Role of environmental agents targeting mitochondria in epigenetic regulation of nuclear gene expression

靶向线粒体的环境因子在核基因表达表观遗传调控中的作用

• 批准号: 10252593
• 负责人: Richard Woychik
• 金额: $ 12.49万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10252593
• 关键词: Acetyl Coenzyme A; Affect; Age-Months; Alleles; Animals; Antioxidants; Automobile Driving; Back; Biochemical Pathway; Biochemical Process; Cell Culture Techniques; Cell Nucleus; Cells; Citric Acid Cycle; Color; Complex; CpG dinucleotide; Cytosine; DNA; DNA Methylation; DNA Modification Methylases; Data; Development; Disease; Engineering; Environmental Exposure; Enzymes; Epigenetic Process; Exposure to; Expression Profiling; Family; Frequencies; Functional disorder; Gene Expression; Gene Expression Regulation; Genes; Genome; Goals; Health; Histone Deacetylase; Histones; Hydroxyl Radical; Hypermethylation; Impairment; In Vitro; Lactation; Lead; Life; Liver; Maintenance; Mediating; Metabolic; Metabolism; Methylation; Mitochondria; Mixed Function Oxygenases; Modeling; Modification; Mus; Mutation; Nuclear; Nucleosomes; Organelles; Oxidative Phosphorylation; Pathology; Perinatal; Pesticides; Pregnancy; Process; Production; Protein Kinase; Proteins; Reactive Oxygen Species; Regulation; Reporting; Respiration; Role; Rotenone; S-Adenosylhomocysteine; alpha ketoglutarate; base; demethylation; environmental agent; epigenetic regulation; epigenome; histone acetyltransferase; histone demethylase; histone methyltransferase; in vivo; methylation pattern; methylome; mitochondrial dysfunction; mitochondrial metabolism; mouse model; mutant; normal aging; offspring; promoter; targeted agent; toxicant; whole genome

The purpose of this project is to determine the role that mitochondria have in the regulation of enzymes responsible for maintenance of the epigenome. The role of mitochondria in generating ATP and reactive oxygen species (ROS) is well recognized. However, less appreciated is the fact that these organelles are also involved in various biochemical pathways in the cells that give rise to a diverse range of metabolic products, including co-factors of proteins that epigenetically regulate the nuclear genome. For instance, mitochondria participate in the metabolism of S-adenosyl-methionine (SAM), which is the substrate used by DNA and histone methyltransferases to methylate CpG dinucleotides and histones, respectively, in the nucleus. Likewise, the production of acetyl-CoA and NAD+ occurs primarily in mitochondria, and these are co-factors of histone acetyltransferases (HATs) and deacetylases (HDACs), respectively, to modify histones. ATP is used by various protein kinases to phosphorylate substrates, including histones, which can change the composition of nucleosomes. Alpha-ketoglutarate, a metabolite from the tricarboxylic acid (TCA) cycle is a co-factor for the Ten-Eleven Translocation (TET) family of hydroxylases involved in hydroxyl-methylation of cytosines. Finally, mitochondrial-generated ROS can inhibit the jumonji (Jmj) demethylases leading to global histone hypermethylation. As modulation of the epigenome regulates gene expression, it follows that environmental agents that target the mitochondria may alter the regulation of gene expression by changing mitochondrial metabolism. Existing evidence indicates that mitochondrial dysfunction can lead to altered DNA methylation patterns in nuclear DNA and hyper-methylation of histones. Mitochondrial impairment can also affect gene expression. However, it still needs to be established whether epigenetic-driven changes in gene expression in the nucleus are a consequence of environmentally-mediated changes in mitochondrial function. In order to determine whether environmental agents that target mitochondria also impart their effects through alteration of the epigenome and gene expression, we exposed a mouse model to a pesticide, rotenone, which is a well-studied mitochondrial toxicant. Specifically, we used the viable yellow agouti mouse (Avy), a powerful epigenetics model that reports on the DNA methylation status of a mutant agouti locus based on the coat color of the animals. When the promoter in the Avy allele is methylated, the animals have a normal agouti coat color (called pseudoagouti). On the other extreme, when the promoter is unmethylated, the coat color of the animals is completely yellow. We found that the offspring of dams exposed to rotenone throughout pregnancy and lactation have an increased frequency of yellow animals, which is indicative of demethylation of the locus. We went ahead and extended this analysis to the liver using whole genome bisulfide sequencing, and found that perinatal rotenone exposure altered the DNA methylation status of thousands of loci throughout the life of the animals. In parallel, we found that gene expression was also altered by this exposure, with some genes having altered expression 6, 12 and even 18 months after rotenone exposure ceased. By employing several approaches, we have been able to establish a strong correlation between differential methylation and changes in gene expression. Finally, we found mitochondrial complex I and II dysfunction as well as impaired antioxidant activities in animals at 12 months of age although we did not identify any pathology in the livers. Collectively, these results show that developmental mitochondrial dysfunction results in changes in the nuclear methylome, including the Avy locus, in a way that remodels the normal aging DNA methylome of the liver. These changes are not only accompanied by altered gene expression profiles but also facilitate mitochondrial dysfunction later in life. These data raise fundamental questions about the long-term impact of changes in mitochondrial metabolism to health and disease, including those induced by environmental exposures. Having confirmed that mitochondrial dysfunction can impact the epigenome in vivo, we have gone back to some in vitro cell culture models in which specific mutations of mitochondrial genes have been engineered. As these mutations affect distinct biochemical processes within the organelle, we aim to better define the extent to which these specific types of mitochondrial dysfunction impact the epigenome.

