| Background and objective:A20 (Tumor Necrosis Factor, alpha-Induced Protein 3, TNFAIP3) is a cytoplasmic zinc finger protein that inhibits nuclear factor kappa-B (NFκB) activity and tumor necrosis factor (TNF) -mediated programmed cell death. It has been shown that A20 can prevent TNF-induced cytotoxicity in a variety of cell types, including fibroblasts, B-lymphocytes, WEHI 164, NIH 3T3 cells and endothelial cells. Overexpression of A20 can inhibit IL-1β-induced production of NO by rat islets, thereby, which can impact positively upon islet graft survival and function. A20 deficient cells, indeed, fail to terminate TNF-induced NF-κB activation. Lee et al generated A20-deficient mice by targeted disruption. A20 -/- mice, born from interbred A20 +/- mice in mendelian ratios, developed runting as early as 1 week of age. Mice deficient for A20 developed severe inflammation and cachexia, were hypersensitive to both lipopolysaccharide and TNF, and died prematurely.In nature, 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.Artificial transcription factors (ATFs) are composed of DNA-binding and functional domains, which can be fused together to create proteins that bind a chosen DNA sequence and regulate expression of a specific gene in vivo. Construction of ATFs in vitro includes construction of DNA-binding and function domains by various methods. It is very important to design a DNA-binding domain that recognizes a specific DNA sequence. Recently there has been a great deal of progress in the development of modular protein domains that recognize specific DNA triplets. The Cys2-His2 zinc finger motif is the ideal structural scaffold on which a sequence-specific protein may be constructed. DNA structural domains of zinc finger proteins (ZFPs) usually consist of 3 or 6 zinc fingers. Artificial ZFP (AZP) technology allows DNA sequences to be selected directionally and a DNA-binding domain to be designed. Along with the rapid development of bioinformatics, the volume of biological information has turned huge in various databases. Most of biological information can be obtained, formerly, only through laboratory research. However, theoretically ideal biological information can be obtained now through efficient use of bioinformatic tools. From this, the research tools for modern genetics, biology, biochemistry and pharmacology have been expanded.It opens a new avenue to developing modern gene therapy to design proteins that do not exist in the natural world but can regulate specific genes through simulating the natural transcription factor structure. The expression of genes is regulated at the transcriptional level primarily by transcription factors that bind to DNA, and is the most economical and effective way to regulate gene expression. To intervene in gene expression, the promoter of the gene of interest must be first studied. A gene promoter contains a series of the DNA sequence elements adjoining the transcriptional start site (TSS), which directly activate or inhibit gene transcription. Hence, the position of a promoter can be located through the TSS, which had been identified. So precise TSS localization is the primary premise to analyze promoters and transcriptional regulation.Main methods1. The presumptive promoter sequence of A20 gene upstream identified and cloned The A20 gene and related sequences were retrieved from the National Center for Biotechnology Information. The A20 gene promoter was analyzed using the sequence processing tools TFSEARCH, TESS, Gene2Promoter and McPromoter. The presumptive promoter sequence of A20 gene upstream was cloned by nested PCR from human genomic DNA and subcloned into pGL3-basic vector. Afterwards, the presumptive promoter activity was analyzed through dual luciferase activity analysis.2. Design of presumptive nucleotide sequence of ZFP and analysis of ZFP expression and activityWe logged on to the Zinc Finger Tools server of the Barbas Laboratory of the Scripps Research Institute, submitted A20 promoter-related sequences, and set up parameters to obtain the specific target site of the A20 gene promoter region and the amino acid sequence of a ZFP. This amino acid sequence was then reverse-translated into a nucleotide sequence, and all codons in that sequence were optimized. The full-length optimized sequence was sent to Shanghai Sangon Company for full-gene synthesis. The characterization and structural analysis of the ZFP were carried out using PepTool Lite 1.1 and ProtScale. Homologous modeling of the ZFP tertiary structure was conducted using Robetta. ZFP gene fragments were amplified by PCR, using artificial ZFP sequence as a template, and which were recombined with the prokaryotic and eukaryotic plasmids so as to constructing recombination expression plasmids. The expression of the artificial zinc finger protein and its biological activity were analyzed by RT-PCR, Western blotting, and EMSA, respectively.3. Construction, expression and activity analysis of artificial transcription factor (ATF) ATF include DNA binding domain (artificial zinc finger protein, ZFP), functional domain (P65 activating domain), nuclear localization signal (NLS), as well as Flag Tag.The expression and biological activity of ATF fusion protein were detected by immunocytology technology, RT-PCR, Western Blot, luciferase activity analysis, and EMSA.4. Analysis of ATF activated A20 gene expression and suppressed the apoptosis of ECV304.ATF recombination expression plasmids were transfected into ECV304 cells, and observed the expression activated of A20 gene, compared with TNF-αand ethanol stimulated. Using FACS, ATF inhibited the apoptosis of the endotoxin-stimulated ECV304 cells was analyzed.Results and conclusion1. The structure and the sequence composition of the A20 gene promoter were analyzed by bioinformatic methods. The TSS and the presumptive promoter of A20 gene were determined by bioinformatics methods. It was showed that the TSS was located in genomic NC000006 138230088, and A20 gene presumptive promoter was determined within A20 gene upstream sequence. Used nested PCR technique, the presumptive promoter sequence was cloned successfully from human genomic DNA.2. The presumptive promoter sequence cloned had relevant promoter biological activity through the detection of relative luciferase activity.3. The target sequences of A20 promoter region were submitted to the on-line ZF Tools server of the Barbas laboratory of the Scripps Research Institute(TSRI) to obtain a specific 18bp target sequence of A20 promoter region and the amino acid sequence of zinc finger protein that acts on specific target sequences of A20 promoter region. The ZFP optimized nucleic acid sequence was obtained by bioinformatics methods.4. Recombinant Prokaryotic and eukaryotic expression vectors of ZFP were constructed and expressed successfully in E. coli ER2566 and COS7 cells, respectively.5. Artificial transcription factor (ATF) was constructed successfully, and which could express normally in the ECV304 cells, and import into the cell nucleus under the NLS guidance. The EMSA results demonstrated that ATF could bind specifically to A20 upstream target sequence, and the fluorescein enzyme activity analysis showed ATF could activate effectively expression of the downstream promoter. Results demonstrate that ATF can express normally in eukaryotic cells and have the relevant biological activity.6. ATF can trigger endogenous A20 gene expression in ECV304 cells.7. ATF can obviously suppress the apoptosis response of the endotoxin-stimulated ECV304 cells... |
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