Mechanisms for the cellular and subcellular effects of ethanol are not well understood. Emerging evidence highlight the importance of histone modifications in gene expressions. We have observed that ethanol increases histone H3 acetylation selectively at Lys 9 (H3-Lys9) but not at Lys 14, 18 or 23. Strikingly, the increases were up to 10 fold and could be detected at as low as 5 mM ethanol. This was noted both in primary cultures of hepatocytes and in the liver of rats administered ethanol in vivo. Based on these promising original data it is hypothesized that 'ethanol increases histone acetylation via modulation of histone acetyl transferase (HAT) leading to transcriptional activation of specific genes' in the liver. The long term objective is to identify the mechanism of the acetylation, determine the identity of the HAT involved and investigate the association of the acetylated histone with specific DMA domains of genes affected by ethanol in the chromatin and correlate these alterations to apoptosis and liver damage. There are three specific aims:
Aim 1 : Determine mechanism and regulation of the histone acetylation: Role of ethanol derived phosphatidylethanol; effect of acetate versus ethanol on acetylation; its correlation to cell damage and apoptosis; role of oxidative stress in acetylation and use of selected knockout (KO) mice [ ADH1 (-/-); SOD 1(-/-); SOD-2 (-/-; )] Aim 2: Determine identity of HAT involved: Immunoprecipitation studies with HAT antibodies; differentiate HAT increase due to upregulation of expression versus its activation; use of SiRNA methodology and selected HAT KO mice (PCAF -/-; GCN5 ).
Aim 3 : Determine significance of the acetylation in transcriptional process: In vitro and in vivo studies; chromatin immunoprecipitation (CHIP) assays to determine site of interaction between the promoter of ADH1 gene and acetylated H3-lys9; analyze association between acetylated histone with specific DMA domains of selected genes (e.g. iNOS, Cyp2E1,TNFa); determine association of acetylated histone with transcription factor(s) (e.g. NFkB, p53); use of TF- protein arrays. This project deals with a new avenue in alcohol research. The data obtained on the mechanisms and consequences of histone acetylations by ethanol will be important in the construction of the 'molecular map' of ethanol induced liver injury and will help develop therapeutic tools targeting specific molecules/steps. Furthermore, study of these epigenetic changes will also have 'lateral impact1 on investigations in other cells/systems affected by ethanol. ? ? ?
