| Ethanol treatment increases gene expression covering a wide spectrum of cellular functions in the liver through mechanisms that are not clearly defined. A growing body of evidence suggests that alcoholic liver disease is associated with these altered gene expressions. Histone acetylation plays an important role in transcriptional activation. The goal of this thesis project was to determine the effects of ethanol on histone acetylation and its role in ethanol-induced gene expression. Immunoblot and immunocytochemical analysis revealed that ethanol significantly increased acetylation of histone H3 at Lys9 with negligible effect at Lys14, 18 and 23 in rat hepatocytes. This ethanol effect was reversible and observed at different glucose and serum levels. Acute in vivo administration of alcohol in rats produced the same results as in vitro observations. Treatment of cells with ethanol metabolizing enzyme inhibitors (4-methylpyrazole, cyanamide) decreased ethanol-induced histone H3 acetylation at Lys9, suggesting that ethanol metabolism is involved in histone acetylation. Inhibitors of MEK (U0126) and JNK (SP600125), but not p38 MAPK inhibitor (SB203580), suppressed ethanol-induced H3 acetylation, indicating that ERK and JNK signaling cascade are involved in ethanol-induced H3 acetylation. Next, we investigated the effect of ethanol on histone acetyltransferase (HAT) using ELISA and immunoblot analysis. Nuclear extracts from ethanol-treated hepatocytes increased acetylation of H3 peptide to a greater extent than extracts from untreated cells, suggesting that ethanol modulates HAT. Use of different H3 peptides indicated that ethanol selectively modulated HAT(s) targeting H3-Lys9. However, ethanol did not affect total histone deacetylase (HDAC) activity. Treatment with acetate, an ethanol metabolite, also increased acetylation of H3-Lys9 and modulated HAT(s) in the same manner as ethanol, suggesting that acetate mediates the ethanol-induced effect on HAT. However, U0126 and SP600125 did not significantly affect ethanol-induced effects on HAT, indicating that ERK and JNK regulate histone acetylation through pathway(s) that do not involve modulation of HAT. (Abstract shortened by UMI.). |
