Regulation Of P53Methylation Through Purα/SET9Protein Complexes

Histone modifications of Epigenetic modification are including methylation,acethlation, phosphorylation and ubiquitination. Most of these modification can changethe status of chromosomes, affecting the transcription factor with DNA sequence binding,and regulation of gene expression by several mechanisms. Lysine methylation is afunctionally complex process, as it can either activate or repress transcription, dependingon sequence-specific lysine methylation sites in histones. Histone methylreansferasesSET9is not limited to histones, it is also able to methylate p53. SET9is shown tomethylate K372resulting in its stability and retention in the mammalian nucleus, thusinfluencing on the regulation of p53mediated gene expression. p53is tumour supperssorfactor that is mutated in approximately50%of human cancers.In normal cells, p53exertsa pivotal role in controlling the apoptosis, cell cycle and DNA repair in response tovarious forms of genotoxic stress. The regulation of p53is complex and occurs mainly atthe post-translational level. Purα is a multifunctional and ubiquitous nucleicacid-binding protein that can bind to both DNA and RNA and functions in theinitiation of DNA replication, control of transcription and mRNA translation.From preliminary experiments we found that Purα interact with SET9. On the baseof the Purα and p53both regulating the DNA repair, we therefore searched for thePurα/SET9protein complex role of p53mthylation. We transformed N-TAP-Purα vectorinto pIRES-Flag-Purα vector, and constructed the293T cell lines of expressionPurα.Search the impact of Purα to p53under the DNA damage conditions, we found addthe doxorubicin or over express Purα that increased p53K372methylatin. Recent studieshave indicated p53K372methylatin related with acetylation of p53at lysines373and382. We found Purα suppress acetylation of p53at lysine373,but no effect to p53atlysine373. We also constructed AD-SET9、AD-Purα、BD-SET9、BD-Purα vectors forfurther Yeast two-hybrid research. This result consisted with the function of Purα, alsoprovided experimental basis with SET9methylation of p53protein for the regulatory mechanism. The mechanism of regulate SET9methylated p53with Purα would be solvedin the follow-up works.