Epigenetic regulation of alcohol tolerance and dependence by methyl CpG binding protein 2

总结 基因表达程序的长期改变被认为是发展的关键， 酒精使用障碍（AUD）。甲基CpG结合蛋白2（MeCP 2）是导致 Rett综合征是一种结合甲基化DNA的蛋白质，反过来，招募转录抑制因子， 持续下调基因表达。我们观察到MeCP 2突变小鼠， 招募转录抑制因子的能力表现出强大的酒精相关表型，其特征为 对酒精镇静作用的敏感性提高，在有限的2瓶选择中减少酒精摄入 以及被动诱导依赖后饮酒没有升级。 最近的证据表明MeCP 2的主要功能是募集转录抑制因子 通过离散的分子结构域在甲基化DNA的位点形成复合物。重要的是，已经发现MeCP 2 调节一组特定的基因--或调节子--而不是广泛地影响基因表达水平， 发现酒精调节基因和MeCP 2调节基因之间存在显著重叠。因此，在本项目中， 我们将测试一个总体假设，即MeCP 2调节的基因是酒精作用的关键， 在酒精依赖的情况下过渡到逐步增加的饮酒。为了检验子假设， MeCP 2募集转录抑制因子是其对饮酒影响的核心，我们将使用MeCP 2 与最近引入的基因突变小鼠相比， MeCP 2突变小鼠具有增加的募集转录抑制因子的能力，以提供最佳的 MeCP 2功能的扰动用于分析MeCP 2调节的基因网络。为了检验子假设 特定的MeCP 2靶基因和调节剂是向饮酒相关的逐步升级过渡的关键， 对于酒精依赖，我们将使用我们最近验证的最先进的系统生物学策略， 脑基因表达全基因组转录相互作用组的重建和研究 数据区.这种方法是集中在公正的识别转录调控关系，从 研究中的扰动对基因表达的影响，而不是从文献中已知的或根据 不同的扰动该系统不是识别一长串差异表达基因， 生物学策略识别并排列了驱动与基因相关的基因签名的少量基因， 表型。因此，获得了关于MeCP 2在酒精作用中的作用的具体机制假设， 然后将在依赖性和非依赖性饮酒的范例中进行实验验证。 最终，这项研究的结果将促进我们对背后分子机制的理解。 过度饮酒的依赖性，并将奠定理由，利用具体的 MeCP 2调节的基因和调节剂用于开发AUD的新治疗概念。

Summary Long-term alterations in gene expression programs are believed to be key to the development and progression of alcohol use disorder (AUD). The methyl CpG binding protein 2 (MeCP2), the causative gene of Rett syndrome, is a protein that binds methylated DNA and, in turn, recruits transcriptional repressors resulting in persistent down-regulation of gene expression. We observed that MeCP2 mutant mice with reduced capacity to recruit transcriptional repressors exhibit a robust alcohol–related phenotype characterized by heightened sensitivity to the sedative effects of alcohol, reduced alcohol intake in limited access 2-bottle choice and lack of escalation of drinking after passive induction of dependence. Recent evidence indicates that MeCP2's primary function is to recruit a transcriptional repressor complex at sites of methylated DNA through a discrete molecular domain. Importantly, MeCP2 has been found to regulate a specific set of genes – or regulon – rather than broadly affecting gene expression levels, and we found significant overlap between alcohol-regulated and MeCP2-regulated genes. Thus, in the present project we will test the overarching hypothesis that MeCP2-regulated genes are key to alcohol's effects and to the transition to escalated alcohol drinking in the setting of alcohol dependence. To test the sub-hypothesis that recruitment of transcriptional repressors by MeCP2 is central to its effects on drinking, we will use MeCP2 mutant mice with reduced capacity to recruit transcriptional repressors in comparison with recently introduced MeCP2 mutant mice with increased capacity to recruit transcriptional repressors, to provide optimal perturbation of MeCP2 function for the analysis of MeCP2 regulated gene networks. To test the sub-hypothesis that specific MeCP2 target genes and modulators are key to the transition to escalated drinking associated with alcohol dependence, we will use a state of the art systems biology strategy that we recently validated for the reconstruction and interrogation of genome-wide transcriptional interactomes from brain gene expression profiles. This approach is centered on unbiased identification of transcriptional regulatory relationships from the gene expression effects of the perturbations under study, rather than what is known from the literature or under different sets of perturbations. Rather than identifying long lists of differentially expressed genes, this systems biology strategy identifies and ranks a small number of genes driving the gene signatures associated with the phenotype. Thus, specific mechanistic hypotheses on the role of MeCP2 in the effects of alcohol are obtained, and will then be experimentally validated in paradigms of dependent and non-dependent alcohol drinking. Ultimately, the results of this study will advance our understanding of the molecular mechanisms behind excessive alcohol drinking in the setting of dependence and will lay the rationale for the exploitation of specific MeCP2-regulated genes and modulators for the development of novel therapeutic concepts for AUD.