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

Abstract

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.

Fig 1

There was no change in SirT1 in the nuclear extracts or the whole homogenates in either the peaks or troughs of the UAL cycle (mean ± SEM, n = 3, p = 0.53 in the nuclear extract).

Fig 2

There was no change in FoxO1 in the liver homogenates or nuclear extracts at the peaks and troughs of the UAL cycle (mean ± SEM, n = 3, p = 0.22 in the nuclear extract).

Fig 3

There was an increase in histone 3 lysine 9 acetylation in the nuclear extracts at the UAL peaks (mean ± SEM), (P vs C : p=0.02; P vs T : p=0.027; n=3).

Fig 4

There was an increase in p300 levels in the nuclear extracts at the peaks of the UAL (mean ± SEM, P vs C : p=0.015; P vs T : p=0.019; ; n = 3).

Fig 5

There was no change in CBP levels in the nuclear extracts at the peaks and troughs of the UAL cycle. (mean ± SEM, n = 3).

Fig 6

There was no change in NFκB p65 levels in the nuclear extracts at the peaks and troughs of the UAL cycle (mean ± SEM, n = 3).

Fig 7

Phospho-c-Jun tended to decrease in the nuclear extracts at the peaks of the UAL cycle (C vs T p = 0.056, C vs P = 0.11, mean ± SEM, n = 3). However there was n o change in c-Jun itself.

Fig. 8

The level of phospho-Akt Th308 in the nuclear extracts was decreased at the trough compared to the controls (C vs T p = 0.013, mean ± SEM, n = 3).

Fig. 9

The levels of phospho ERK1/phosphoERK2 in the liver in the nuclear extracts were decreased at the peaks of the UAL (C vs P p= 0.04 C vs T p= 0.022, mean ± SEM, n=3).

Fig 10

The levels of p38 MAPK were decreased in the nuclear extracts at the peaks and troughs of the UAL cycle (mean ± SEM, n = 3), p<0.02 P vs C, p<0.047 vs C).

Fig. 11

The levels of phospho-JNK was decreased in the nuclear extracts at both the peaks and troughs of the UAL cycle (C vs P p= 0.031 C vs T p=0.040, mean ± SEM, n = 3).

Fig 12

The level of β-catenin was increased in the nuclear extracts at both the peaks and troughs of the UAL cycle (C vs T p=0.013, C vs P p=0.006, mean ± SEM, n = 3).