AIM: To examine the effects of ethanol-induced proteasome inhibition, and the effects of proteasome inhibition in the regulation of epigenetic mechanisms. histone acetylation, and it is hypothesized that this proteasome proteolytic activity regulated histone modifications by controlling the stability of histone modifying enzymes, and, therefore, regulated the chromatin structure, allowing easy access to chromatin by RNA polymerase, and, thus, proper gene expression. Proteasome inhibition by PS-341 increased histone acetylation similar to chronic ethanol feeding. In addition, proteasome inhibition caused dramatic changes in hepatic remethylation reactions as there was a significant decrease in the enzymes responsible for the regeneration of S-adenosylmethionine, and, in particular, a significant decrease in the betaine-homocysteine methyltransferase enzyme. This suggested that hypomethylation was associated with proteasome inhibition, as indicated by the decrease in histone methylation. CONCLUSION: The role of proteasome inhibition in regulating epigenetic mechanisms, and its link to liver injury in alcoholic liver disease, is thus a promising approach to study liver injury 67392-87-4 due to chronic ethanol consumption. the methyltransferases, such as glycine N-methyltransferase (GNMT). In this study, BHMT gene expression was markedly decreased by proteasome inhibition. BHMT is an essential enzyme in the remethylation pathway, and is involved in the recovery of SAMe. Betaine, a choline derivative which has been used clinically to treat, with some success, patients with methylenetetrahydrofo-late reductase deficiency[11,12], acts as a substrate for BHMT, and serves as an alternative methyl donor for remethylation of homocysteine in the liver and kidney[13]. Therefore, betaine supplementation may cover the down-regulation of gene expression induced by proteasome inhibition, and correct the deregulation of hepatic transmethylation reactions due to the proteasome inhibition-induced decrease in BHMT activity. MATERIALS AND METHODS Animals Male Wistar rats (Harleco, Hollister, CA, USA), weighing 250-300 g, were fed ethanol using the Tsukamoto-French intragastric model[14,15]. PS-341 was administered intraperitoneally, 24 h before sacrifice[16,17]. The rats were maintained according to the Guidelines of Animal Care, as described by the National Academy of Sciences and published by the National Institute of Health (1996). Nuclei isolation Histones were isolated from the nuclei, according to the method of Umlauf et al[18]. Liver tissues, frozen in isopentane and immersed in liquid nitrogen, were homogenized in a Dounce homogenizer with 10 strokes. Homogenates were centrifuged for 10 min at 6000 on a sucrose cushion. The pellets contained the nuclei. Histones were VPS15 isolated from the nuclei, according to the method of Shechter et al[19]. Isolated nuclei were mixed with 0.2 mol/L H2SO4, and incubated on a rotator for 30 min at 4C. Samples were spun in a microcentrifuge at 16000 values were determined by one-way ANOVA and Student-Newman Keuls for multiple group comparisons (Sigma-Stat software, San Francisco, CA, USA). 0.05 was used to establish significant differences. Correlation of data was done by linear regression analysis using Pearsons period momentum method. RESULTS Microarray analysis of liver samples from rats fed ethanol showed that a large number of genes (about 1300) were up-regulated and down-regulated due to chronic ethanol feeding[8]. Microarray analyses of liver samples from rats given PS-341 (Bortezomib, Velcade?) also showed dramatic changes in gene expression (about 2082 genes changed) affecting several functional pathways (Physique ?(Figure11). Physique 1 Kegg functional pathway changes in gene expression induced by proteasome inhibition. Both ethanol feeding and proteasome 67392-87-4 inhibition affected almost all pathways. The present study was based on the observation that this inhibition of proteasome, caused by chronic ethanol feeding, participated in the development of liver injury due to ethanol by altering the mechanisms through which normal epigenetic regulation occurs. The consequence of this was a marked change in the gene expression of several functional pathways in liver cells (Physique ?(Figure11). Data mining and gene specific pathway clustering showed that, similar to ethanol feeding, several transcriptional factors, such as cell cycle, histone modifying enzymes, and the remethylation pathway, were significantly changed by proteasome inhibition. Proteasome inhibition by PS-341 thus proved to be a powerful tool to investigate the role of proteasome activity in epigenetic mechanisms. To verify the hypothesis that gene expression changes are regulated by nuclear proteasome activity, where inhibition is usually caused by chronic ethanol feeding[20], proteasome activity was measured in isolated nuclei from the liver of rats fed ethanol chronically, and from the liver of rats given 67392-87-4 PS-341. Figure ?Physique22 shows that chronic ethanol feeding caused a significant decrease in proteasome chymotrypsin-like activity in isolated liver nuclei. Physique 2 Nuclear proteasome chymotrypsin-like activity. 20S proteasome chymotrypsin-like activity was measured in isolated nuclei from the liver of rats fed ethanol chronically and from the liver of rats given PS-341. To further investigate the role of proteasome activity in regulating epigenetic mechanisms, histone acetylation was analyzed in the liver of rats given PS-341, and compared to histone acetylation in the liver of rats fed ethanol chronically. Physique ?Figure3A3A shows that acetylated histone 3 lysine 9 (AcH3K9) was increased in the liver of.