Abstract
BackgroundArtificial transcription factors (ATFs) are composed of DNA-binding and functional domains. These domains can be fused together to create proteins that can bind a chosen DNA sequence. To construct a valid ATF, it is necessary to design suitable DNA-binding and functional domains. The Cys2-His2 zinc finger motif is the ideal structural scaffold on which to construct a sequence-specific protein. A20 is a cytoplasmic zinc finger protein that inhibits nuclear factor kappa-B activity and tumor necrosis factor (TNF)-mediated programmed cell death. A20 has been shown to prevent TNF-induced cytotoxicity in a variety of cell types including fibroblasts, B lymphocytes, WEHI 164 cells, NIH 3T3 cells and endothelial cells.ResultsIn order to design a zinc finger protein (ZFP) structural domain that binds specific target sequences in the A20 gene promoter region, the structure and sequence composition of this promoter were analyzed by bioinformatics methods. The target sequences in the A20 promoter were submitted to the on-line ZF Tools server of the Barbas Laboratory, Scripps Research Institute (TSRI), to obtain a specific 18 bp target sequence and also the amino acid sequence of a ZFP that would bind to it. Sequence characterization and structural modeling of the predicted ZFP were performed by bioinformatics methods. The optimized DNA sequence of this artificial ZFP was recombined into the eukaryotic expression vector pIRES2-EGFP to construct pIRES2-EGFP/ZFP-flag recombinants, and the expression and biological activity of the ZFP were analyzed by RT-PCR, western blotting and EMSA, respectively. The ZFP was designed successfully and exhibited biological activity.ConclusionIt is feasible to design specific zinc finger proteins by bioinformatics methods.
Highlights
Artificial transcription factors (ATFs) are composed of DNA-binding and functional domains
Gene expression is regulated at the transcriptional level primarily by transcription factors that bind to DNA
The results indicated many trans-acting factors binding the relevant elements in the gene sequence, with several HSF binding sites at the distal end and 6 SP1 and 2 NF-κB binding sites in the GC-rich region at the proximal end
Summary
Artificial transcription factors (ATFs) are composed of DNA-binding and functional domains These domains can be fused together to create proteins that can bind a chosen DNA sequence. Gene expression is regulated at the transcriptional level primarily by transcription factors that bind to DNA Many of these transcription factors consist of two essential yet separable modules: a DNA-binding domain and a functional domain [1,2,3]. Artificial transcription factors (ATFs) are composed of DNA-binding and functional domains [4,5,6], which can be fused together to create proteins that bind a chosen DNA sequence and regulate expression of a specific gene in vivo [1,2,4,7,8]. Artificial ZFP (AZP) technology allows DNA sequences to be selected directionally and a DNA-binding domain to be designed [10,11,12,13]
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