Role of epigenetic crosstalks in directing locus sensitivity to arsenic

项目总结 本应用程序中提供的研究的总体目标是了解 基因组基因座比其他基因座更容易受到环境化学扰动的影响。使用无机砷 (IAS)作为与人类高度相关的模型环境毒物，我们将寻求机械地调查 表观遗传串扰如何决定特定位点对砷的敏感性。 IAS是一种模型表观遗传毒物，因为它对全球DNA低甲基化的影响众所周知 与用于DNA和组蛋白的通用甲基供体SAM水平的下降不谋而合 甲基化。然而，这种IAS表观遗传机制的模型在很大程度上被认为 不能令人满意，因为(1)即使在全球DNA低甲基化的背景下，一些基因座也表现出超甲基化 而其他基因则没有变化；(2)对组蛋白甲基化的影响是不一致的，许多甲基化 组蛋白标记显示增加，而其他标记显示减少。在这里，我们建议在令人信服的基础上 通过高度定量的质谱学和代谢组学研究获得的初步数据表明，在 在亚砷酸钠不会导致ROS水平显著增加的情况下，小鼠ESCs，a SAM、DNA甲基化和几个组蛋白标记，如H3K36me2/3显著减少 观察到的。然而，H3K27me3水平增加，而H3K9me3水平不变。此外， RNA-seq研究揭示，即使在深刻的转录变化的背景下，重复的元件也是 在亚砷酸钠暴露后，被H3K9me3沉积抑制的基因在转录上保持沉默。 因此，我们假设表观遗传串扰可以差异化地竞争减少的SAM池 由IAS暴露引起，从而驱动轨迹敏感性。 为了验证这一假设，我们将使用串扰特征很好的小鼠ESCs。在目标1中，我们将 描述全基因组DNA甲基化和3个不同的组蛋白PTM的变化。我们还将测试 IAS引起的这些表观遗传改变是否需要砷的代谢活性 甲基转移酶AS3MT。在目标2中，我们将使用击倒、过度表达和剖析的组合 机械审问方法在砷暴露背景下的作用已有充分表征 不同基因组座位上DNA甲基化和组蛋白PTM之间的串扰。最后，在目标3中，我们将 检查砷诱导的表观遗传学改变的可重编程性，因为ESCs分化为早期 进入生殖细胞阶段，经历深刻的表观遗传重塑浪潮。 在完成这些目标后，我们将建立DNA变化的全面概况 砷暴露后甲基化和4个组蛋白PTM。我们还将确定表观遗传学 串扰调节位点特异性对砷的敏感性，以及它们在PGCs中重新编程的能力。这部作品 将坚定地确立表观遗传相声在应对环境侮辱中的核心作用。

PROJECT SUMMARY The overarching goal of the research presented in this application is to understand what make some genomic loci more susceptible than others to environmental chemical perturbation. Using inorganic arsenic (iAs) as a model environmental toxicant of high human relevance, we will seek to mechanistically investigate how epigenetic crosstalks dictate locus-specific sensitivity to arsenic. iAs is a model epigenetic toxicant owing to its well described impact on global DNA hypomethylation coinciding with a reduction in the levels of the universal methyl donor SAM, used towards DNA and histone methylation. However, this model of epigenetic mechanism of iAs has been acknowledged as largely unsatisfactory since (1) even in the context of global DNA hypomethylation, some loci show hypermethylation while others show no change, and (2) the effect on histone methylation are non-uniform with many methylated histone marks showing increases while others show a decrease. Here, we propose to build on compelling preliminary data obtained through highly quantitative Mass Spec and metabolomic studies that show that in mouse ESCs, at levels where sodium arsenite does not cause a significant increase in ROS levels, a pronounced decrease in SAM, DNA methylation, and in several histone marks, such as H3K36me2/3, are observed. However, H3K27me3 levels are increased while H3K9me3 levels are unchanged. Furthermore, RNA-seq studies revealed even in the context of profound transcriptional changes, repetitive elements that are repressed by deposition of H3K9me3 remain transcriptionally silenced following sodium arsenite exposure. Thus, we hypothesize that epigenetic crosstalks can differentially compete for the reduced SAM pool caused by iAs exposure, thereby driving locus sensitivity. To test this hypothesis, we will use mouse ESCs where crosstalks are well characterized. In aim 1, we will characterize the genome-wide changes in DNA methylation and in 3 distinct histone PTMs. We will also test whether these epigenetic alterations caused by iAs require the metabolic activity of the arsenic methyltransferase AS3MT. In aim 2, we will use a combination of knock-down, over-expression, and profiling approaches to mechanistically interrogate in the context of arsenic exposure the role of the well-characterized crosstalks between DNA methylation and histone PTMs at distinct genomic loci. Finally, in aim 3, we will examine the reprogrammability of arsenic-induced epigenetic alterations as ESCs are differentiated into early stage germ cells and go through profound waves of epigenetic remodeling. At the completion of these aims, we will have established the comprehensive profile of changes in DNA methylation and 4 histone PTMs following arsenic exposure. We will also have determined how epigenetic crosstalks mediate locus-specific sensitivity to arsenic and their ability to be reprogrammed in PGCs. This work will firmly establish the central role of epigenetic crosstalks in the response to environmental insults.