| Tumor suppressor p53 is known as "Genome Guardian" and "Molecular Police", the gene with highest mutation frequency in human tumor discovered so far. P53 could be activated by many stress signals to decide cell fate, but how could p53 pro-tein precisely control the particularly complicated downstream signaling pathways, is still a mystery have not been completely uncovered until now. It’s believed that mechanism research about a large amount of p53 isoforms found in the recent years, will be helpful for answering this puzzle. Δ113p53 (zebrafish)/Δ133p53 (human) is a N-terminal truncated p53 isoform, Studies have proved that it antagonizes p53-mediated apoptotic activity, but the molecular mechanism of Δ113p53/Δ133p53 protein function is still not so clear.First part of my subject was investigating the molecular mechanism of Δ113p53/Δ133p53 antagonizes apoptosis, and digging new Δ113p53/Δ133p53 function, following are the three sub-parts of detailed results:First, thanks to two different zebrafish p53 protein antibodies:Zfp53-A7C10 and used an zebrafish antibody Zfp53-N(only recognizes full length p53) to do im-munoprecipitation(IP), another zebrafish antibody Zfp53-A7C10 (could recognizes both p53 and Δ113p53) to detect IP-protein, for the first time we proved that en-dogenous p53 and Δ113p53 could form protein complex under treatment of the DNA damage drug. We further constructed single amino acid mutation in Δ113p53 oligomerization domain(OD), two mutants lost the binding ability to p53 protein were found. With the research in these two mutants, we revealed that protein inter-action between p53 and Δ113p53 was required for the anti-apoptotic function.Second, through single overexpression of p53 or the Δ133p53,or co-expression of them in human lung adenocarcinoma cells, and the detection their gene expres-sion by real-time fluorescent quantitative PCR technique, we found that Δ133p53 repressed the transcription expression level of Gls2、TIGAR and Sestrinl in a p53 dependent manner, especially for Gls2 (glutaminase 2) gene, expression amount of which has been stable decreased repeatedly to half. These results implied that Δ133p53 may play important roles in oxidative phosphorylation inhibition, glycoly-sis Facilitation and mTOR mediated cell grow promotion.Third, we were surprising to find that Δ113p53 could be induced to a large amount by γ-irradiation rather than ultraviolet light and heat shock in wild type zebrafish embryos, further study demonstrated that Δ113p53 can directly bind to the de novo response element in the promoter of DSB repair related genes such as rad51, rad52 and ligase4, then promote the raise of transcription lever of these three genes. This is the first time and first p53 isoform proved to be functional in a p53-independent manner.Secondary part, in addition to the identity as the important tumor suppressor genes, P53 is also a classic transcription factor, so it mainly functions as specific binding to the promoter of downstream target gene through its own DNA binding domain, and then carries out the transcription regulation function. Because of the traditional classical research means of protein-DNA binding is difficult to meet our requirements, we constructed a new method named in vitro chromatin precipitation (in vitro ChIP). This project first explored the optimum condition of p53 specific DNA-binding in vitro by using purified protein from E. coli and two different plas-mids with or without known p53 binding site. Next we incubated zebrafish embryo nuclear extract and sonicated genomic DNA in vitro under these conditions, then directly subjected the complex to immune precipitation by tag antibody without crosslink, the success of the experiment had been confirmed by using the positive primers to qPCR amplify the purified enriched DNA, finally we sent the enriched DNA for high-throughput sequencing collected by this method, and now we are waiting the data analysis. The successfully established of in vitro ChIP technology not only has the vital significant of the study of interaction between transcription factors and DNA, but also laid a novol and convennient technology foundation for study the whole complex network of transcriptional regulation of all the transcrip-tion factor in zebrafish. |
PDF Full Text Request