Designing Transcription Factor Architectures for Drug Discovery

Blancafort, Pilar; Segal, David J.; Barbas, Carlos F.

doi:10.1124/mol.104.002758

Cited by 164 publications

(124 citation statements)

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“…These results clearly demonstrate that the observed effects were not due to special properties of ZFs Zif268 and PBSII, but can be achieved using other custom ZFs, such as PE1A. This feature suggests that SEERs can be rapidly designed to detect virtually any desired DNA sequence using custom ZF DNA-binding technology (14,15).In summary, we have demonstrated the use of TEM-1 β-lactamase to improve the sequenceenabled enzyme reactivation method for the detection of double-stranded DNA. The assay can detect its target sequence rapidly and with high specificity.…”

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confidence: 61%

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Sequence-Enabled Reassembly of β-Lactamase (SEER-LAC): A Sensitive Method for the Detection of Double-Stranded DNA

et al. 2006

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This work describes the development of a new methodology for the detection of specific doublestranded DNA sequences. We previously showed that two inactive fragments of green fluorescent protein, each coupled to engineered zinc finger DNA-binding proteins, were able to reassemble an active reporter complex in the presence of a predefined DNA sequence. This system, designated SEquence-Enabled Reassembly (SEER), was demonstrated in vitro to produce a DNAconcentration-dependent signal. Here we endow the SEER system with catalytic capability using the reporter enzyme TEM1 β-lacatamase. This system could distinguish target from non-target DNA in less than 5 minutes, representing a more than 1,000-fold improvement over our previous SEER design. A single base pair substitution in the DNA binding sequence reduced the signal to nearly background levels. Substitution of a different custom zinc finger DNA-binding domain produced a signal only on the new cognate target. Signal intensity was not affected by genomic DNA when present in equal mass to the target DNA. These results present SEER as a rapid and sensitive method for the detection of double-stranded DNA sequences.Keywords zinc finger; beta-lactamase; diagnostics; biosensors Virtually all scientific methods for reading the sequence information of DNA rely on the hybridization properties of complementary nucleic acid molecules. Such methods, including PCR, Sanger sequencing, DNA microarray, Southern and Northern blotting, and in situ hybridization, all consequently require denaturation of the native DNA double helix into single strands and subsequent renaturation with specific primers or probes under carefully controlled conditions. In contrast, nature frequently relies on sequence-specific DNA-binding proteins to read the sequence information of DNA, such as occurs during the processes of transcription initiation, intron homing, and defense against invasive DNA by restriction endonucleases. In the human genome, DNA-binding transcription factors comprise one of the largest classes of † D.S. was supported by American Cancer Society IRG7400125 and NIH P50CA95060. C.I.S. was supported by an NIH training grant. AUTHOR EMAIL ADDRESSES: segal@pharmacy.arizona.edu, ghosh@email.arizona.edu * TO WHOM CORRESPONDENCE SHOULD BE ADDRESSED: David J. Segal, djsegal@ucdavis.edu Indraneel Ghosh, Tel: 520-621-6331, E-mail: ghosh@email.arizona.edu § PRESENT ADDRESS: Department of Pharmacology/Genome Center University of California, Davis Davis, CA 95616 BRIEFS: A split protein system composed of the reporter enzyme β-lactamase and designed zinc-finger DNA-binding domains can detect specific double-stranded DNA sequences in less than five minutes. SUPPORTING INFORMATION AVAILABLE: Details of the SEER-LAC protein fragment design, construction, and an annotated sequence, as well a full description of the oligonucleotide DNA targets used in this study and additional information on assay background are available free of charge via the Internet at http://pubs.acs.org. Here we describe...

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“…These custom-made ZF proteins can be linked to functional domains to generate novel chimeric proteins that produce the desired activity at specific DNA sequences. This approach has been employed in designing targeted transcription factors (14,16), targeted endonucleases (17), and targeted integrases (18).…”

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Sequence-Enabled Reassembly of β-Lactamase (SEER-LAC): A Sensitive Method for the Detection of Double-Stranded DNA

et al. 2006

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“…Each ZF domain specifically recognizes 3 base pairs (bps) of DNA (Pavletich and Pabo, 1991). Thus, a designed 6ZF ATF will recognize 18-bp, providing high degree of specificity (Blancafort et al, 2004;Beltran et al, 2006). Indeed, previous reports have described that highly specific 6ZF ATFs are able to target unique sites in the human genome (Tan et al, 2003).…”

