Rates of histone turnover and functional significance in fetal alcohol syndrome

 描述（由申请人提供）：妊娠期间饮酒是一个重大的公共卫生问题，并可能导致对胎儿的连续不良后果，称为胎儿酒精谱系障碍（FASD）。这些疾病的范围从生长迟缓到神经行为改变，包括心理健康问题、社会适应能力差和压力耐受力差。胎儿酒精缺陷的范围从大脑区域大小的缩小到神经元和神经胶质细胞损失水平的显微结构病理学、神经元和神经胶质的异位位置或纤维束缺陷。乙醇在发育过程中的神经毒性作用使海马神经元、皮质神经元、大脑颗粒和浦肯野神经元以及下丘脑神经元的数量减少。我们的工作和其他人的工作表明，酒精代谢释放出双产物，如乙醛和活性氧（ROS），这可能导致DNA损伤，并在DNA修复缺陷的条件下可能导致基因组不稳定和细胞死亡。最近的调查可能涉及DNA甲基化和组蛋白修饰的中介酒精对胎儿大脑的不良影响的表观遗传机制提供了一个很有前途的方法来了解复杂的可观察到的特征与FAS。不幸的是，到目前为止，所有用于研究导致许多胎儿酒精综合征表型的基因表观遗传修饰的方法都依赖于静态测量。我们相信，定量组蛋白周转可能是一个重要的参数，可以有助于更好地了解发育中的大脑基因表达的表观遗传调控。我们的中心假设是，组蛋白营业额可能是一个重要的参数，可以有助于乙醇介导的表观遗传改变，导致基因表达和细胞存活的缺陷。我们建议使用一种新的稳定同位素示踪方法，我们最近开发的，它使测量蛋白质的合成和营业额在体内，回答这个问题。本提案中概述的研究将标准蛋白质组学分析与我们使用重水标记和LC-MS/MS的动力学方法相结合，以同时测量核心组蛋白，组蛋白变体及其后修饰异构体的半衰期和丰度，并确定组蛋白周转对基因表达和蛋白质网络的功能意义，特别强调DNA损伤反应途径。拟议的研究具有高度创新性，因为它们将是第一个表征发育中大脑中组蛋白动力学的研究，它们还可以确定治疗干预的新领域以及胎儿酒精谱系障碍的标志物。

 DESCRIPTION (provided by applicant): Alcohol consumption during pregnancy is a significant public health problem and can result in a continuum of adverse outcomes to the fetus known as fetal alcohol spectrum disorders (FASD). These disorders range from growth retardation to neurobehavioral alterations including mental health problems, poor social adjustment and poor stress tolerance. Fetal alcohol defects range from diminution in the size of the brain region to microstructural pathology at the level of loss of neurons and glial cells, ectopic locations of neurons and glia, or defects in fiber tracts. The neurotoxic actions of ethanol during development reduce the number of hippocampal neurons, cortical neurons, cerebral granule and Purkinje neurons and hypothalamic neurons. Our work and that of others have shown that alcohol metabolism releases bi-products, such as acetaldehyde and reactive oxygen species (ROS), which can cause DNA damage and in conditions of DNA repair deficiencies can lead to genomic instability and cell death. Recent investigation of possible epigenetic mechanisms involving DNA methylation and histone modifications as mediators of alcohol's adverse effects on the brain of the fetus provides a promising approach for understanding the complex observable characteristics associated with FAS. Unfortunately, to this point, all of the methods that have been used to study epigenetic modifications of genes causing many fetal alcohol syndrome phenotypes rely on static measurements. We believe that quantifying histone turnover might be an important parameter that can contribute to better understanding of epigenetic regulation of gene expression in developing brain. Our central hypothesis is that histone turnover might be an important parameter that can contribute to the ethanol-mediated epigenetic alterations leading to defects in gene expression and cell survival. We propose to use a novel stable isotope tracer method that we recently developed, which enables measurements of protein synthesis and turnover in vivo, to answer this question. The studies outlined in this proposal will couple standard proteomic analyses with our kinetic method using heavy water labeling and LC-MS/MS to simultaneously measure the half-life and abundance of core histones, histone variants and their post-translationally modified isoforms and determine the functional significance of histone turnover on gene expression and protein network, with specific emphasis on DNA damage response pathways. The proposed studies are highly innovative as they would be the first to characterize histone kinetics in the developing brain, they may also identify new areas for therapeutic intervention as well as markers for fetal alcohol spectrum disorders.