Her with significant reductions in SAM and the SAM:SAH methylation ratio, all of which were prevented by supplementing the ethanol diet with the methyl donor betaine (Fig. 2 and Table 1). Contrasting genotype effects, plasma homocysteine levels were significantly increased in the ethanol fed heterozygotes compared to those in the wild type mice (Table 1), as has been shown by others who developed this model (Watanabe et al., 1995). These data showing greater perturbation of methionine metabolism among heterozygotes provide a rationale for the use of this genotype for several of the subsequent analyses, including MethylC-seq genomic and pyrosequencing methylation analyses. Unexpectedly, betaine supplementation did not significantly reduce ALT levels in either genotype despite the improvement of histological scores of inflammation and steatosis in both genotypes. A prior study on the effects of betaine supplementation in a similar intragastric mouse model with 6 weeks of ethanol feeding showed decreased serum homocysteine and prevention of the steatosis response to ethanol but non-significant reduction in ALT levels (Ji and Kaplowitz, 2003). We may speculate that longer dietary exposure in our mouse model could have been associated with greater histopathology in the ethanol groups and potential reductions in ALT in the betaine supplemented groups. Second, there were no significant changes in global DNA methylation according to dot-blot analyses of all samples from both genotypes and all three diets. These findings are consistent with those from our prior study (Esfandiari et al., 2010) which used the liquid chromatography-tandem mass spectrometry (LC-TMS) method to calculate percentage of methyl-C among total cytosine residues (Quinlivan and Gregory, 2008). Both methylation dot blot and MethylC-seq specifically quantitate CpG site methylation, while LC-TMS measures all cytosine methylation. According to prior studies in animal order Acadesine models of other liver diseases, global DNA methylation may be regulated in part by underlying hepatic inflammation (Gonda et al., 2012; Medici et al., 2013; Stenvinkel et al., 2007; Wierda et al., 2010), which was minimal in our studies. Third, our genome analysis of heterozygote samples by MethylC-seq showed that DNA hypomethylation was induced by ethanol feeding in each of 19 autosomes and was prevented in each autosome by betaine supplementation (Table 2). Furthermore, the results of this analysis showed that hypomethylation occurred exclusively in gene bodies, whereasNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptAlcohol Clin Exp Res. Author manuscript; available in PMC 2015 June 01.Medici et al.Pageno diet induced methylation effects were found in gene promoter regions (Fig. 3). These novel findings highlight the need to examine gene body methylation patterns when examining DNA methylation changes in other models of ASH. Our data are consistent with other SKF-96365 (hydrochloride) site genome-wide studies of DNA methylation patterns, in which CpG island promoters are predominantly un-methylated, in contrast to the high levels of methylation within gene bodies (Aran et al., 2011; Ball et al., 2009; Lister et al., 2009; Zemach et al., 2010). Recent studies indicate that gene body methylation levels are likely to be determined by the greater accessibility of their DNA to methyl donors and methyltransferases (Jjingo et al., 2012). Fourth, among the 21 selected genes related to liver injury and methionine metabolism, on.Her with significant reductions in SAM and the SAM:SAH methylation ratio, all of which were prevented by supplementing the ethanol diet with the methyl donor betaine (Fig. 2 and Table 1). Contrasting genotype effects, plasma homocysteine levels were significantly increased in the ethanol fed heterozygotes compared to those in the wild type mice (Table 1), as has been shown by others who developed this model (Watanabe et al., 1995). These data showing greater perturbation of methionine metabolism among heterozygotes provide a rationale for the use of this genotype for several of the subsequent analyses, including MethylC-seq genomic and pyrosequencing methylation analyses. Unexpectedly, betaine supplementation did not significantly reduce ALT levels in either genotype despite the improvement of histological scores of inflammation and steatosis in both genotypes. A prior study on the effects of betaine supplementation in a similar intragastric mouse model with 6 weeks of ethanol feeding showed decreased serum homocysteine and prevention of the steatosis response to ethanol but non-significant reduction in ALT levels (Ji and Kaplowitz, 2003). We may speculate that longer dietary exposure in our mouse model could have been associated with greater histopathology in the ethanol groups and potential reductions in ALT in the betaine supplemented groups. Second, there were no significant changes in global DNA methylation according to dot-blot analyses of all samples from both genotypes and all three diets. These findings are consistent with those from our prior study (Esfandiari et al., 2010) which used the liquid chromatography-tandem mass spectrometry (LC-TMS) method to calculate percentage of methyl-C among total cytosine residues (Quinlivan and Gregory, 2008). Both methylation dot blot and MethylC-seq specifically quantitate CpG site methylation, while LC-TMS measures all cytosine methylation. According to prior studies in animal models of other liver diseases, global DNA methylation may be regulated in part by underlying hepatic inflammation (Gonda et al., 2012; Medici et al., 2013; Stenvinkel et al., 2007; Wierda et al., 2010), which was minimal in our studies. Third, our genome analysis of heterozygote samples by MethylC-seq showed that DNA hypomethylation was induced by ethanol feeding in each of 19 autosomes and was prevented in each autosome by betaine supplementation (Table 2). Furthermore, the results of this analysis showed that hypomethylation occurred exclusively in gene bodies, whereasNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptAlcohol Clin Exp Res. Author manuscript; available in PMC 2015 June 01.Medici et al.Pageno diet induced methylation effects were found in gene promoter regions (Fig. 3). These novel findings highlight the need to examine gene body methylation patterns when examining DNA methylation changes in other models of ASH. Our data are consistent with other genome-wide studies of DNA methylation patterns, in which CpG island promoters are predominantly un-methylated, in contrast to the high levels of methylation within gene bodies (Aran et al., 2011; Ball et al., 2009; Lister et al., 2009; Zemach et al., 2010). Recent studies indicate that gene body methylation levels are likely to be determined by the greater accessibility of their DNA to methyl donors and methyltransferases (Jjingo et al., 2012). Fourth, among the 21 selected genes related to liver injury and methionine metabolism, on.
