Component 2: Zhao & Valenzuela

调控神经元基因表达的重要机制是DNA甲基化介导的

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..