To create a universal system for the control of gene expression, we have studied methods for the construction of novel polydactyl zinc finger proteins that recognize extended DNA sequences. Elsewhere we have described the generation of zinc finger domains recognizing sequences of the 5-GNN-3 subset of a 64-member zinc finger alphabet. Here we report on the use of these domains as modular building blocks for the construction of polydactyl proteins specifically recognizing 9-or 18-bp sequences. A rapid PCR assembly method was developed that, together with this predefined set of zinc finger domains, provides ready access to 17 million novel proteins that bind the 5-(GNN) 6 -3 family of 18-bp DNA sites. To examine the efficacy of this strategy in gene control, the human erbB-2 gene was chosen as a model. A polydactyl protein specifically recognizing an 18-bp sequence in the 5-untranslated region of this gene was converted into a transcriptional repressor by fusion with Krüppel-associated box (KRAB), ERD, or SID repressor domains. Transcriptional activators were generated by fusion with the herpes simplex VP16 activation domain or with a tetrameric repeat of VP16's minimal activation domain, termed VP64. We demonstrate that both gene repression and activation can be achieved by targeting designed proteins to a single site within the transcribed region of a gene. We anticipate that genespecific transcriptional regulators of the type described here will find diverse applications in gene therapy, functional genomics, and the generation of transgenic organisms.

We have taken a comprehensive approach to the generation of novel DNA binding zinc finger domains of defined specificity. Herein we describe the generation and characterization of a family of zinc finger domains developed for the recognition of each of the 16 possible 3-bp DNA binding sites having the sequence 5-GNN-3. Phage display libraries of zinc finger proteins were created and selected under conditions that favor enrichment of sequence-specific proteins. Zinc finger domains recognizing a number of sequences required refinement by site-directed mutagenesis that was guided by both phage selection data and structural information. In many cases, residues not expected to make basespecific contacts had effects on specificity. A number of these domains demonstrate exquisite specificity and discriminate between sequences that differ by a single base with >100-fold loss in affinity. We conclude that the three helical positions ؊1, 3, and 6 of a zinc finger domain are insufficient to allow for the fine specificity of the DNA binding domain to be predicted. These domains are functionally modular and may be recombined with one another to create polydactyl proteins capable of binding 18-bp sequences with subnanomolar affinity. The family of zinc finger domains described here is sufficient for the construction of 17 million novel proteins that bind the 5-(GNN) 6 -3 family of DNA sequences. These materials and methods should allow for the rapid construction of novel gene switches and provide the basis for a universal system for gene control.

Chimeric nucleases that are hybrids between a nonspecific DNA cleavage domain and a zinc finger DNA recognition domain were tested for their ability to find and cleave their target sites in living cells. Both engineered DNA substrates and the nucleases were injected into Xenopus laevis oocyte nuclei, in which DNA cleavage and subsequent homologous recombination were observed. Specific cleavage required two inverted copies of the zinc finger recognition site in close proximity, reflecting the need for dimerization of the cleavage domain. Cleaved DNA molecules were activated for homologous recombination; in optimum conditions, essentially 100% of the substrate recombined, even though the DNA was assembled into chromatin. The original nuclease has an 18-amino-acid linker between the zinc finger and cleavage domains, and this enzyme cleaved in oocytes at paired sites separated by spacers in the range of 6 to 18 bp, with a rather sharp optimum at 8 bp. By shortening the linker, we found that the range of effective site separations could be narrowed significantly. With no intentional linker between the binding and cleavage domains, only binding sites exactly 6 bp apart supported efficient cleavage in oocytes. We also showed that two chimeric enzymes with different binding specificities could collaborate to stimulate recombination when their individual sites were appropriately placed. Because the recognition specificity of zinc fingers can be altered experimentally, this approach holds great promise for inducing targeted recombination in a variety of organisms.Procedures and reagents that allow the directed alteration of genes in situ constitute a powerful toolbox for experimental genetics and potentially for agricultural and therapeutic applications. In many organisms, however, and particularly in higher eukaryotes, the efficiency of recombination between an introduced DNA and the homologous chromosomal target is discouragingly low. For example, such events typically occur in mammalian cells at a frequency of only about 1 for each 10 6 cells treated (3, 31). We are interested in developing procedures that would substantially improve the frequency of gene targeting.A major impediment to efficient gene replacement is the status of the chromosomal target. Increasing the number of target sequences has little or no effect on targeting efficiency (54, 60). In contrast, making an intentional double-strand break (DSB) in the target DNA increases the yield of specific homologous recombination events up to 1,000-fold or more (10,11,14,44,46). It is believed that exonucleases act at broken ends to generate single-stranded tails that are recombinagenic in any of several pathways. In particular, the singlestrand annealing mechanism (33), by which homologous recombination involving exogenous DNA usually occurs in higher eukaryotes (53), cannot proceed unless both the donor and target have ends (5, 48).Whatever the mechanism of recombination, it is clear that the frequency of targeted recombination can be substantially improved by i...

