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Promoter-Targeted Phage Display Selections with Preassembled Synthetic Zinc Finger Libraries for Endogenous Gene Regulation

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Promoter-Targeted Phage Display Selections with Preassembled Synthetic Zinc Finger Libraries for Endogenous Gene Regulation doi:10.1016/j.jmb.2004.04.057 J. Mol. Biol. (2004) 340, 599–613 Promoter-targeted Phage Display Selections with Preassembled Synthetic Zinc Finger Libraries for Endogenous Gene Regulation Caren V. Lund, Pilar Blancafort, Mikhail Popkov and Carlos F. Barbas III* The Skaggs Institute for Regulation of endogenous gene expression has been achieved using Chemical Biology and synthetic zinc finger proteins fused to activation or repression domains, Department of Molecular zinc finger transcription factors (TFZFs). Two key aspects of selective gene Biology, The Scripps Research regulation using TFZFs are the accessibility of a zinc finger protein to its Institute, 10550 North Torrey target DNA sequence and the interaction of the fused activation or repres- Pines Road, La Jolla, CA 92037 sion domain with endogenous proteins. Previous work has shown that USA predicting a biologically active binding site at which a TFZF can control gene expression is not always straightforward. Here, we used a library of preassembled three-finger zinc finger proteins (ZFPs) displayed on fila- mentous phage, and selected for ZFPs that bound along a 1.4 kb promoter fragment of the human ErbB-2 gene. Following affinity selection by phage display, 13 ZFPs were isolated and sequenced. Transcription factors were prepared by fusion of the zinc finger proteins with a VP64 activation domain or a KRAB repression domain and the transcriptional control imposed by these TFZFs was evaluated using luciferase reporter assays. Endogenous gene regulation activity was studied following retroviral delivery into A431 cells. Additional ZFP characterization included DNase I footprinting to evaluate the integrity of each predicted pro- tein:DNA interaction. The most promising TFZFs able to both up-regulate and down-regulate ErbB-2 expression were extended to six-finger pro- teins. The increased affinity and refined specificity demonstrated by the six-finger proteins provided reliable transcriptional control. As a result of studies with the six-finger proteins, the specific region of the promoter most accessible to transcriptional control by VP64-ZFP and KRAB-ZFP fusion proteins was elucidated and confirmed by DNase I footprinting, flow cytometric analysis and immunofluorescence. The ZFP phage display library strategy disclosed here, coupled with the growing availability of genome sequencing information, provides a route to identifying gene-regu- lating TFZFs without the prerequisite of well-defined promoter elements. q 2004 Elsevier Ltd. All rights reserved. Keywords: phage display selection; zinc finger; gene regulation; ErbB-2; *Corresponding author immunofluorescence Introduction and chromatin remodeling proteins associate with the promoter regions of genes.1,2 The presence or Transcription factors (TFs), mediator proteins, absence of these proteins determines the timing Supplementary data associated with this article can be found at doi: 10.1016/j.jmb.2004.04.057 Abbreviations used: TF, transcription factor; ZFP, zinc finger protein; TFZF, zinc finger transcription factor; UTR, untranslated region; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence-activated cell sorting; KRAB, Kruppel-associated box; HA, hema-glutinin; DAPI, 40,6-diamidino-2-phenylindole dihydro-chloride; P/PMR, polypurine/polypyrimidine mirror repeat; GFP, green fluorescent protein; VP64, tetrameric repeat of herpes simplex VP16’s minimal activation domain. E-mail address of the corresponding author: [email protected] 0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. 600 Phage Display Selection of Zinc Finger Transcription Factors and duration of gene transcription. One of the scriptional control is far from complete for any most common classes of TFs contains the DNA- promoter. binding zinc finger transcription factors.3,4 In Here, we have studied the application of phage order to impart artificial control on gene transcrip- display to provide ZFP-based TFs that bind tion, synthetic zinc finger proteins (ZFPs) have throughout a given promoter sequence with the been engineered to bind specific DNA sequences. aim of applying these proteins to endogenous A subset of these engineered proteins (TFZFs) has gene regulation. This strategy resulted in the been shown to control endogenous gene identification of a site at which a three-finger or a expression selectively when fused to activation or six-finger TFZF could provide regulation of ErbB-2 repression domains.5–17 The prediction of success- expression in human epidermoid carcinoma A431 ful target sites for TFZFs in endogenous promoters cells. The site identified would not have been pre- has been based on ZFP affinity, position within the dicted from the linear sequence of the promoter, 50 untranslated region (UTR), and their location known TF binding sites, or chromatin characteri- within DNase I-hypersensitive regions.6–8,10 Appli- zation, suggesting that this approach can be used cation of these criteria, however, has not resulted