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Fu, F. et al. Zinc Finger Database (ZiFDB): a repository for information on C2H2 zinc fingers and engineered zinc-finger arrays. Nucleic Acids Res. 37, D279-D283

Department of Genetics, Development & Cell Biology, 1043 Roy J. Carver Co-Laboratory, Iowa State University, Ames, Iowa 50011, USA.
Nucleic Acids Research (Impact Factor: 9.11). 10/2008; 37(Database issue):D279-83. DOI: 10.1093/nar/gkn606
Source: PubMed

ABSTRACT Zinc fingers are the most abundant DNA-binding motifs encoded by eukaryotic genomes and one of the best understood DNA-recognition domains. Each zinc finger typically binds a 3-nt target sequence, and it is possible to engineer zinc-finger arrays (ZFAs) that recognize extended DNA sequences by linking together individual zinc fingers. Engineered zinc-finger proteins have proven to be valuable tools for gene regulation and genome modification because they target specific sites in a genome. Here we describe ZiFDB (Zinc Finger Database; http://bindr.gdcb.iastate.edu/ZiFDB), a web-accessible resource that compiles information on individual zinc fingers and engineered ZFAs. To enhance its utility, ZiFDB is linked to the output from ZiFiT--a software package that assists biologists in finding sites within target genes for engineering zinc-finger proteins. For many molecular biologists, ZiFDB will be particularly valuable for determining if a given ZFA (or portion thereof) has previously been constructed and whether or not it has the requisite DNA-binding activity for their experiments. ZiFDB will also be a valuable resource for those scientists interested in better understanding how zinc-finger proteins recognize target DNA.

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    • "Because C2H2-ZFs are the most prevalent DNA-binding domain in metazoans, high accuracy predictions of their DNA-binding specificities would be a great aid in expanding our knowledge of regulatory networks. Further, due to their ability to bind a wide-range of DNA targets, they have proven to be a powerful platform for genome engineering (45,46), and tools for predicting their DNA-binding specificities can be leveraged for designing proteins to bind specific DNA regions amenable to targeting (47). Previous approaches, both bioinformatics (10–13) and molecular mechanics based (15,48–51), have had limited testing, primarily on the Zif268 model system and a small number of natural C2H2-ZF proteins. "
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    ABSTRACT: Proteins with sequence-specific DNA binding function are important for a wide range of biological activities. De novo prediction of their DNA-binding specificities from sequence alone would be a great aid in inferring cellular networks. Here we introduce a method for predicting DNA-binding specificities for Cys2His2 zinc fingers (C2H2-ZFs), the largest family of DNA-binding proteins in metazoans. We develop a general approach, based on empirical calculations of pairwise amino acid-nucleotide interaction energies, for predicting position weight matrices (PWMs) representing DNA-binding specificities for C2H2-ZF proteins. We predict DNA-binding specificities on a per-finger basis and merge predictions for C2H2-ZF domains that are arrayed within sequences. We test our approach on a diverse set of natural C2H2-ZF proteins with known binding specificities and demonstrate that for >85% of the proteins, their predicted PWMs are accurate in 50% of their nucleotide positions. For proteins with several zinc finger isoforms, we show via case studies that this level of accuracy enables us to match isoforms with their known DNA-binding specificities. A web server for predicting a PWM given a protein containing C2H2-ZF domains is available online at http://zf.princeton.edu and can be used to aid in protein engineering applications and in genome-wide searches for transcription factor targets.
    Nucleic Acids Research 10/2013; 42(1). DOI:10.1093/nar/gkt890 · 9.11 Impact Factor
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    • "Many more copies of the human consensus sequence are found in the genome at non-hotspot sites than at the hotspots themselves [18], and Berg et al. [10] showed that human hotspots possessing or not possessing this consensus sequence are equally dependent on PRDM9. In mice, the consensus sequence originally predicted for the mouse PRDM9Cst variant [6] is more commonly present in non-hotspot than in hotspot regions, and the ZF prediction programs used in various studies [20-22] also predict that the mouse Dom2 variant of PRDM9 (PRDM9Dom2) should bind to hotspots that genetic studies have shown are not activated by this allele. Nevertheless, when tested experimentally, longer oligonucleotides containing buried sequences matching the predicted recognition motifs of two human PRDM9 alleles have been shown to bind PRDM9 protein expressed in cell cultures [6,19]. "
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    ABSTRACT: Background Meiotic recombination ensures proper segregation of homologous chromosomes and creates genetic variation. In many organisms, recombination occurs at limited sites, termed 'hotspots', whose positions in mammals are determined by PR domain member 9 (PRDM9), a long-array zinc-finger and chromatin-modifier protein. Determining the rules governing the DNA binding of PRDM9 is a major issue in understanding how it functions. Results Mouse PRDM9 protein variants bind to hotspot DNA sequences in a manner that is specific for both PRDM9 and DNA haplotypes, and that in vitro binding parallels its in vivo biological activity. Examining four hotspots, three activated by Prdm9Cst and one activated by Prdm9Dom2, we found that all binding sites required the full array of 11 or 12 contiguous fingers, depending on the allele, and that there was little sequence similarity between the binding sites of the three Prdm9Cst activated hotspots. The binding specificity of each position in the Hlx1 binding site, activated by Prdm9Cst, was tested by mutating each nucleotide to its three alternatives. The 31 positions along the binding site varied considerably in the ability of alternative bases to support binding, which also implicates a role for additional binding to the DNA phosphate backbone. Conclusions These results, which provide the first detailed mapping of PRDM9 binding to DNA and, to our knowledge, the most detailed analysis yet of DNA binding by a long zinc-finger array, make clear that the binding specificities of PRDM9, and possibly other long-array zinc-finger proteins, are unusually complex.
    Genome biology 04/2013; 14(4):R35. DOI:10.1186/gb-2013-14-4-r35 · 10.47 Impact Factor
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    • "We built the ZFPs using modular assembly methods, the B2H-based OPEN platform, and a hybrid method that combines the two protocols.14,15 This resulted in several candidate ZFPs for every target half-site.42 Therefore, we used several different screens to identify the most appropriate ZFP that has the ability to recognize and direct activity to the cognate target site, whether as a ZFTF or a ZFN (Table 2 and Supplementary Table S1). "
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    ABSTRACT: The ability to direct human telomerase reverse transcriptase (hTERT) expression through either genetic control or tunable regulatory factors would advance not only our understanding of the transcriptional regulation of this gene, but also potentially produce new strategies for addressing telomerase-associated disease. In this work, we describe the engineering of artificial zinc finger transcription factors (ZFTFs) and ZF nucleases (ZFNs) to target sequences within the hTERT promoter and exon-1. We were able to identify several active ZFTFs that demonstrate a broadly tunable response when screened by a cell-based transcriptional reporter assay. Using the same DNA-binding domains, we generated ZFNs that were screened in combinatorial pairs in cell-based extrachromosomal single-strand annealing (SSA) assays and in gene-targeting assays using stably integrated constructs. Selected ZFN pairs were tested for the ability to induce sequence changes in a Cel1 assay and we observed frequencies of genomic modification up to 18.7% at the endogenous hTERT locus. These screening strategies have pinpointed several ZFN pairs that may be useful in gene editing of the hTERT locus. Our work provides a foundation for using engineered ZF proteins (ZFPs) for modulation of the hTERT locus.Molecular Therapy - Nucleic Acids (2013) 2, e87; doi:10.1038/mtna.2013.12; published online 23 April 2013.
    04/2013; 2(4):e87. DOI:10.1038/mtna.2013.12
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