The Effection Of DNA Demethylation On Expression Of HFâ…§ Integrated Into Human RDNA Locus

As a kind of gene expression regulation mechanism,DNA methylation play an important role in many biology processes,including embryonic development,genetic imprinting,cell differentiation, tumorigenesis and so on.pHrneo,a human ribosomal DNA-targeting vector developed by our group,can target exogenous gene to human ribosomal DNA locus.The exogenous genes carried by pHrneo could stably express in some cell lines,such as HL7702(human hepatocyte) and HT1080(human fibrosarcoma).But the advanced efficiency of expression is still our aim.The factors affecting interested site-directed integration gene expression may involve copy numbers of interested gene, DNA methylation modification,histone acetylation modification and so on.In this study,our focus is on the relation between interested site-directed integration gene expression and DNA methlation modification.Objects:In this research,we investigated the relation between interested site-directed integration gene expression and DNA methlation modification by comparing human coagulation factor FⅧexpression level of site-directed integration cell between demethylation agent 5-aza-2'-deoxycytidine treatment group and control group,and the methylation pattern of interested gene promoter.Methods:Three concentrations of 5-aza-2'-deoxycitidine(5μM, 10μM,20μM)were respectively applied to treat site-directed integration cell pHrneoSK39-25 for 72h and 120h.Wild site-directed integration cell pHrneoSK39-25,HT1080 cell treated by 5-aza-2'-deoxycitidine(5μM, 10μM,20μM)and three concentrations of 5-aza-2'-deoxycitidine(5μM, 10μM,20μM)in identical conditions were employed as control groups. The expression of human coagulation factor FⅧwere defined by ELISA detecting cell supernatant of each group.Furthermore,the methylation pattern of CMV promoter was detected by bisulfite sequencing PCR.Results:The result indicated that the expression of human coagulation factor FⅧin 5-aza- 2'-deoxycytidine treatment group were respectively advanced about 4 times and 9 times,compared to wild site-directed integration cell pHrneoSK39-25.But the methylation point wasn't found in CMV promoter.Conclusions:DNA methlytion modification could play an important role in interested site-directed integration gene expression,but which may be not involved in the alteration of site-directed integration gene promoter. It was implicated that our result may be attributed to the change of chromatin structure of rDNA region caused by DNA demethylation.