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.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
5R01AA016347-03
Application #
7619302
Study Section
Hepatobiliary Pathophysiology Study Section (HBPP)
Program Officer
Orosz, Andras
Project Start
2007-05-15
Project End
2012-04-30
Budget Start
2009-05-01
Budget End
2010-04-30
Support Year
3
Fiscal Year
2009
Total Cost
$333,078
Indirect Cost
Name
University of Missouri-Columbia
Department
Pharmacology
Type
Schools of Medicine
DUNS #
153890272
City
Columbia
State
MO
Country
United States
Zip Code
65211
Park, Pil-Hoon; Lim, Robert W; Shukla, Shivendra D (2012) Gene-selective histone H3 acetylation in the absence of increase in global histone acetylation in liver of rats chronically fed alcohol. Alcohol Alcohol 47:233-9
Aroor, Annayya R; Jackson, Daniel E; Shukla, Shivendra D (2012) Dysregulated phosphorylation and nuclear translocation of cyclic AMP response element binding protein (CREB) in rat liver after chronic ethanol binge. Eur J Pharmacol 679:101-8
James, Taryn T; Aroor, Annayya R; Lim, Robert W et al. (2012) Histone H3 phosphorylation (Ser10, Ser28) and phosphoacetylation (K9S10) are differentially associated with gene expression in liver of rats treated in vivo with acute ethanol. J Pharmacol Exp Ther 340:237-47
Nichols, Lanita A; Jackson, Daniel E; Manthey, John A et al. (2011) Citrus flavonoids repress the mRNA for stearoyl-CoA desaturase, a key enzyme in lipid synthesis and obesity control, in rat primary hepatocytes. Lipids Health Dis 10:36
Aroor, Annayya R; Jackson, Daniel E; Shukla, Shivendra D (2011) Elevated activation of ERK1 and ERK2 accompany enhanced liver injury following alcohol binge in chronically ethanol-fed rats. Alcohol Clin Exp Res 35:2128-38
Choudhury, Mahua; Pandey, Ravi S; Clemens, Dahn L et al. (2011) Knock down of GCN5 histone acetyltransferase by siRNA decreases ethanol-induced histone acetylation and affects differential expression of genes in human hepatoma cells. Alcohol 45:311-24
Aroor, Annayya R; James, Taryn T; Jackson, Daniel E et al. (2010) Differential changes in MAP kinases, histone modifications, and liver injury in rats acutely treated with ethanol. Alcohol Clin Exp Res 34:1543-51
Choudhury, Mahua; Park, Pil-Hoon; Jackson, Daniel et al. (2010) Evidence for the role of oxidative stress in the acetylation of histone H3 by ethanol in rat hepatocytes. Alcohol 44:531-40
Choudhury, Mahua; Shukla, Shivendra D (2008) Surrogate alcohols and their metabolites modify histone H3 acetylation: involvement of histone acetyl transferase and histone deacetylase. Alcohol Clin Exp Res 32:829-39
Pal-Bhadra, Manika; Bhadra, Utpal; Jackson, Daniel E et al. (2007) Distinct methylation patterns in histone H3 at Lys-4 and Lys-9 correlate with up- &down-regulation of genes by ethanol in hepatocytes. Life Sci 81:979-87