Component 2: Zhao & Valenzuela

神经元基因表达调控的一个重要机制是DNA甲基化 表观遗传调节MeCP 2蛋白与甲基化DNA结合并调节基因转录 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/或函数的更改 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。 这种蛋白质在环境损伤引起的智力迟钝综合征中的作用。鉴于 发育期接触酒精是智力迟钝的主要环境原因， 研究MeCP 2在胎儿酒精谱系障碍中的作用。我们最近报道了急性乙醇暴露 有效地刺激大鼠新生海马体的CAS区域中的网络活动（J Neurochem. 94：1500-11，2005），并且文献表明这种作用可以调节MeCP 2水平和/或活性。 因此，我们用海马切片进行了初步的体外实验，发现急性 暴露于乙醇迅速增加MeCP 2表达水平，并降低磷酸-MeCP 2/总 MeCP 2比率。基于这些体外初步数据，我们假设， 妊娠晚期等效物将在体内产生相同的效果，增加MeCP 2与其DNA靶标的结合。 在目标#1中，我们将评估乙醇是否影响体内总MeCP 2或磷酸-MeCP 2水平。大鼠母鼠 并且它们各自的新生后代将在吸入室中暴露于乙醇。然后我们将 使用Western blot研究乙醇对总MeCP 2和磷酸化MeCP 2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 暴露会影响MeCP 2与其DNA靶标的结合。我们将初步评估乙醇是否会影响MeCP 2 使用染色质免疫沉淀测定（ChIP）结合BDNF启动子。我们亦会研究 EtOH是否影响MeCP 2与其他靶DNA的结合，使用最先进的ChIP-微阵列（ChIP-onchip） 比色法这些试验性研究的结果将为今后详细的 乙醇对MeCP 2表达和/或功能的作用。说明：该试点项目将调查 与遗传性精神发育迟滞综合征相关的蛋白质在作用机制中的作用 酒精在发展过程中;发展酒精暴露是主要的环境原因， 精神发育迟滞在美国。

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..