TitleStructure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases active, two subunits of these zinc finger nucleases (ZFN) are typically assembled at the 5 cleavage site. Presumably due to cleavage at off-target sites, the use of ZFNs is often associated with significant cytotoxicity. Here, we describe a structure-based approach to reduce ZFN-induced toxicity. Rational redesign of the FokI dimer interface aiming at destabilizing dimerization in combination with preventing homodimerization of the ZFN subunits was based on protein modeling and energy calculations. Cell-based recombination 10 assays confirmed that the modified ZFNs elicit significantly reduced cytotoxicity without compromising on performance. Our results present a critical step towards the therapeutic application of the ZFN technology.

RNA-directed DNA methylation (RdDM) in Arabidopsis thaliana depends on the upstream synthesis of 24-nucleotide small interfering RNAs (siRNAs) by RNA POLYMERASE IV (Pol IV)1,2 and downstream synthesis of non-coding transcripts by Pol V. Pol V transcripts are thought to interact with siRNAs which then recruit DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) to methylate DNA3-7. The SU(VAR)3-9 homologs SUVH2 and SUVH9 act in this downstream step but the mechanism of their action is unknown8,9. Here we show that genome-wide Pol V association with chromatin redundantly requires, SUVH2 and SUVH9. Although SUVH2 and SUVH9 resemble histone methyltransferases a crystal structure reveals that SUVH9 lacks a peptide-substrate binding cleft and lacks a properly formed S-adenosyl methionine (SAM) binding pocket necessary for normal catalysis, consistent with a lack of methyltransferase activity for these proteins8. SUVH2 and SUVH9 both contain SET- and RING-ASSOCIATED (SRA) domains capable of binding methylated DNA8, suggesting that they function to recruit Pol V through DNA methylation. Consistent with this model, mutation of DNA METHYLTRANSFERASE 1 (MET1) causes loss of DNA methylation, a nearly complete loss of Pol V at its normal locations, and redistribution of Pol V to sites that become hypermethylated. Furthermore, tethering SUVH2 with a zinc finger to an unmethylated site is sufficient to recruit Pol V and establish DNA methylation and gene silencing. These results suggest that Pol V is recruited to DNA methylation through the methyl-DNA binding SUVH2 and SUVH9 proteins, and our mechanistic findings suggest a means for selectively targeting regions of plant genomes for epigenetic silencing.

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 ␤␤␣ ...

Zinc-finger proteins of the Cys 2 -His 2 type represent a class of malleable DNA-binding proteins that may be selected to bind diverse sequences. Typically, zinc-finger proteins containing three zinc-finger domains, like the murine transcription factor Zif268 and the human transcription factor Sp1, bind nine contiguous base pairs. To create a class of proteins that would be generally applicable to target unique sites within complex genomes, we have utilized structurebased modeling to design a polypeptide linker that fuses two three-finger proteins. Two six-fingered proteins were created and demonstrated to bind 18 contiguous bp of DNA in a sequence-specific fashion. Expression of these proteins as fusions to activation or repression domains allows transcription to be specifically up-or down-modulated within human cells. Polydactyl zinc-finger proteins should be broadly applicable as genome-specific transcriptional switches in gene therapy strategies and the development of novel transgenic plants and animals.From the simplest of organisms to the most complex, transcriptional regulation is achieved primarily by proteins that bind nucleic acids. The advent of genomic sequencing and the availability of the complete sequences of several genomes provide new opportunities to study biology and to develop therapeutic strategies through specific modulation of the transcription of target genes. Of the DNA-binding motifs that have been manipulated by design or selection, the TFIIIA-related Cys 2 -His 2 zinc-finger proteins have demonstrated the greatest potential for manipulation into general and specific transcription factors (1, 2). Each Cys 2 -His 2 zinc-finger domain consists of approximately 30 amino acids and typically binds 3 base pairs of double-stranded DNA sequence (3, 4).Specific delivery of a DNA-binding protein to a single site within a genome as complex as that found in humans, 3.5 billion bp, requires an address of at least 16 bp. Statistically assuming random base distribution, a unique 16-bp sequence will occur only once in 4 16 or 4.3 billion nucleotides, roughly the same size of a human genome (3.5 ϫ 10 9 bp). An 18-bp address would be specific within 68 billion base pairs of sequence. The 18-bp address could be specified by a protein containing six zinc fingers if the periodicity of the protein domains could match that of the DNA over this extended sequence. An address of this length would be sufficient to uniquely specify any locus within all known genomes. Although natural proteins containing long polydactyl arrays of zinc-finger domains have been inferred from sequence, no zinc-finger proteins have been demonstrated to bind such a long, contiguous DNA sequence. Structural studies of the five-finger human glioblastoma (GLI) protein-DNA complex (5) and biochemical studies of the nine-finger protein TFIIIA (6, 7) have demonstrated that DNA binding in these polydactyl proteins is dominated by the interactions of a select few fingers. Sequence-specific binding of more than three contiguous zinc-fin...

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