One carbon metabolism modifies histone methylation and acetylation in human colonic epithelial cells

Colorectal cancer is one of the most frequent malignancies in Western countries and the third leading cause of cancer-related deaths in men and women in the United States. In the past several years comprehensive studies have focused on the genetic basis of colorectal cancer; thus, this disease provides an excellent model to understand the molecular basis of carcinogenesis. Besides genetic alterations, colorectal cancer arises as a consequence of the accumulation of epigenetic alterations such as aberrant DNA methylation and histone modification. For epigenetic changes, nutrients in one-carbon transfer reactions such as methionine, folate, choline and B vitamins are essential. The purpose of this thesis is to investigate the mechanism by which derangement of one-carbon metabolism affects epigenetic phenomena and subsequent gene expression of tumor suppressor p16. For this thesis we used NCM460, a non-malignant human colonic epithelial cell line. First we determined an in vitro methyl-deficiency model in NCM460 for 30 days, further investigated the epigenetic changes in methyl deficiency. There was no significant difference in histone H3 modification, DNA methylation and p16 gene expression between cells in a control condition and methyl deficiency, indicating that methyl deficiency by itself is not sufficient to change those epigenetic phenomena in colonic epithelial cells. The focus of the second study was to inhibit S-adenosylmethionine (AdoMet)-dependent methyltransferases and subsequently inhibit methylation reactions. In this study we had two strategies to inhibit methylation reactions: (1) we added extracellular S-adenosylhomocysteine (AdoHcy), which is the product inhibitor of methyltransferases, to the culture media; (2) we used adenosine dialdehyde (AdOx) to increase endogenous AdoHcy. AdOx is a potent inhibitor of AdoHcy hydrolase and known to be an indirect inhibitor of methylation reactions. AdoHcy increased H3K9me3 at low (10--100nmol/L) and high (300micromol/L) doses, and 100micromol/L of AdOx decreased H3K9ac time-dependently (P=0.004). Neither AdoHcy nor AdOx affected DNA methylation status and p16 gene expression. This study demonstrates that inhibiting methylation reactions by AdoHcy and AdOx reveals site-specific changes in both histone acetylation and histone methylation. In the third study, we aimed to combine the first and second parts of the thesis using ethanol to inhibit one-carbon metabolism and reduce methyl group availability. Specifically, we investigated the impact of ethanol on epigenetic changes and p16 gene expression. H3K9ac and H3K4me3, both of which are increased in genes that are actively transcribed, showed opposite responses to 100mmol/L of ethanol; ethanol decreased H3K9ac (P<0.0001) and increased H3K4me3 (P=0.005) in time-dependent fashion. The changes in histone modification produced by ethanol were paralleled by increased p16 expression (P for time was 0.003). Neither DNA methylation nor p16-specific histone modification at H3 tail was changed by ethanol. To the best of our knowledge, this is the first report of ethanol-induced post-translational deacetylation of histone H3 at Lys9 in colonic epithelial cells. In conclusion, derangements in one-carbon metabolism induced various changes in global histone modifications in conjunction with changes in p16 expression. These findings may have significant implications for understanding the causes and roles of epigenetic alterations in colonic carcinogenesis, and they may be helpful to produce more effective prevention strategies and therapeutics for patients with colorectal cancer.