Regulation of endogenous gene expression using small molecule-controlled engineered zinc-finger protein transcription factors

Dent, Carolyn L.; Lau, Gee W.; Drake, Elizabeth A.; Yoon, A-Rum; Case, Colette; Gregory, Philip D.

doi:10.1038/sj.gt.3302985

Cited by 24 publications

(23 citation statements)

References 46 publications

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“…Previous studies demonstrated that chemically iTF ZF s, prepared using single-chain estrogen receptor homodimers and retinoid X/ecdysone receptor heterodimers with engineered LBDs that are not sensitive to endogenous hormones, worked to regulate transcription of reporter genes in response to bioavailable ligand homologs that are considered safe. 12 Dent et al 27 used the progesterone receptor in conjunction with a ZF and the p65 activation domain to regulate endogenous VEGF-A. Here we have expanded the gene switch toolbox to show that iTF ZF s prepared using engineered estrogen receptor and retinoid X/ecdysone receptor can both activate and repress the transcription of endogenous genes.…”

Section: Discussionmentioning

confidence: 99%

Drug-inducible and simultaneous regulation of endogenous genes by single-chain nuclear receptor-based zinc-finger transcription factor gene switches

2008

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Chemically inducible gene switches that regulate expression of endogenous genes have multiple applications for basic gene expression research and gene therapy. Single-chain zinc-finger transcription factors that utilize either estrogen receptor homodimers or retinoid X receptor-a/ecdysone receptor heterodimers are shown here to be effective regulators of ICAM-1 and ErbB-2 transcription. Using activator (VP64) and repressor (Krü ppel-associated box) domains to impart regulatory directionality, ICAM-1 was activated by 4.8-fold and repressed by 81% with the estrogen receptor-inducible transcription factors. ErbB-2 was activated by up to threefold and repressed by 84% with the retinoid X receptor-a/ecdysone receptor-inducible transcription factors. The dynamic range of these proteins was similar to the constitutive system and showed negligible basal regulation when ligand was not present. We have also demonstrated that the regulation imposed by these inducible transcription factors is dose dependent, sustainable for at least 11 days and reversible upon cessation of drug treatment. Importantly, these proteins can be used in conjunction with each other with no detectable overlap of activity enabling concurrent and temporal regulation of multiple genes within the same cell. Thus, these chemically inducible transcription factors are valuable tools for spatiotemporal control of gene expression that should prove valuable for research and gene therapy applications.

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Section: Discussionmentioning

confidence: 99%

Drug-inducible and simultaneous regulation of endogenous genes by single-chain nuclear receptor-based zinc-finger transcription factor gene switches

2008

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“…In conclusion, we have demonstrated that BXRb can function, in conjunction with ZFs, as a gene switch in the context of reporter plasmids and endogenous genes. These BLB and RLB ZF TF s complement the previously described nuclear receptor-based gene switches 5,6 by providing a system activated by small, inexpensive ligands. The BLB and RLB ZF TF systems can be readily adapted through engineering of the BXR LBD to bind derivatives of the known ligands as has been previously done with RXR.…”

Section: Resultsmentioning

confidence: 93%

“…2,3 Using nuclear receptor ligand-binding domains (LBDs) as effector domains has the added desired elements of tunable exogenous and reversible control. [4][5][6] Selected ZF domains specifically recognize three bases of DNA. 7 ZFs are modular proteins that can be combined to create chains that recognize longer DNA sequences.…”

Section: Introductionmentioning

confidence: 99%

“…6 In an alternative approach, Dent and coworkers used a truncated progesterone receptor and the nuclear factor-kB p65 activation domain linked to the VZ+434 ZF to create a smallmolecule-activated gene switch that relies on translocation of the protein to the nucleus after ligand binding. 5 Here, we report a new tool for the gene-switch toolbox that uses the benzoate X receptor-b (BXRb) as a gene switch. The BXRb from Xenopus laevis is a member of the nuclear receptor superfamily of ligand-activated transcription factors.…”

Section: Introductionmentioning

confidence: 99%

“…Ligands for other nuclear receptor used in gene switches include tamoxifen, mifepristone and ponasterone-A. 5,6 These compounds are expensive and, with the exception of ponasterone-A, have biological activity in humans. The ligands for BXRb, ethyl-4-hydroxybenzoate (E-4HB) and propyl-4-hydroxybenzoate (P-4HB), are benzoates and are commonly known as parabens.…”

Section: Introductionmentioning

confidence: 99%

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Benzoate X receptor zinc-finger gene switches for drug-inducible regulation of transcription

2011

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Targeted zinc-finger (ZF) DNA-binding domains in conjunction with nuclear receptor ligand-binding domains (LBDs) produce chemically inducible gene switches that have applications in gene therapy and proteomic and genomic research. The benzoate X receptor-b (BXRb) LBD was used to construct homodimer and single-chain ZF transcription factors (ZF TF s). These ZF TF s specifically regulated the transcription of target genes in response to two ligands, ethyl-4-hydroxybenzoate and propyl-4-hydroxybenzoate, in a dose-dependent manner. The ZF TF s also regulated the expression of endogenous intercellular adhesion molecule-1 in response to either ligand. The advantage of BXRb-based ZF TF s is that the ligands are inexpensive and easily synthetically modified, making the system a base for creation of orthogonal ligand-receptor pairs and expanding the gene-switch toolbox.

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Orthogonal control of endogenous gene expression in mammalian cells using synthetic ligands

Liang

McLachlan

Zhao

2013

Biotech & Bioengineering

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Gene switches have wide utility in synthetic biology, gene therapy, and developmental biology, and multiple orthogonal gene switches are needed to construct advanced circuitry or to control complex phenotypes. Endogenous vascular endothelial growth factor (VEGF-A) is crucial to angiogenesis, and it has been shown that multiple alternately spliced VEGF-A isoforms are necessary for proper blood vessel formation. Such a necessity limits the utility of direct transgene delivery, which can provide only one splice variant. To overcome this limitation, we constructed a gene switch that can regulate the (VEGF-A) locus in mammalian cells by combining an engineered estrogen receptor (ER) ligand-binding domain (LBD), a p65 activation domain, and an artificial zinc-finger DNA binding domain (DBD). Our gene switch is specifically and reversibly controlled by 4,4'-dyhydroxybenzil (DHB), a small molecule, non-steroid synthetic ligand, which acts orthogonally in a mammalian system. After optimization of the gene switch architecture, an endogenous VEGF-A induction ratio of >100-fold can be achieved in HEK293 cells at 1 µM DHB, which is the highest endogenous induction reported to date. In addition, induction has been shown to be reversible, repeatable, and sustainable. Another advantage is that the ligand response is tunable by varying the clonal composition of a stably integrated cell line. The integration of our findings with the technology to change ligand specificity and DNA binding specificity will provide the framework for generating a wide array of orthogonal gene switches that can control multiple genes with multiple orthogonal ligands.

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Regulation of endogenous gene expression using small molecule-controlled engineered zinc-finger protein transcription factors

Cited by 24 publications

References 46 publications

Drug-inducible and simultaneous regulation of endogenous genes by single-chain nuclear receptor-based zinc-finger transcription factor gene switches

Drug-inducible and simultaneous regulation of endogenous genes by single-chain nuclear receptor-based zinc-finger transcription factor gene switches

Benzoate X receptor zinc-finger gene switches for drug-inducible regulation of transcription

Orthogonal control of endogenous gene expression in mammalian cells using synthetic ligands

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