Preliminary Epigenetic Regulation Of SOX9 Promoter By WDR5

WDR5 is an important member of the WD-40 protein family.The coding sequence of WDR5 is composed of 333 amino acids.The peptide chain contains 7WD repeat structures,and forms a total of 7 beta folds,which constitute the main functional domain of WDR5.As a member of the WD40 family,which is involved in a wide range of regulation of gene expression in vertebrate development.It plays an important role in the development of vertebrate,tumor formation,VISA signal complex assemble,the activation of IRF3 and NF-kappa B.So far,many of the biological mechanisms of WDR5 remain to be elucidated.In the chapter two We construct a recombinant vector containing human WDR5 by gene recombination technique and establish LNCaP cell strain with stable expression of WDR5.WDR5 gene was amplified by PCR and was cloned into eukaryotic expression vector pCI-neo.The resulted pCI-neo-WDR5 plasmid was verified by restriction enzyme digestion and DNA sequencing.LNCaP cells were transfected with pCI-neo-WDR5 by liposome.The expression of WDR5 in the cells was examined by Real-time PCR.Restriction enzyme digestion and sequencing analysis showed that WDR5 cDNA was correctly cloned into pCI-neo vector.After transfection of LNCa P cells with the recombinant plasmids,the monoclonal cell strain of stably expressed were obtained by G418 screening,which showed expressions of WDR5 m RNAs detected by Real Time-PCR.It was concluded that the recombinant of PCI-neo-WDR5 stably express in the transfected LNCaP cells,which laid a foundation of further study on WDR5.During mammalian development,DNA methylation plays important roles insex determination and differentiation.Recently,we have confirmed that WDR5 and SRY can be co-located on the promoter of SOX9.SRY and WDR5 synergetically activated the expression of SOX9,but the expression of beta-Catenin is inhibited by SRY and WDR5,which could be important for sex determination.However,it is not clear how WDR5 regulates the expression of SOX9 gene.In the chapter three,using Chromosome immunoprecipitation(ChIP)assay demonstrated the initial epigenetic regulation of WDR5 promoter on SOX9 gene.