To establish whether MeCP2 upstream regulatory elements (termed R1-R3) or intron 1 (R4-R6) are targeted by ethanol, targeted pyrosequencing was used to analyze DNA methylation at selected CpG sites. Briefly, genomic DNA was treated with bisulfite, selected regions were amplified, and the sequence was determined by pyrosequencing. Non-bisulfite-treated samples served as control. Methylation of MeCP2 promoter CpG regions was not specifically altered, but during the chronic exposure there was slight hypermethylation in region 6 of intron 1 (4.5%, p>0.05). Since previous work showed that even slight changes in DNA methylation caused significant changes in MeCP2 expression (Liyanage et al. 2013), this result was judged to be biologically meaningful. However, bisulfite-pyrosequencing does not distinguish between 5mC and 5-hydroxymethyl cytosine (5hmC). Further analysis differentiated which type of methylation (5mC vs. 5hmC) was present on these regulatory elements using 5hmC-specific antibody to immunoprecipitate enriched DNA fragments (hMeDIP) or 5mC-specific MeDIP. 5hmC levels were enriched in R1,R3 and R5 in the chronic ethanol exposure. 5mC enrichment levels (associated with diminished expression) were reduced in R3 and R6. Similar effects were seen in the withdrawal
