Histone acetyltransferase p300 modulates gene expression in an epigenetic manner at high blood alcohol levels.

When rats are fed ethanol intragastrically at a constant rate for 1 month, the urinary alcohol level (UAL) cycles over 7-9 day intervals. At the peak UAL, the liver is hypoxic shifting the redox state to a reduced rate. Microarray analysis done on livers at the UAL peaks shows changes in approximately 1300 gene expression compared to the pair-fed controls. To determine the mechanism of the gene expression changes, histone acetylation regulation was investigated in liver nuclear extracts at the peaks and troughs of the UAL and their pair-fed controls. No change occurred in SirT-1. P300, a histone acetyltransferase (HAT), which acetylates histone H3 on lysine 9, was increased at the peaks. Histone 3 acetylated at lysine 9 was also increased at the peaks. This indicates that the up regulated genes at the UAL peaks resulted from an increase in p300 transcription regulation, epigenetically. P300 activates transcription of numerous genes in response to signal transcription factors such as H1F 1alpha, increased in the nucleus at UAL peaks. Signal transduction pathways, such as NFkappaB, AP-1, ERK, JNK, and p38 were not increased at the peaks. beta-Catenin was increased in the nuclear extract at the UAL troughs, where increased gene expression was absent. The increase in gene expression at the peaks was due, in part, to increased acetylation of histone 3 at lysine 9.

The proteasome inhibitor, PS-341, causes cytokeratin aggresome formation.

Mallory body (MB) experimental induction takes 10 weeks of drug ingestion. Therefore, it is difficult to study the dynamics and mechanisms involved in vivo. Consequently, an in vitro study was done using primary tissue culture of hepatocytes from drug-primed mice livers in which MBs had already formed. The hypothesis to be tested was that MBs are cytokeratin aggresomes, which form when hepatocytes have a defective ubiquitin-proteasome pathway by which turnover of cytokeratin proteins is prevented. To test this hypothesis, primary tissue cultures of the hepatocytes from normal and MB-forming livers were incubated with the proteasome inhibitor PS-341 and then the cytokeratin filaments and the filament connecting proteins, that is, beta-actin, and ZO1, were visualized by immunofluorescence microscopy. PS-341 caused detachment of the cytokeratins from the cell surface plasma membrane. The cytokeratin filaments retracted toward the nucleus and cytokeratin aggresomes formed. In human livers, MBs showed colocalization of cytokeratin-8 (CK-8) with ubiquitin but not with beta-actin or ZO1. Mouse hepatoma cell lines were studied using PS-341 to induce cytokeratin aggresome formation. In these cell lines, the cytokeratin filaments first retracted toward the nucleus then formed cytokeratin-ubiquitin aggresomes polarized at one side of the nucleus. At the same time, the cells became dissociated from each other, however. The results simulated MB formation. MBs differ from cytokeratin aggresomes both morphologically and in ultrastructure.