A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter

Isalan, Mark; Klug, A.; Choo, Yen

doi:10.1038/90264

Cited by 178 publications

(162 citation statements)

References 30 publications

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“…The Zif268 DNA-binding domain is currently the main focus of a field of research whose aim is to create proteins that can bind specifically to any chosen DNA sequence. Already, variants of the Zif268 domain have been created that recognize naturally occurring sequences in the genomes of higher eukaryotes, and these have been used to target chimeric proteins to specific genomic loci (22,23). It should therefore be quite straightforward to adapt the system described here to promote recombination at many novel sites, by replacing the Zif268 domain of Z-resolvase with one of these variants.…”

Section: Discussionmentioning

confidence: 99%

Chimeric recombinases with designed DNA sequence recognition

Akopian

Boocock

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

107

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Site-specific recombination typically occurs only between DNA sequences that have co-evolved with a natural recombinase enzyme to optimize sequence recognition, catalytic efficiency, and regulation. Here, we show that the sequence recognition and the catalysis functions of a recombinase can be specified by unrelated protein domains. We describe chimeric recombinases with a catalytic domain from an activated multiple mutant of the bacterial enzyme Tn3 resolvase, fused to a DNA recognition domain from the mouse transcription factor Zif268. These proteins catalyze efficient recombination specifically at synthetic target sites recognized by two Zif268 domains. Our results demonstrate the functional autonomy of the resolvase catalytic domain and open the way to creating ''custom-built'' recombinases that act at chosen natural target sequences.

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Section: Discussionmentioning

confidence: 99%

Chimeric recombinases with designed DNA sequence recognition

Akopian

Boocock

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

107

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“…The ZFP itself was assembled from an archive of two-finger modules described in ref. 20, wherein the amino acid residues of the helical regions (from the Ϫ1 to ϩ6 positions) responsible for specific DNA binding are F1, RSDHLSR; F2, DNRDRTK; F3, DRKTLIE; F4, TSSGLSR; F5, RSDHLSE; and F6, TSSDRTK.…”

Section: Mapping Of Dnase I-accessible Chromatin Regions In Chk2 Locusmentioning

confidence: 99%

“…HS1 contained the major start site of transcription, as determined by rapid amplification of cDNA ends (or RACE) (data not shown). The sequence of the HS1 site was therefore used to design a six-finger ZFP TF (ZFP-5475) recognizing the site 5Ј-ACCCGGGTTCCCCTCGGG-3Ј constructed from an archive of zinc-finger DNA-binding modules (20). This ZFP TF consisted of a string of three two-finger units, which was demonstrated to have increased specificity over more conventional poly-zinc-finger peptide units in vitro (18).…”

Section: Retroviral Constructs Virus Preparation and Generation Of mentioning

confidence: 99%

Zinc-finger protein-targeted gene regulation: Genomewide single-gene specificity

Tan

Guschin

Davalos

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

137

126

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Zinc-finger protein transcription factors (ZFP TFs)D efects in transcriptional regulation underlie numerous disease states, most notably cancer (1). A major goal of current strategies for correcting such defects is to achieve sufficient specificity of action (2). Designed zinc-finger protein transcription factors (ZFP TFs) emulate natural transcriptional control mechanisms and therefore provide an attractive tool for precisely regulating gene expression. Accurate control of gene expression is important for understanding gene function (target validation) and for developing therapeutics to treat disease (3). We and others have used engineered ZFP TFs to either activate or repress a variety of endogenous gene targets (4-11). For these proteins, or any other gene-regulation technology, to succeed as tools in drug discovery or direct agents in the clinic, their specificity of action within the genome must be precise, a challenging criterion to meet given the size and complexity of the human genome. Recent studies with small interfering RNA (12, 13) and antisense DNA͞RNA (14) have illuminated the magnitude of the task of achieving single-gene specificity in regulating the human genome.We focus here on the use of ZFP TFs in the area of oncology and specifically on the emerging role of checkpoint kinase 2 (CHK2). CHK2 acts as a key integrator of DNA-damage signals regulating cell-cycle progression, DNA repair, and cell death by phosphorylating a variety of substrates, including the p53 tumor suppressor protein (15, 16). Here we show that a designed ZFP TF targeted to a unique 18-bp recognition sequence in the promoter of the CHK2 gene binds the intended site within chromatin and represses CHK2 transcription in vivo. Moreover, repression of CHK2 by this engineered ZFP TF occurs with remarkable specificity, while simultaneously reducing CHK2 protein to levels that functionally ablate the action of this kinase. Finally, we show that constitutive expression of the ZFP TF in telomerase-immortalized, untransformed human fibroblasts provides stable repression of the CHK2 gene and results in loss of DNA-damage-induced CHK2-dependent phosphorylation of p53 on Ser-20. These data demonstrate that ZFP TFs can be exquisitely specific, yet potent repressors of gene expression and, therefore, are potentially powerful reagents for target validation and therapeutic interventions in vivo.

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“…Cys 2 -His 2 ZFPs with the desired DNA sequence specificity were assembled by joining three two-finger units, where each two-finger unit was chosen from a pool of premade ZFP libraries selected via phage display [18]. Within each unit, zinc finger domains were connected with the canonical TGEKP linker; the two-finger units were connected with extended linkers [19] to enhance DNA-binding specificity.…”

Section: Zfp Design Analysis and Plasmid Constructionmentioning

confidence: 99%

Engineered Zinc Finger Proteins for Controlling Stem Cell Fate

et al. 2003

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Stem cells are functionally defined as progenitor cells that can self-renew and differentiate. Critical transitions in these cells are controlled via signaling pathways and subsequent transcriptional regulation. Technologies capable of modulating the levels of gene expression, especially those of transcription factors, represent powerful tools for research and could potentially be used in therapeutic applications. In this study, we evaluated the ability of synthetic zinc finger protein transcription factors (ZFPTFs) to cause the differentiation of embryonic stem (ES) cells. We constructed ZFP-TFs that target the mouse Oct-4 gene (which is a major regulator of ES cell pluripotency and self-renewal). These designed transcription factors were able to regulate the transcription of Oct-4, affecting the expression of downstream genes and thus regulating ES cell differentiation.

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A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter

Cited by 178 publications

References 30 publications

Chimeric recombinases with designed DNA sequence recognition

Chimeric recombinases with designed DNA sequence recognition

Zinc-finger protein-targeted gene regulation: Genomewide single-gene specificity

Engineered Zinc Finger Proteins for Controlling Stem Cell Fate

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