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confidence: 99%

Re-activation of a dormant tumor suppressor gene maspin by designed transcription factors

et al. 2006

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The controlled and specific re-activation of endogenous tumor suppressors in cancer cells represents an important therapeutic strategy to block tumor growth and subsequent progression. Other than ectopic delivery of tumor suppressor-encoded cDNA, there are no therapeutic tools able to specifically re-activate tumor suppressor genes that are silenced in tumor cells. Herein, we describe a novel approach to specifically regulate dormant tumor suppressors in aggressive cancer cells. We have targeted the Mammary Serine Protease Inhibitor (maspin) (SERPINB5) tumor suppressor, which is silenced by transcriptional and aberrant promoter methylation in aggressive epithelial tumors. Maspin is a multifaceted protein, regulating tumor cell homeostasis through inhibition of cell growth, motility and invasion. We have constructed artificial transcription factors (ATFs) made of six zinc-finger (ZF) domains targeted against 18-base pair (bp) unique sequences in the maspin promoter. The ZFs were linked to the activator domain VP64 and delivered in breast tumor cells. We found that the designed ATFs specifically interact with their cognate targets in vitro with high affinity and selectivity. One ATF was able to re-activate maspin in cell lines that comprise a maspin promoter silenced by epigenetic mechanisms. Consistently, we found that this ATF was a powerful inducer of apoptosis and was able to knock down tumor cell invasion in vitro. Moreover, this ATF was able to suppress MDA-MB-231 growth in a xenograft breast cancer model in nude mice. Our work suggests that ATFs could be used in cancer therapeutics as novel molecular switches to re-activate dormant tumor suppressors.

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“…Typically, three zinc fingers are combined to bind to a specific 9-bp DNA sequence with the nanomolar affinity required to be biologically useful, but additional fingers can be incorporated to confer increased specificity (8-11). Zinc-finger fusions to various functional domains have been used to create artificial transcription factors and DNA-modifying proteins (12,13).When attached to the FokI nuclease domain, zinc fingers can direct cleavage to specific DNA sequences. The nuclease domain must dimerize to cleave DNA (14), and because the dimer interface is weak (15), two nuclease domains are typically brought into close proximity by pairs of zinc-finger sets binding to neighboring sites.…”

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Induction and repair of zinc-finger nuclease-targeted double-strand breaks in Caenorhabditis elegans somatic cells

Morton

Davis

Jørgensen

et al. 2006

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

169

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Zinc-finger nucleases are chimeric proteins consisting of engineered zinc-finger DNA-binding motifs attached to an endonuclease domain. These proteins can induce site-specific DNA double-strand breaks in genomic DNA, which are then substrates for cellular repair mechanisms. Here, we demonstrate that engineered zinc-finger nucleases function effectively in somatic cells of the nematode Caenorhabditis elegans. Although gene-conversion events were indistinguishable from uncut DNA in our assay, nonhomologous end joining resulted in mutations at the target site. A synthetic target on an extrachromosomal array was targeted with a previously characterized nuclease, and an endogenous genomic sequence was targeted with a pair of specifically designed nucleases. In both cases, Ϸ20% of the target sites were mutated after induction of the corresponding nucleases. Alterations in the extrachromosomal targets were largely products of end-filling and blunt ligation. By contrast, alterations in the chromosomal target were mostly deletions. We interpret these differences to reflect the abundance of homologous templates present in the extrachromosomal arrays versus the paucity of such templates for repair of chromosomal breaks. In addition, we find evidence for the involvement of error-prone DNA synthesis in both homologous and nonhomologous pathways of repair. DNA ligase IV is required for efficient end joining, particularly of blunt ends. In its absence, a secondary end-joining pathway relies more heavily on microhomologies in producing deletions.DNA repair ͉ gene targeting ͉ nematodes ͉ nonhomologous end joining T he ability to make targeted double-strand breaks in chromosomal DNA has several important uses. It allows the detailed study of DNA repair mechanisms; it leads to localized mutagenesis at the break site; and it enhances the efficiency of targeted gene replacement through homologous recombination. We have been exploring the capabilities of one class of targetable cleavage reagents, the zinc-finger nucleases (ZFNs).ZFNs are chimeric proteins composed of DNA-binding Cys 2 His 2 zinc fingers fused to the nonspecific nuclease domain of the restriction enzyme FokI (1). Each finger makes contact primarily with a separate DNA triplet (2, 3). Natural and artificial zinc fingers have been characterized that bind to all 5Ј-GNN-3Ј, many ANN and CNN, and some TNN triplets (4-7). Furthermore, the modular nature of the zinc fingers allows them to be joined in essentially arbitrary combinations. Typically, three zinc fingers are combined to bind to a specific 9-bp DNA sequence with the nanomolar affinity required to be biologically useful, but additional fingers can be incorporated to confer increased specificity (8-11). Zinc-finger fusions to various functional domains have been used to create artificial transcription factors and DNA-modifying proteins (12,13).When attached to the FokI nuclease domain, zinc fingers can direct cleavage to specific DNA sequences. The nuclease domain must dimerize to cleave DNA (14), and because the di...

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Designing Transcription Factor Architectures for Drug Discovery

Cited by 164 publications

References 98 publications

Sequence-Enabled Reassembly of β-Lactamase (SEER-LAC): A Sensitive Method for the Detection of Double-Stranded DNA

Sequence-Enabled Reassembly of β-Lactamase (SEER-LAC): A Sensitive Method for the Detection of Double-Stranded DNA

Re-activation of a dormant tumor suppressor gene maspin by designed transcription factors

Induction and repair of zinc-finger nuclease-targeted double-strand breaks in Caenorhabditis elegans somatic cells

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