09-071 Sigma-AldrichAnti-G9a (BAT8) Antibody

The transient exposure of immature rodents to ethanol during postnatal day 7 (P7), comparable to a time point within the third trimester of human pregnancy, induces neurodegeneration. However, the molecular mechanisms underlying the deleterious effects of ethanol on the developing brain are poorly understood. In our previous study, we showed that a high dose administration of ethanol at P7 enhances G9a and leads to caspase-3-mediated degradation of dimethylated H3 on lysine 9 (H3K9me2). In this study, we investigated the potential role of epigenetic changes at G9a exon1, G9a-mediated H3 dimethylation on neurodegeneration and G9a-associated proteins in the P7 brain following exposure to a low dose of ethanol. We found that a low dose of ethanol induces mild neurodegeneration in P7 mice, enhances specific acetylation of H3 on lysine 14 (H3K14ace) at G9a exon1, G9a protein levels, augments the dimethylation of H3K9 and H3 lysine 27 (H3K27me2). However, neither dimethylated H3K9 nor K27 underwent degradation. Pharmacological inhibition of G9a activity prior to ethanol treatment prevented H3 dimethylation and neurodegeneration. Further, our immunoprecipitation data suggest that G9a directly associates with DNA methyltransferase (DNMT3A) and methyl-CpG-binding protein 2 (MeCP2). In addition, DNMT3A and MeCP2 protein levels were enhanced by a low dose of ethanol that was shown to induce mild neurodegeneration. Collectively, these epigenetic alterations lead to association of G9a, DNMT3A and MeCP2 to form a larger repressive complex and have a significant role in low-dose ethanol-induced neurodegeneration in the developing brain.

Cocaine-mediated repression of the histone methyltransferase (HMT) G9a has recently been implicated in transcriptional, morphological and behavioral responses to chronic cocaine administration. Here, using a ribosomal affinity purification approach, we found that G9a repression by cocaine occurred in both Drd1-expressing (striatonigral) and Drd2-expressing (striatopallidal) medium spiny neurons. Conditional knockout and overexpression of G9a within these distinct cell types, however, revealed divergent behavioral phenotypes in response to repeated cocaine treatment. Our studies further indicated that such developmental deletion of G9a selectively in Drd2 neurons resulted in the unsilencing of transcriptional programs normally specific to striatonigral neurons and in the acquisition of Drd1-associated projection and electrophysiological properties. This partial striatopallidal to striatonigral 'switching' phenotype in mice indicates a new role for G9a in contributing to neuronal subtype identity and suggests a critical function for cell type-specific histone methylation patterns in the regulation of behavioral responses to environmental stimuli.

Rodent exposure to binge-like ethanol during postnatal day 7 (P7), which is comparable to the third trimester of human pregnancy, induces neuronal cell loss. However, the molecular mechanisms underlying these neuronal losses are still poorly understood. Here, we tested the possibility of histone methylation mediated by G9a (lysine dimethyltransferase) in regulating neuronal apoptosis in P7 mice exposed to ethanol. G9a protein expression, which is higher during embryogenesis and synaptogenic period compared to adult brain, is entirely confined to the cell nuclei in the developing brain. We found that ethanol treatment at P7, which induces apoptotic neurodegeneration in neonatal mice, enhanced G9a activity followed by increased histone H3 lysine 9 (H3K9me2) and 27 (H3K27me2) dimethylation. In addition, it appears that increased dimethylation of H3K9 makes it susceptible to proteolytic degradation by caspase-3 in conditions in which ethanol induces neurodegeneration. Further, pharmacological inhibition of G9a activity prior to ethanol treatment at P7 normalized H3K9me2, H3K27me2 and total H3 proteins to basal levels and prevented neurodegeneration in neonatal mice. Together, these data demonstrate that G9a mediated histone H3K9 and K27 dimethylation critically regulates ethanol-induced neurodegeneration in the developing brain. Furthermore, these findings reveal a novel link between G9a and neurodegeneration in the developing brain exposed to postnatal ethanol and may have a role in fetal alcohol spectrum disorders.

PPARγ promotes adipogenesis while Wnt proteins inhibit adipogenesis. However, the mechanisms that control expression of these positive and negative master regulators of adipogenesis remain incompletely understood. By genome-wide histone methylation profiling in preadipocytes, we find that among gene loci encoding adipogenesis regulators, histone methyltransferase (HMT) G9a-mediated repressive epigenetic mark H3K9me2 is selectively enriched on the entire PPARγ locus. H3K9me2 and G9a levels decrease during adipogenesis, which correlates inversely with induction of PPARγ. Removal of H3K9me2 by G9a deletion enhances chromatin opening and binding of the early adipogenic transcription factor C/EBPβ to PPARγ promoter, which promotes PPARγ expression. Interestingly, G9a represses PPARγ expression in an HMT activity-dependent manner but facilitates Wnt10a expression independent of its enzymatic activity. Consistently, deletion of G9a or inhibiting G9a HMT activity promotes adipogenesis. Finally, deletion of G9a in mouse adipose tissues increases adipogenic gene expression and tissue weight. Thus, by inhibiting PPARγ expression and facilitating Wnt10a expression, G9a represses adipogenesis.