulate transcription of genes.…”

Re-activation of a dormant tumor suppressor gene maspin by designed transcription factors

“…(b) a-Helical ZF sequences engineered to bind their corresponding target DNA triplets. These ZF sequences were chosen based on the available specific ZF lexicons (Segal et al, 1999;Dreier et al, 2000Dreier et al, , 2001Dreier et al, , 2005. The 6ZF ATFs were constructed by overlapping PCR; Supplementary methods).…”

Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases

“…A single zinc finger motif consists of 28 amino acids and includes an α-helix that contacts three bases in the major groove of DNA. Through modular assembly of such zinc finger modules, artificial DNA-binding domains can be created to bind to a wide 15 variety of DNA sequences [10][11][12][13][14] . A DNA-binding domain consisting of three zinc finger motifs has hence the potential to bind 9 bp of DNA.…”

“…A DNA-binding domain consisting of three zinc finger motifs has hence the potential to bind 9 bp of DNA. Many engineered 3-finger proteins have been shown to have excellent binding specificity in vitro, while others were less specific [10][11][12][13][14][15] .…”