Study On The Transcriptional Regulatory Function Of Mutant P53

In more than 50% of the tumor tissues, TP53 gene is mutated. The vast majority of p53 mutants are missense mutation, occurring in the DNA binding domain. The mutant p53 not only loses the tumor suppressor function, but also gains new oncogenic function. Gain-of-function of mutant p53 plays an important role in promoting the occurrence and development of tumor at all stages. Mutant p53 may regulate new target genes through interaction with other proteins, but the specific mechanism is still not clear. Study on the transcriptional regulatory network of mutant p53, can deepen the understanding of the occurrence and development of tumor, providing new ideas and strategy for cancer prevention and therapy.We used chromatin immunoprecipitation combined with microarray technology(Ch IP-on-chip) to identify genome-widely the target genes of p53-R273 H in the breast cancer cell line MDA-MB-468(the 273 rd amino acid of the p53 protein in the cell is mutated from arginine to histidine, i.e., p53R273H). Through analysis of Ch IP-on-chip data by MEME-chip program, we obtained the p53-R273 H potential binding motif(GGCGG[CT]GGCGGCGGC[GT]GCGG[CG]). This motif is different with that of wild-type p53, but is overlapping with that of Sp1. We also carried out bioinformatics analysis(genome mapping, gene annotation, KEGG pathway analysis and interaction network construction) to identify the roles of p53 target genes, and founded that the target genes of p53R273 H distributes equally in almost all chromosomes, except that there are few genes in chromosome Y and chromosome 21. Most of the target genes of p53R273 H are those related to the process of gene proliferation and cell cycle, involved in Wnt, TGF-beta and MAPK signal pathway. In the international network of p53 target genes, the hub genes with high degree of connection are mainly those involved in Wnt, TGF-beta and MAPK signal pathway genes.As we found that the binding motifs of p53 R273 H and Sp1 are overlapping, we hypothesized that p53R273 H may regulate gene transcription via mediation of Sp1 or co-operation with Sp1. Then, we conducted endogenous and exogenous co-immunoprecipitation experiments to detect the interaction of Sp1 and p53R273 H, and found that p53R273 H interacts with Sp1. Next, we constructed the transcriptional activity reporter with 4 Sp1-binding sites, and co-transfected with wild type p53 or p53R273 H expression plasmid. The results showed that p53R273 H increase the luciferase activity in a dose-dependent way, and luciferase activity in the presence of Sp1 is significantly higher than that in the absence of Sp1. But transfection of wild-type p53 decreases the luciferase activity. All these results demonstrated that wild type p53 inhibits the activity of the transcription factor Sp1, while mutant p53R273 H can significantly enhance the activity of the transcription factor Sp1.In a word, our results suggest that p53R273 H may co-operate with Sp1 to regulate the key genes involved in Wnt, TGF-beta and MAPK pathways, and then promote the occurrence and development of cancers.