Chromatin remodeling due to histone deacetylase (HDAC)-induced histone modifications has been implicated in the pathophysiology of psychiatric disorders. We investigated the role of HDAC-2 isoform-linked chromatin remodeling in anxiety and alcoholism. More specifically, it was found that nuclear, but not cytosolic, HDAC activity was higher in the amygdala of alcohol-preferring (P) rats when compared with non-preferring (NP) rats. This correlated with higher protein levels of HDAC-2 (class I HDAC), but not with that of HDAC-4 (class II HDAC) in the central (CeA) and medial amygdala (MeA) of P compared with NP rats. We also found that acetylation of histones (H3-K9 and H4-K8) was lower and dimethylation of H3 (K-9) was higher in the CeA and MeA of P rats compared with NP rats. Treatment with trichostatin A (TSA), an HDAC inhibitor, inhibited HDAC activity and decreased HDAC-2, but not HDAC-4 protein levels, and also corrected deficits in histone acetylation in the CeA and MeA of P rats. TSA treatment attenuated anxiety-like behaviors and decreased voluntary alcohol-drinking behaviors in P rats, but had no effect on these behaviors in NP rats. It has been shown that HDAC-2 regulates synaptic plasticity; therefore, we examined changes in HDAC-2 and dendritic spine density (DSD) during ethanol exposure. Acute ethanol exposure normalized nuclear HDAC activity, HDAC-2 protein levels, histone (H3) methylation and histone (H3) acetylation in the amygdaloid structures of P, but not NP rats. Acute ethanol corrected the deficits in DSD and activity-regulated cytoskeleton-associated protein (Arc) gene expression and also increased the levels of acetylated H3 in the Arc gene promoter region in amygdaloid structures and produced anxiolytic effects in P, but not in NP rats. Interestingly, decreasing HDAC-2 expression in the CeA by siRNA produced anxiolytic effects and attenuated alcohol intake in P rats and corrected deficits in histone acetylation and DSD in the CeA of P rats. Taken together, these results indicate that upregulation of HDAC-2 in the neurocircuitry of the amygdala may be responsible for the abnormal chromatin architecture that may be involved in the comorbidity of anxiety and alcoholism. (This study was supported by NIH-NIAAA grants and VA Merit and Career Scientist Grants to SCP.) # S09.3 FETAL ALCOHOL-INDUCED HYPERMETHYLATION OF PROOPIOMELANOCORTIN GENE IN THE HYPOTHALAMUS TRANSMITS THROUGH GERMLINE {#article-title-2} Our study aimed at determining whether the stress axis abnormality in fetal alcohol-exposed offspring is caused by epigenetic marks on POMC gene, since a product of this gene β-endorphin (BEP) has been shown to control the stress axis function. We found that the reduced BEP neuronal function in the hypothalamus is associated with low expression of proopiomelanocortin (POMC) mRNA, increased DNA methylation of the POMC gene promoter and increased production of histone and DNA methyltransferases in BEP neurons of fetal alcohol-exposed offspring. Further support of the notion that fetal alcohol exposure caused POMC gene promoter hypermethylation and reduced gene expression was evident with additional finding that supplementation of choline or treatment with DNMT or HDAC blockers prevented DNA hypermethylation, POMC gene suppression and/or the stress axis abnormalities in the alcohol-exposed offspring. The alcohol-induced abnormalities in DNA methylation, POMC gene expression and stress axis function were long-lasting and transferred via male germ line to three subsequent generations. These data support that fetal alcohol exposures incite transgenerational alteration in the epigenetic programming of the POMC gene to induced stress axis dysfunction. (This study was supported by NIH Grant AA016695 and R37 AA08757.) # S09.4 EPIGENETIC MECHANISMS IN THE BRAIN OF HUMAN ALCOHOLICS: ADAPTATIONS IN THE OPIOID GENES {#article-title-3} Genetic, epigenetic and environmental factors may influence the risk for neuropsychiatric disease through their effects on gene transcription. These effects may be integrated through methylation of CpGs overlapping with SNPs associated with a disorder. We addressed this hypothesis by analyzing prodynorphin ( PDYN ) CpG-SNPs, which are associated with alcohol dependence, in human alcoholics. Analysis of postmortem human brain demonstrated that PDYN expression is activated in the dorsolateral prefrontal (dl-PFC) in alcoholics. This activation may contribute to neurocognitive dysfunctions relevant for ‘preoccupation/anticipation’ stages of addiction and disrupted inhibitory control. Three PDYN SNPs associated with alcohol dependence overlap with CpGs. Methylation of these CpG-SNPs was analyzed by pyrosequencing followed by bisulfite treatment in the dl-PFC and motor cortex (MC), showing PDYN upregulation or no expression changes, respectively, in 14 alcoholics and 14 controls. In the dl-PFC but not MC of alcoholics, methylation levels of the C, non-risk variant of 3′-untranslated region (3′-UTR) SNP (rs2235749; C > T) were increased ( P