Objective: To investigate the epigenetic regulation of neprilysin (NEP) gene in mouse neuroblastoma Neuro-2a (N2a) cells and further determine the interaction between DNA methylation and histone acetylation.
Methods: N2a cells were treated with DNA methylation inhibitor, 5-aza-deoxycytidine (5-Aza-dc) at 3 and 5 μmol/L for 48 hours and histone deacetylase inhibitor, trichostatin A (TSA) at 300, 500 and 700 nmol/L for 24 hours. The expression of NEP after the treatment was evaluated by reverse transcription PCR(RT-PCR) and Western blotting. Bisulfite sequencing PCR (BSP) assay was utilized to detect the methylation status of NEP gene promoter regions. The level of acetylated histone H3 on NEP promoter was measured by chromatin immunoprecipitation (ChIP) assay.
Results: 5-Aza-dc induced the demethylation of NEP gene and significantly increased NEP expression in a dose-dependent manner (P<0.01). TSA treatment significantly enhanced NEP expression by elevating the acetylation of histone H3 on NEP promoter (P<0.01, P<0.05). However, methylation status of NEP promoter was not altered by TSA treatment (P>0.05).
Conclusion: The expression of NEP gene is regulated by DNA methylation and histone acetylation in N2a cells. Histone acetylation has no effect on DNA methylation.