Epigenetic Effects of Prenatal Ethanol Exposure

描述(由申请者提供)：胎儿酒精综合症(FAS)是全球范围内导致智力低下和出生缺陷的重要原因。然而，乙醇导致Fas的发育异常和神经毒性特征的机制尚不清楚。我们假设，怀孕期间通过母体摄入长期接触乙醇会导致发育中的胎儿发生特殊的表观遗传学变化，特别是在神经系统。关键基因子集正常调控的这些稳定变化将导致Fas的发育异常。我们进一步假设，子宫内乙醇暴露的表观遗传学变化也会导致后代在以后的生活中出现长期的健康问题。该项目的目标是确定在子宫内乙醇暴露后发生表观遗传变化并导致大脑基因表达异常的基因子集。我们将在整个怀孕期间通过液体饮食将怀孕小鼠暴露在乙醇中，然后比较实验和对照(未接触乙醇)幼鼠的脑组织，以了解小鼠基因组启动子区DNA甲基化模式的变化。这一策略将使用甲基化DNA免疫沉淀(MeDIP)与Affymetrix的商业可用平铺阵列一起用于meDIP芯片上的脑DNA分析。首先，通过免疫沉淀从酒精暴露的小鼠和对照小鼠的基因组DNA中获得高度丰富的甲基化DNA序列的一部分脑DNA，使用抗5-甲基胞嘧啶的抗体。然后，这些DNA片段将被扩增、标记，并与包含小鼠基因组中所有已知启动子区域的Affymetrix微阵列杂交。通过平行表达微阵列研究整个基因组对乙醇的反应，在特定启动子上检测到的DNA甲基化的任何变化都将与相应基因的mRNA表达水平的变化相关联。DNA甲基化水平和mRNA表达水平因产前酒精暴露而发生变化的基因将成为候选基因，其大脑中异常的表观遗传调控可能与Fas表型有关。我们提出的研究应该在分子水平上提供对乙醇影响的新见解，并潜在地识别与产前酒精暴露的病理有关的基因。 公共卫生相关性：研究报告胎儿酒精综合征(FAS)是人类人口中出生缺陷和智力低下的主要原因。1979年至1993年间，在美国出生的患有Fas的儿童的发病率急剧上升，几乎增加了七倍。孕妇在怀孕期间摄入乙醇的有害影响很多，包括严重的生长发育障碍以及形态、行为和神经异常。然而，Fas的分子基础仍不清楚。该项目将探索这样一种假设，即胎儿在宫内接触酒精后的表观遗传学变化，特别是在大脑中，有助于Fas的临床表型。对Fas潜在机制的了解可能有助于开发治疗Fas的新疗法。

DESCRIPTION (provided by applicant): Fetal alcohol syndrome (FAS) is a significant cause of mental retardation and birth defects worldwide. However, the mechanisms by which ethanol causes the developmental anomalies and neurotoxicity characteristic of FAS are poorly understood. We hypothesize that chronic exposure to ethanol during gestation via maternal intake during pregnancy causes specific epigenetic changes in the developing fetus, particularly in the nervous system. These stable changes in the normal regulation of a subset of critical genes would contribute to the developmental abnormalities seen in FAS. We further postulate that the epigenetic changes in response to ethanol exposure in utero also contribute to long-term health problems of the offspring later in life. The goal of this project is to identify a subset of genes that undergo epigenetic changes in response to ethanol exposure in utero and result in aberrant gene expression in the brain. We will expose pregnant mice to ethanol via a liquid diet throughout gestation, then compare brain tissue from experimental and control (non-ethanol exposed) pups for changes in DNA methylation patterns at promoter regions in the mouse genome. This strategy will employ methylated DNA immunoprecipitation (meDIP) in conjunction with commercially available tiling arrays from Affymetrix in meDIP-on-chip assays of brain DNA. First, a fraction of brain DNA highly enriched in methylated DNA sequences will be generated by immunoprecipitation of sheared genomic DNA from ethanol-exposed and control mouse pups using antibodies against 5-methylcytosine. This DNA fraction then will be amplified, labeled, and hybridized to Affymetrix microarrays containing all known promoter regions in the mouse genome. Any changes in DNA methylation detected at specific promoters due to prenatal ethanol exposure will be correlated with changes in mRNA expression levels of the corresponding genes by parallel expression microarray studies of changes in brain mRNA levels across the genome in response to ethanol. Genes that exhibit changes in both DNA methylation and mRNA expression levels in response to prenatal ethanol exposure will be candidate genes whose aberrant epigenetic regulation in the brain may contribute to the FAS phenotype. Our proposed studies should provide novel insight into the effects of ethanol at a molecular level not previously explored, and potentially identify genes that contribute to the pathology of prenatal ethanol exposure. PUBLIC HEALTH RELEVANCE: Research Statement Fetal alcohol syndrome (FAS) is a major cause of birth defects and mental retardation in the human population. The incidence of children born in the United States with FAS has risen dramatically, nearly seven fold, between 1979 and 1993. The detrimental effects of maternal ethanol ingestion during pregnancy are numerous, and include severe growth impairment, and morphological, behavioral and neurological abnormalities. However, the molecular basis for FAS remains unclear. This project will explore the hypothesis that epigenetic changes in the fetus in response to ethanol exposure in utero, particularly in the brain, contribute to the clinical phenotype of FAS. An understanding of the underlying mechanisms of FAS may permit the development of novel therapies for treatment of FAS.