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

Abstract

Considerable progress has been made in recent years in the design of transcription factors for the directed regulation of endogenous genes. Although many strategies involve selection methods that must be applied for each new target sequence, we have developed an approach based on linkage of predefined zinc finger domains that each recognize a three-base pair DNA sequence to construct artificial transcription factors that bind to a desired sequence. These domains can be assembled to recognize unique 18-base pair DNA sequences with high specificity. Here we report the development and characterization of zinc finger domains that bind to 15 of the 16 5-CNN-3 subsites. These domains were created through a combination of phage display selection, site-directed mutagenesis, and de novo design. Furthermore, these domains were used to generate a highly specific six-finger protein targeting the ERBB-2 promoter. When fused to regulatory domains, this protein was capable of up-and down-regulating the expression of the endogenous ERBB-2 gene. With the addition of this collection of predefined zinc finger domains, most 5-CNN-3-, 5-GNN-3-, and 5-ANN-3-containing sequences can now be rapidly targeted for directed gene regulation and nuclease cleavage.The ability to rapidly prepare proteins with predefined specificities for DNA sequences could enable a wide range of technologies that might be used, for example, to direct the expression of genes or to physically modify genes and genomes. To develop a universal system for gene regulation, much effort has been applied to the development of artificial transcription factors based on polydactyl zinc finger proteins (1-3). Such a system might have considerable impact on biology and biotechnology and offer a new approach for treatment of diseases based on directed gene regulation. It has now been shown that gene expression can be specifically altered using artificial transcription factors based on polydactyl zinc finger proteins that bind to 18-bp target sites (1, 2). Targeting of sites as small as 9 bp can also provide some degree of regulatory specificity presumably through the aid of chromatin occlusion (4 -6). In addition to transcriptional regulation, novel zinc finger DNA binding specificities show tremendous promise in directing homologous recombination through their fusion with the Fok I nuclease domain (7,8).Zinc finger domains of the type Cys 2 -His 2 are a unique and promising class of proteins for the recognition of extended DNA sequences due to their modular nature. Each domain consists of ϳ30 amino acids folded into a ␤␤␣ structure stabilized by hydrophobic interactions and chelation of a zinc ion by the conserved Cys 2 -His 2 residues (9, 10). To date, the best-characterized protein of this family of zinc finger proteins is the mouse transcription factor Zif268. Each of the three zinc finger domains of Zif268 binds to a 3-bp subsite by insertion of the ␣-recognition helix into the major groove of the DNA double helix (11,12). To facilitate the rapid construction of DNA-bin...

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

“…A BLAST-Search on the human genome has revealed that the three 18-bp targeted sites in the maspin promoter are unique (data not shown). Targets À126 and À97 were selected based on their close proximity to the transcription start site and because of the highly specific interactions predicted for the DNA triplets and the available ZF lexicons (Segal et al, 1999;Dreier et al, 2000Dreier et al, , 2002Dreier et al, , 2005. In addition to the proximal ATF sites, we have designed an ATF recognizing an 18-bp site comprising the hormone response element (HRE) sequence, which interacts with the hormone receptor both in breast (Khalkhali-Ellis et al, 2004) and prostate (Zhang et al, 1997) cells.…”

“…(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] .…”

Benzoate X receptor zinc-finger gene switches for drug-inducible regulation of transcription

“…[8][9][10] To date, ZF domains that recognized almost all of the GNN, ANN and CNN DNA triplets have been identified. [10][11][12][13][14] Approaches other than modular assembly also provide access to polydactyl ZF proteins. 15 Artificial transcription factors that use ZFs for DNA recognition typically use six ZFs to recognize 18 bases of DNA; this length of sequence is theoretically unique in the genome.…”