A key step in the regulation of networks that control gene expression is the sequence-specific binding of transcription factors to their DNA recognition sites. A more complete understanding of these DNA-protein interactions will permit a more comprehensive and quantitative mapping of the regulatory pathways within cells, as well as a deeper understanding of the potential functions of individual genes regulated by newly identified DNA-binding sites. Here we describe a DNA microarray-based method to characterize sequence-specific DNA recognition by zinc-finger proteins. A phage display library, prepared by randomizing critical amino acid residues in the second of three fingers of the mouse Zif268 domain, provided a rich source of zinc-finger proteins with variant DNA-binding specificities. Microarrays containing all possible 3-bp binding sites for the variable zinc fingers permitted the quantitation of the binding site preferences of the entire library, pools of zinc fingers corresponding to different rounds of selection from this library, as well as individual Zif268 variants that were isolated from the library by using specific DNA sequences. The results demonstrate the feasibility of using DNA microarrays for genomewide identification of putative transcription factor-binding sites.A n understanding of the sequence specificity of DNAprotein interactions has resulted from studies of the effects of mutations in the DNA-binding sites and the amino acid residues implicated in sequence-specific binding. The zinc-finger transcription factors are among the best understood families in terms of sequence-specific DNA binding. Rational zinc-finger design by using structure-based (1) and database-guided (2) approaches has permitted some progress in revealing certain rules that govern these discriminating contacts (3-6). In addition, phage display has emerged as a powerful tool to select for zinc fingers that recognize given target DNA sites (5, 7-9). Although this technology has permitted millions of protein variants to be sampled simultaneously, the effects of individual mutants have had to be measured one at a time by using nitrocellulose-binding assays (10), gel mobility-shift analysis (11), ELISA (12), Southwestern blotting (13), or reporter constructs (14). Because these methods are generally too laborious to be used for the analysis of a large number of DNA-protein interactions, it has not been possible to gather data on vast collections of variant DNA-protein pairings. Although in vitro selections (15) and ''binding-site signatures'' (4) have permitted the sampling of multiple DNA-binding sites for a given DNAbinding protein, these in vitro selections provide only a partial view of binding-site specificity, because only the tightest binding interactions are selected, whereas information about suboptimal interactions is lost in the experimental process. It is possible that these lower-affinity DNA sites are functionally significant in transcriptional regulation of gene expression. Therefore, we have taken advantage of...

We have used two selection techniques to study sequence-specific DNA recognition by the zinc finger, a smail, modular DNA-binding domain. We have chosen zinc fingers because they bind as independent modules and so can be linked together in a peptlde designed to bind a predetermined DNA site. In this paper, we describe how a library of zinc fingers displayed on the surface of bacteriophage enables selection of fingers capable of binding to given DNA triplets. The amino acid sequences of selected fingers which bind the same triplet are compared to e n how sequence-specific DNA recognition occurs. Our results can be rationalized in terms of coded interactions between zinc fingers and DNA, involving base contacts from a few a-helical positions. In the paper following this one, we describe a complementary technique which confirs the Identity of amino acids capable of DNA sequence discrimination from these positions.