这个项目的目的是确定线粒体在负责维持表观基因组的酶的调节中所起的作用。线粒体在产生三磷酸腺苷和活性氧(ROS)中的作用是公认的。然而，人们不太了解的是，这些细胞器还参与了细胞中的各种生化途径，这些途径产生了一系列不同的代谢产物，包括对核基因组进行表观遗传调节的蛋白质的辅助因子。例如，线粒体参与S-腺苷-蛋氨酸的代谢，组蛋白甲基转移酶和脱氧核糖核酸甲基转移酶使用该底物分别甲基化细胞核中的CpG二核苷酸和组蛋白。同样，乙酰辅酶A和NAD+的产生主要发生在线粒体中，它们分别是组蛋白乙酰转移酶(HATS)和脱乙酰基酶(HDACs)的辅助因子，以修饰组蛋白。ATP被各种蛋白激酶用来磷酸化底物，包括组蛋白，这可以改变核小体的组成。α-酮戊二酸是三羧酸(TCA)循环的代谢物，是参与胞嘧啶羟甲基化的Ten-Eleven易位(Tet)羟基酶家族的辅助因子。最后，线粒体产生的ROS可以抑制JMJ(JMJ)去甲基酶，从而导致组蛋白的全局性高甲基化。 由于表观基因组的调节调节基因的表达，因此以线粒体为靶标的环境因子可能通过改变线粒体代谢来改变基因表达的调节。现有证据表明，线粒体功能障碍可导致核DNA甲基化模式的改变和组蛋白的高甲基化。线粒体损伤也会影响基因表达。然而，表观遗传驱动的核基因表达变化是否是环境介导的线粒体功能变化的结果仍需确定。 为了确定针对线粒体的环境因子是否也通过改变表观基因组和基因表达来传递它们的影响，我们将一种杀虫剂鱼藤酮暴露于小鼠模型，鱼藤酮是一种研究得很好的线粒体毒物。具体地说，我们使用了存活的黄色刺鼠(Avy)，这是一个强大的表观遗传学模型，根据动物的毛色报告突变的刺鼠基因座的DNA甲基化状态。当Avy等位基因中的启动子甲基化时，动物会有正常的刺鼠毛色(称为假刺鼠)。在另一个极端，当启动子没有甲基化时，动物的毛色完全是黄色的。我们发现，在整个怀孕和哺乳期暴露于鱼藤酮的母鸡的后代出现黄色动物的频率增加，这表明该基因座去甲基化。我们继续使用全基因组二硫化物测序将这一分析扩展到肝脏，发现围产期接触鱼藤酮会改变动物一生中数千个基因座的DNA甲基化状态。与此同时，我们发现这种暴露也改变了基因表达，一些基因在鱼藤酮暴露停止后6个月、12个月甚至18个月改变了表达。通过采用几种方法，我们已经能够在差异甲基化和基因表达变化之间建立强烈的相关性。最后，我们在12个月大的动物中发现线粒体复合体I和II功能障碍以及抗氧化剂活性受损，尽管我们没有在肝脏中发现任何病理。总而言之，这些结果表明，发育中的线粒体功能障碍会导致包括Avy基因在内的核甲基组的变化，从而重塑肝脏正常老化的DNA甲基组。这些变化不仅伴随着基因表达谱的改变，而且还有助于线粒体在以后的生活中功能障碍。这些数据提出了线粒体新陈代谢变化对健康和疾病的长期影响的根本问题，包括环境暴露引起的影响。 在确认了线粒体功能障碍可以影响体内的表观基因组后，我们回到了一些体外细胞培养模型中，在这些模型中已经设计了线粒体基因的特定突变。由于这些突变影响细胞器内不同的生化过程，我们的目标是更好地定义这些特定类型的线粒体功能障碍对表观基因组的影响程度。