Bohnsack John Peyton, Patel Vraj K, Morrow A Leslie
Department of Pharmacology (J.P.B., A.L.M.), Department of Psychiatry (A.L.M.), and Bowles Center for Alcohol Studies (J.P.B., V.K.P., A.L.M.), University of North Carolina, Chapel Hill, North Carolina.
Department of Pharmacology (J.P.B., A.L.M.), Department of Psychiatry (A.L.M.), and Bowles Center for Alcohol Studies (J.P.B., V.K.P., A.L.M.), University of North Carolina, Chapel Hill, North Carolina
J Pharmacol Exp Ther. 2017 Oct;363(1):1-11. doi: 10.1124/jpet.117.242446. Epub 2017 Aug 10.
-Aminobutyric acid A receptors (GABA-Rs) mediate the majority of inhibitory neurotransmission in the adult brain. The 1-containing GABA-Rs are the most prominent subtype in the adult brain and are important in both homeostatic function and several disease pathologies including alcohol dependence, epilepsy, and stress. Ethanol exposure causes a decrease of 1 transcription and peptide expression both in vivo and in vitro, but the mechanism that controls the transcriptional regulation is unknown. Because ethanol is known to activate epigenetic regulation of gene expression, we tested the hypothesis that ethanol regulates 1 expression through histone modifications in cerebral cortical cultured neurons. We found that class I histone deacetylases (HDACs) regulate ethanol-induced changes in 1 gene and protein expression as pharmacologic inhibition or knockdown of HDAC1-3 prevents the effects of ethanol exposure. Targeted histone acetylation associated with the promoter using CRISPR (clustered regularly interspaced palindromic repeat) dCas9-P300 (a nuclease-null Cas9 fused with a histone acetyltransferase) increases histone acetylation and prevents the decrease of expression. In contrast, there was no effect of a mutant histone acetyltransferase or generic transcriptional activator or targeting P300 to a distant exon. Conversely, using a dCas9-KRAB construct that increases repressive methylation (H3K9me3) does not interfere with ethanol-induced histone deacetylation. Overall our results indicate that ethanol deacetylates histones associated with the promoter through class I HDACs and that pharmacologic, genetic, or epigenetic intervention prevents decreases in 1 expression in cultured cortical neurons.
γ-氨基丁酸A受体（GABA-Rs）介导成年大脑中大部分抑制性神经传递。含γ1的GABA-Rs是成年大脑中最主要的亚型，在稳态功能以及包括酒精依赖、癫痫和应激在内的多种疾病病理过程中都很重要。乙醇暴露在体内和体外均会导致γ1转录和肽表达下降，但控制转录调控的机制尚不清楚。由于已知乙醇会激活基因表达的表观遗传调控，我们测试了乙醇通过组蛋白修饰在大脑皮质培养神经元中调节γ1表达的假说。我们发现I类组蛋白去乙酰化酶（HDACs）调节乙醇诱导的γ1基因和蛋白表达变化，因为HDAC1 - 3的药理学抑制或敲低可防止乙醇暴露的影响。使用CRISPR（成簇规律间隔短回文重复序列）dCas9 - P300（一种与组蛋白乙酰转移酶融合的无核酸酶Cas9）靶向与γ1启动子相关的组蛋白乙酰化可增加组蛋白乙酰化并防止γ1表达下降。相比之下，突变的组蛋白乙酰转移酶或通用转录激活剂或将P300靶向远处外显子则没有效果。相反，使用增加抑制性甲基化（H3K9me3）的dCas9 - KRAB构建体不会干扰乙醇诱导的组蛋白去乙酰化。总体而言，我们的结果表明乙醇通过I类HDACs使与γ1启动子相关的组蛋白去乙酰化，并且药理学、遗传学或表观遗传学干预可防止培养的皮质神经元中γ1表达下降。
