Figure 3. CpG-2 demethylation up-regulates SNCA expression through dopamine receptor signaling.

A: Dose dependent up-regulation of alpha-synuclein by dopamine. RNA was extracted from cells treated with the indicated dopamine concentration for 48 h and quantitative PCR was performed. The relative amount of alpha-synuclein mRNA against beta-actin was normalized to dopamine = 0. * P<0.05 vs. DA = 0. B: Dopamine does not affect cell viability. MTS assay was performed after addition of dopamine for 48 h. P = 0.1559 with ANOVA. C: Methylation of CpG-2 suppresses downstream gene expression. Luciferase assays were performed after transfection of a CpG-free firefly luciferase vector harboring the CpG-2 region at the 5′ of the luciferase gene. The vector was treated with (+) or without (-) CpG methyltransferase prior to transfection. Sea pansy luciferase was co-transfected as an internal control. Relative luciferase activity was calculated by dividing firefly luciferase activity by sea pansy luciferase activity and normalized to methylation (−) as 100%. Bars indicate standard error. * P = 0.0025. D: 293 cells express D1 and D2 dopamine receptors. Expression of DRD1, −2 and −3 were analyzed in HCN-2, SH-SY5Y, and 293 cells by quantitative RT-PCR. Relative amounts of each receptor are normalized to HCN-2 = 1. E: A D2 receptor antagonist ameliorates the demethylation effect of dopamine. Dopamine (DA)- and haloperidol-treated 293 cells were lysed and methylated CpGs were enriched by MBD2 protein. Methylated CpG-2 was detected by PCR before and after enrichment.