Study On TEAD1 Gene Function And Validation Of New TEAD1 Regulated Target Genes

TEAD1 (TEA domain family member 1) is an essential transcription factor in regulating the expression of muscle-specific gene, which contains a TEA domain and binds the M-CAT domain in the promoter region of genes. TEAD1 regulates the expression of a large number of muscle-specific genes, which play very important role in the process muscle development. The members of TEAD1 family function as a crucial role in development myocardium, cardiac hypertrophy, cardiac arrhythmia, smooth muscle development and myofibroblast formation, skeletal muscle hypertrophy and regeneration and the development of Neural Crest Development.Gene transcription regulation is the most important and efficient progress of gene expression regulation in regulating protein abundance and protein function, and trans-acting factors interact with cis-acting element is the main form of regulating gene transcription. Transcription factors regulating gene expression exhibits the "one to many" features, which means one transcription factor can regulate many genes expression. Previous study on the transcription factor TEAD1 only focused on a single target gene in one paper, and there is no information of TEAD1 target genes within the whole genome.This research is in-depth and progress of the preliminary research in our laboratory. Early research done by colleagues in our laboratory found that TEAD1 gene was up-regulated in pig embryonic development, and a mutation in the 5’untranslated region of the gene was significantly associated with average daily gain of pigs. Dr. Qiu Haifang used high-throughput technique, CHIP-on-chip, to screen TEAD1 regulating target genes in mice muscle, and identified 745 potential genes regulated by TEAD1. In this study, based on chip data, we carried on the bioinformatics analysis, promoter analysis and expression analysis, EMSA assay to identify and validate the new target genes, and the function and the expression regulation of target gene was preliminarily studied. The expression patterns of TEAD1 in mouse tissues and differentiated C2C12 cells, and the TEAD1 gene function in C2C12 was elaborated in our study for the first time. From Rigorous experimental design and a series of experiments, the research can draw some conclusions as bellows:1 Gene function analysis of TEAD1:The expression pattern of TEAD1 gene in mouse various tissues was detected by semi-quantitative RT-PCR, the results showed that TEAD1 gene expressed highly and ubiquitously in all tissues in mouse; and the expression of the gene in differentiated C2C12 cells was detected by Quantitative PCR, TEAD1 gene was up-regulated during differentiation. TEAD1 gene overexpression vector was constructed and then transfected into C2C12 cells, this vector can effectively overexpress TEAD1 gene in C2C12 cells. Cell cycle tested by flow cytometry showed no significant changes in the cell cycle after transfecting the overexpreesion vector in C2C12 cell. Quantitative PCR was used to detect the expression of C2C12 cell differentiation marker gene myh4 in TEAD1 gene overexpression cells, the result showed that TEAD1 gene overexpression can cause myh4 upregulation expression in C2C12 cell which indicated that TEAD1 gene may function in C2C12 differentiation.2 Validation of target genes:Bioinformatics analysis software TESS predicted that Mrpl21, Ndufa6, Ccnel and Ankrd42 gene promoter region contained a TEAD1 protein binding sites, and these four genes are expressed in cardiac or skeletal muscle. The quantitative PCR was employed to detect expression of candidate target genes Mrpl21, Ndufa6, Ccnel and Ankrd42 in TEAD1 overexpression C2C12 cells, we found that Ankrd42 was upregulated, while the expression Ndufa6, Ccnel gene was downregulated. The promoters of mice gene Mrpl21, Ndufa6, Ccnel and Ankrd42 were cloned, and were built into the PGL-3 basic vector. TEAD1 overexpression vector and target gene promoter vector were co-transfected into C2C12 cells, then promoter activity were measured. Comparing with the control group (co-transfect the empty vector and promoter vector into C2C12 cells), promoter activity of Mrpl21 and Ankrd42 gene significantly increased, while the promoter activity of Ndufa6, Ccnel gene was significantly decreased. In vitro, EMSA experiment directly confirmed the interaction between TEAD1 protein and Mrpl21 and Ndufa6 promoter because TEAD1 can bind the specific probes of these two genes. and TEAD1 protein can not directly bind Ankrd42 gene specific probe. TEAD1 can directly regulated gene expression Mrpl21 and Ndufa6, while the regulation of Ankrd42 promoter activity and expression was indirect.3 The quantitative PCR was used to detect the expression trend of Mrpl21, Ndufa6, Ccnel in differentiated C2C12 cell, we found that Mrpl21 and Ndufa6 were significantly upregulated, while Ccnel gene was significantly downregulated in the differentiation process.4 We constructed Mrpl21 gene promoter length mutant vectors, and measured the promoter activity in the proliferation and differentiated C2C12 cells, we found the promoter activity in differentiated cell was higher than in the proliferative cell, and promoter activity of mutant containing the TEAD1 binding site was higher than those without the binding site.The above study revealed the function of transcription factor TEAD1 in C2C12 cell and verified the two new direct target genes,Mrpl21 and Ndufa6, regulated by TEAD1 gene, and an indirect regulated gene, Ankrd42. Whether Ccnel was a direct regulated gene needed to be further confirmed by EMSA experiment. Our study deepened the understanding of TEAD1 gene function and the regulation of its target genes, added new content to process of regulation of muscle development.