Component 2: Zhao & Valenzuela

DNA甲基化是控制神经元基因表达的重要机制 表观遗传调控。MeCP2蛋白与甲基化的DNA结合并调节基因的转录 对正常的神经发育很重要，比如脑源性神经营养因子。表达式和/或功能的更改 MeCP2的突变会导致Rett综合征，一种遗传性精神发育迟滞综合征。然而，人们对此知之甚少 该蛋白在环境侮辱引起的智力低下综合征中的作用。考虑到 发育过程中接触酒精是导致智力低下的主要环境原因，重要的是 探讨MeCP2在胎儿酒精谱障碍中的作用。我们最近报告说，急性酒精暴露 有效地刺激新生大鼠海马区CAS区的网络活动(J Neurochem. 94：1500-11,2005)，并且文献表明这种效应可以调节MeCP2水平和/或活性。 因此，我们对海马片进行了初步的体外实验，发现急性 酒精暴露迅速增加MeCP2的表达水平，并降低磷酸化的MeCP2/总 MeCP2比值。基于这些体外初步数据，我们假设急性乙醇暴露在 相当于晚期妊娠的药物在体内也会产生同样的效果，增加MeCP2与其DNA靶点的结合。 在目标1中，我们将评估乙醇是否影响体内总MeCP2或磷酸化MeCP2水平。老鼠坝 它们各自的新生后代将在吸入室中暴露在乙醇中。到时候我们会的 用免疫印迹技术研究乙醇对总MeCP2和磷酸化MeCP2表达水平的影响 免疫印迹和免疫组织化学技术。在目标2中，我们将研究体内乙醇是否 暴露影响MeCP2与其DNA靶标的结合。我们将初步评估乙醇是否会影响MeCP2 用染色质免疫沉淀分析(CHIP)与BDNF启动子结合。我们还将检查 使用最先进的ChlP-微阵列(ChlP-onChip)，乙醇是否影响MeCP2与其他靶DNA的结合 化验。这些初步研究的结果将构成未来详细的机理研究的基础。 乙醇对MeCP2表达和/或功能的影响版面描述：该试点项目将调查 一种与遗传性智力低下综合征相关的蛋白质在作用机制中的作用 发育过程中的酒精暴露；发育性酒精暴露是导致 美国的精神发育迟滞

An important mechanism for the control of gene expression in neurons is DNA methylation-mediated epigenetic regulation. The protein MeCP2 binds to methylated DNA and regulates the transcription of genes that are important for normal neurodevelopment such as BDNF. Alterations in the expression and/or function of MeCP2 cause Rett syndrome, a genetic mental retardation syndrome. However, little is known about the role of this protein in mental retardation syndromes caused by environmental insults. Given that developmental exposure to alcohol is a leading environmental cause of mental retardation, it is important to study the role of MeCP2 in fetal alcohol spectrum disorder. We recently reported that acute ethanol exposure potently stimulates network activity in the CAS region of the rat neonatal hippocampus (J Neurochem. 94:1500-11, 2005) and the literature indicates that this effect could modulate MeCP2 levels and/or activity. We therefore performed preliminary in vitro experiments with hippocampal slices and found that acute exposure to ethanol rapidly increases MeCP2 expression levels and decreases the phospho-MeCP2/total MeCP2 ratio. Based on these in vitro preliminary data, we hypothesize that acute EtOH exposure during the third trimester equivalent will produce the same effects in vivo, increasing MeCP2 binding to its DNA targets. In aim #1, we will assess whether ethanol affects total MeCP2 or phospho-MeCP2 levels in vivo. Rat dams and their respective neonatal offspring will be exposed to ethanol in inhalation chambers. We will then investigate the effect of ethanol on total MeCP2 and phospho-MeCP2 expression levels using Western immunoblotting and immunohistochemical techniques. In aim #2, we will investigate whether in vivo ethanol exposure affects MeCP2 binding to its DNA targets. We will initially assess whether ethanol affects MeCP2 binding to the BDNF promoter using a chromatin immunoprecipitation assay (ChIP). We will also examine whether EtOH affects MeCP2 binding to other target DNAs using a state-of-art ChlP-microarray (ChlP-onchip) assay. The results of these pilot studies will form the basis for future detailed mechanistic studies of ethanol's action on MeCP2 expression and/or function. Lay Description: This pilot project will investigate the role of a protein that has been linked to genetic mental retardation syndromes in the mechanism of action of alcohol during development; developmental alcohol exposure is the leading environmental cause of mental retardation in the U.S.A..