to synthesize gene regulators without prior charac- consistently in specific gene regulation. With the terization of a target gene’s promoter. roles of endogenous TFs, mediator proteins, and chromatin remodeling proteins largely uncharac- terized for any given promoter, a more empirical Results method of sampling binding sites across a pro- moter is required to achieve robust, imposed gene Phage display optimization and selection regulation. against an ErbB-2 promoter fragment Synthetic ZFPs have been derived from the framework of naturally occurring zinc finger TFs, Previous studies of zinc finger proteins using including Sp1 and the murine Zif268, which have phage display have focused on the selection of 5 three zinc finger domains. The C2H2 zinc finger zinc finger variants of altered specificity. These domain consists of an a helix packed against two studies have utilized short pieces of DNA as the antiparallel b sheets and stabilized via coordination target for selection, typically oligonucleotides that of a zinc ion. The side-chains of amino acid resi- present the 9 bp binding site for a three-finger dues at the terminus of the b-sheet and continuing protein.9,28 – 30 Our goal was to select zinc fingers into the a helix, constitute the DNA reading-head that bound optimally within a 1.4 kb promoter of the domain that is responsible for sequence- fragment. The phage display library used in this specific interaction with the DNA. A single zinc study contained 9177 three-finger ZFPs, each finger domain typically recognizes 3 bp of DNA, capable of recognizing 9 bp of DNA. The construc- primarily through major groove interactions. tion of the library was based on a combinatorial Synthetic ZFPs typically consist of a repeat of assembly of a subset of defined zinc-finger DNA three to six zinc finger domains, allowing the sequences recognizing GNN, ANN and TNN resulting polydactyl proteins to recognize DNA triplets.9,14,30,31 There were only a few TNN sequences of 9–18 bp. Transcription factors have domains available at the time of the library’s been constructed by fusion of an activation or construction, so the library is biased towards repression domain to the ZFP, creating TFZFs. recognition of RNN triplets, where R ¼ Gor Naturally occurring activators and repressors, or A. Theoretically, the double-stranded human 14 variants thereof, are commonly used with ZFPs, genome contains 750 million (RNN)3 sites. In particularly the VP16 activation domain and the addition, many promoters contain GNN-rich Kruppel associated box (KRAB).5,18 – 20 regions of DNA, which makes the library particu- The target of imposed gene regulation is not larly applicable to the isolation of promoter bind- simply DNA, but rather the three-dimensional ing proteins.32 landscape of chromatin structure constructed from As the length of the DNA target was far in excess DNA:protein interactions and protein:protein of any used previously, we first evaluated the interactions along a promoter. The linear sequence ability of the previously established phage display of DNA provides little indication of a promoter’s protocol to recover ZFPs. A six-finger protein, dynamic topology. Eukaryotic gene promoter E2C, with a dissociation constant of 0.75 nM for sequences often stretch over many thousands of an 18 bp site in the ErbB-2 promoter (see Figure bases, and include both positive and negative 2), was used as a positive control.5 Using phage regulatory elements. Genome-wide characteri- expressing the six-finger protein E2C on its surface, zation of promoters employing deletion constructs, test selections were done to compare the recovery linker scanning, computational TF binding site of E2C phage by a biotinylated 1.4 kb promoter or analyses, and TF isolation, is ongoing.21 – 25 In a biotinylated 18 bp hairpin oligonucleotide. Both addition, experiments have begun to address selections had the E2C phage diluted into control the chromatin state of promoters, the location phage bearing a different selective marker at a of nucleosomes, and the nature of histone ratio of 1:10,000. The 1.4 kb promoter DNA yielded modifications.26,27 In spite of this information, the 90% fewer phage compared to the number of understanding of the precise mechanism of tran- phage recovered using the 18 bp oligonucleotide. Phage Display Selection of Zinc Finger Transcription Factors 601 Panning conditions were subsequently optimized, of panning was subcloned into a retroviral vector and phage selection using E2C phage was and expressed as fusions with the activation improved to 85% relative to the phage recovered domain, VP64, which is a tetrameric repeat of the with the 18 bp oligonucleotide. minimal activation domain of herpes simplex The library of preassembled three-finger proteins virus VP16 activator protein.5,33 The ability of each was then selected over four rounds of panning pool of proteins to activate ErbB-2 expression in using the 1.4 kb ErbB-2 promoter fragment. The A431 cells was evaluated using fluorescence- first and second rounds of panning contained activated cell sorting (FACS) analysis (Figure 1C). 125 nM promoter DNA per binding reaction. The The unselected library did show some activation third and fourth rounds contained 62 nM and of ErbB-2 expression.
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