The Effect Of DNA Demethylation On Antibody Gene During The Process Of Antibody Production

Objects: The effect of DNA demethylation on antibody gene transcription wasstudied, and its mechamism was preliminarily investaged in this work.Methods: We used DNA methyltransferase inhibitor,5-azacytidine(5-Aza), toinhibit DNA methylation of genome of mice, methylation-sensitive restrictionendonuclease (MS-RE)-PCR and silver staining to detect the level of globaldemethylation of Antibody gene, and sulfite modificationm combined with realtime-PCR to detect the demethylation of specifical sites of promoter domain of heavyand κ light chain of antibody gene. ELISA assay was used to determine antibody titer,and PCR and DNA sequencing were used to detect mutation and rearrangement of thevariable, diversity and joining (V-D-J) regions of antibody gene. MTT assay and flowcytometry were used to assay the effect of5-Aza on the growth of spleen cells in mice.real time-PCR was used to detect demethylation of promoter domain of interleukin(IL)-2, IL-4, IL-6, Toll-like receptor(TLR)4, TLR9, major histocompatibilitycomplex(MHC)Ⅰ, MHCⅡ and interferon gamma(IFN)-γ, and RT-PCR was used todetect expression level of mRNA of IL-2, IL-4, IL-6, TLR4, TLR9, MHC Ⅰ, MHC Ⅱ,IFN-γ, DNA methyltransferase (Dnmt)1, Dnmt3a and Dnmt3b.Results:(1) The antibody titer of mice treated with5-Aza was higher than that of control.(2) The methylation level of promoter domain of heavy and κ light chain of antibodygene of mice treated with5-Aza was lower than that of control, as well as the globallevel of methylation of antibody gene.(3) DNA demethylation can lead to a high site mutation frequency with the similarmutation sites.(4) DNA demethylation had no effect on V(D)J rearrangement of antibody gene in themouse spleen.(5)5-Aza had no effect on transcription of DNA methyltransferase.(6)5-Aza can inhibit the growth of spleen cells, induce apoptosis of them, and blockcell cycle. (7) The methylation level of IL-2、IL-4、TLR9and IFN-γ of mice treated with5-Azawas lower than that of control, but their transcription level was higher than that ofcontrol.Conclusions:5-Aza can induce DNA demethylation, and demethylation ofantibody gene can promote antibody production and increase mutation frequencyduring the process of antibody production.5-Aza-mediated improving antibody levelwas not due to that5-Aza stimulate B-lymphocytes to differentiate, but possibly dueto that DNA demethylation of promoter domainof IL2、IL4、TLR9、IFN-γ up regulatedtheir transcription level, then stimulated B-lymphocytes to differentiate, and finallyimproved antibody production.