Measuring the impact of prenatal alcohol exposure on the fetal epigenome

描述（由申请人提供）：产前暴露于酒精严重影响胎儿的生长和分化，并导致广泛的发育异常。这些严重程度各不相同，从几乎无法检测到严重的身体和认知障碍，统称为胎儿酒精谱系障碍（FASD）。酒精敏感的发育窗口与DNA甲基化和组蛋白重塑的主要时期的相关性表明了FASD表型中令人困惑的异质性的潜在表观遗传机制。胚胎DNA甲基化和翻译后组蛋白修饰谱的遗传性、持久性变化改变了胎儿的发育程序，并有可能导致严重的发育异常。尽管有大量的观察数据证实了产前酒精暴露对胚胎表观基因组产生负面影响的能力，但解释其潜在机制的信息却很少。研究表观遗传编程的最佳模型是基因组印记、X失活和反转录转座子沉默。由于前两个过程是专门的转录调控机制，后者的研究提供了机会，洞察一般表观遗传基因调控，以及它是如何受到产前酒精暴露的影响。 该项目的长期目标是开发和验证一种体内模型，以监测表观遗传编程，并检查胎儿表观基因组在酒精暴露后的组织特异性变化。在这个建议中要测试的假设是，产前暴露于酒精在不同的发育窗口诱导胎儿表观基因组的组织特异性改变，这可能与FASD的不同表型。该方案的创新之处在于基于刺鼠IAP元件开发了一种含有逆向元件报告基因的绿色荧光蛋白（GFP）。暴露于乙醇的组织将释放IAPGFP报告基因的抑制，导致逆转录元件激活和GFP表达。因此，受影响的器官和组织将变成绿色。我们假设，已知在特定发育窗口期对酒精暴露敏感的组织将根据酒精暴露的剂量和时间改变颜色。在我们的模型的验证中，我们将GFP表达与组织特异性基因表达的已知变化以及IAPGFP报告基因和候选基因的表观遗传修饰相关联。总之，该报告者将能够深入分析乙醇在开发过程中的表观遗传影响，同时提供一个平台来检查治疗干预的有效性。鉴于表观遗传学在维持干细胞身份和分化方面的关键作用，了解如何，何时以及何种剂量的酒精暴露足以改变胎儿的发育程序是我们更好地了解FASD表观遗传基础的关键一步。 公共卫生相关性：早孕是儿童发育的关键窗口。该提案将研究子宫内接触酒精可能导致反复流产和儿童疾病发展的基本机制。了解与怀孕期间饮酒相关的精神和身体残疾是如何产生的，对于制定预防措施和治疗至关重要。

DESCRIPTION (provided by applicant): Prenatal exposure to alcohol severely affects growth and differentiation of the fetus and causes a wide range of developmental abnormalities. These vary in severity, from barely detectable to crippling physical and cognitive disabilities that are collectively referred to as fetal alcohol spectrum disorders (FASDs). Correlation of alcohol sensitive developmental windows with major periods of DNA methylation and histone remodeling suggest an underlying epigenetic mechanism to the bewildering heterogeneity in FASD phenotype. Heritable, long lasting changes to the DNA methylation and post-translational histone modification profile of the embryo alter the developmental program of the fetus, and have the potential to result in severe developmental abnormalities. Despite the wealth of observational data confirming the capacity of prenatal alcohol exposure to negatively affect the embryonic epigenome, there is a paucity of information explaining the underlying mechanism. The best models for investigating epigenetic programming are genomic imprinting, X-inactivation and retrotransposon silencing. As the first two processes are specialized transcriptional regulatory mechanisms, study of the latter offers opportunities for insight into general epigenetic gene regulation and how it is influenced by prenatal alcohol exposure. The long-term goal of this project is to develop and validate an in vivo model to monitor epigenetic programming and examine tissue specific changes to the fetal epigenome in response to alcohol exposure. The hypothesis to be tested in this proposal is that prenatal exposure to alcohol during different developmental windows induce tissue specific alterations in the fetal epigenome which can be correlated with the diverse phenotypes of FASDs. The innovative aspect of this proposal is the development of a Green Fluorescent Protein (GFP) containing retro-element reporter based on the agouti-IAP element. Tissues exposed to ethanol will loose repression of the IAPGFP reporter leading retro-element activation and GFP expression. As a result, affected organs and tissues will turn green. We hypothesize that tissues known to be sensitive to alcohol exposure during specific developmental windows will change color according to dosage and timing of ethanol exposure. In validation of our model we will correlate GFP expression with known changes in tissue specific gene expression and established epigenetic modifications of both the IAPGFP reporter and candidate genes. In summary, this reporter will enable in-depth analysis of the epigenetic influence of ethanol during development while at the same time providing a platform to examine the efficacy of therapeutic interventions. Given the critical role of epigenetics in maintaining stem cell identity and differentiation, understanding how, when and what dose of alcohol exposure is sufficient to alter the developmental program of the fetus is crucial step in our goal to better understand the epigenetic basis of FASDs. PUBLIC HEALTH RELEVANCE: Early pregnancy is a critical window in child development. This proposal will examine basic fundamental mechanisms by which exposure to alcohol in the womb can result in recurrent pregnancy loss and development of childhood diseases. Understanding how mental and physical disabilities associated with drinking during pregnancy arise is critical for developing preventative measures and treatments.