Similar bisulfite pyrosequencing analysis at D8 indicated that the three Mecp2 promoter regions (R1 to R3) and intron 1 regions (R4 to R6) were remethylated and DNA methylation was almost re-established following decitabine withdrawal (Figure 6C). Analyzing the average DNA methylation over the Mecp2 promoter R2 and the entire intron 1 (which were demethylated at D2), we observed no significant differences in DNA methylation between D8 control and decitabine-treated cells (Figure 6D). Despite the fact that DNA remethylation is expected to restore the gene expression levels, expression of both Mecp2 isoforms were significantly downregulated. This observation implies that at D8, other regulatory mechanisms apart from promoter/intron 1 DNA methylation might be involved in downregulating Mecp2 expression.
