Epigenomic Regulation of Gene Expression in Diet Induced Obesity

描述(申请人提供)：肥胖及其相关疾病，包括2型糖尿病、代谢综合征和心血管疾病，在美国已经达到流行水平。虽然遗传易感性为肥胖和2型糖尿病的获得性代谢缺陷的表达提供了背景，但这些代谢缺陷中的大多数只有在能量失衡，即能量供应过剩和/或能量消耗减少时才会变得明显。因此，基因-环境的相互作用在这些疾病的发展中具有重要意义，越来越多的证据表明，这些影响是在表观遗传学水平上通过生物学手段介导的，表观遗传学是一系列对基因表达的基本上不依赖于序列的调控影响，包括DNA甲基化。表观遗传学对表型的影响最好的例证可能是最近的研究表明，宫内环境，无论是营养不良还是营养过剩，通过基因的差异甲基化影响基因表达，导致代谢表型改变，从而增加后代肥胖和肥胖相关疾病的风险。然而，人们对成年后能量代谢和肥胖的表观遗传调控的可能性知之甚少。一种有趣的可能性是，长期暴露在易使人肥胖和糖尿病的环境中，如高脂肪的西方饮食和久坐不动的生活方式，会导致DNA甲基化改变，代谢活跃组织的表型改变。肝脏是新陈代谢调节的中心，由于它在解剖上靠近肠道，是大多数摄取营养物质的关键分配者，是测试营养过剩引起的表观遗传学变化的理想候选者。我们建议检验这一假设，即成年后肥胖的发展改变了肝脏中的基因表达和能量代谢，至少部分是通过DNA甲基化的变化。目标1是对暴露于高脂肪饮食后的肝脏DNA甲基组进行全基因组定量分析，这将使用甲基敏感切割计数(MSCC)来完成。目标2是对暴露在高脂肪饮食中的肝脏转录组进行全基因组定量分析，这将使用全基因组5‘固体鼠尾草来完成。目的3是确定DNA甲基化和基因表达之间的机制关系，这将使用最先进的计算方法来识别差异甲基化和表达的基因，然后选择的基因将通过qRT-PCR和单基因甲基化分析进行验证。最后，转录因子募集和转录速率之间的机制关系将通过染色质免疫沉淀和核跑动来确定。该项目有可能提供有关肥胖表观遗传学的突破性信息。 公共卫生相关性：肥胖及其相关疾病、代谢综合征和心血管疾病往往具有潜在的遗传成分，只有在营养过剩和/或久坐不动的生活方式下才会显现出来。这表明环境在肥胖的发展中起着重要的作用，从生物学上讲，这可能表现为一种表观遗传现象；即，遗传受到DNA序列以外的水平的影响。我们建议检验这一假设，即成年后肥胖的发展改变了肝脏中的基因表达和能量代谢，至少部分是通过DNA甲基化的变化。在高脂肪饮食后，将在整个基因组水平和高分辨率下测量DNA甲基化和基因表达。使用最先进的生物信息学方法和复杂的分子分析，然后我们将识别和验证在高脂肪饮食后甲基化和表达发生变化的单个基因，并将确定DNA甲基化如何改变转录因子的招募和转录速度。这个项目有可能提供有关环境(在这种情况下是高脂肪饮食)如何影响肥胖遗传的开创性信息。

DESCRIPTION (provided by applicant): Obesity and its related disorders, including type 2 diabetes mellitus, metabolic syndrome, and cardiovascular disease, have reached epidemic levels in the United States. While genetic predisposition provides a background for the expression of acquired metabolic defects in obesity and type 2 diabetes, the majority of these metabolic defects become apparent only through energy imbalance, that is, energy oversupply and/or decreased energy expenditure. Therefore, gene- environment interactions are significant in the development of these diseases and there is increasing evidence that these effects are mediated biologically at the level of epigenetics, a spectrum of largely sequence-independent regulatory influences on gene expression including DNA methylation. Epigenetic influences on phenotype are perhaps best exemplified by recent studies demonstrating that the intrauterine environment, either malnutrition or overnutrition, influences gene expression through differential methylation of genes that lead to an altered metabolic phenotype that increases the risk for obesity and obesity-related disorders in the offspring. However, very little is known about the potential for epigenetic regulation of energy metabolism and obesity in adult life. An intriguing possibility is that chronic exposure to an environment that predisposes people to obesity and diabetes, such as a high fat Western diet and sedentary lifestyle, leads to altered DNA methylation and altered phenotype of metabolically active tissues. The liver is central to metabolic regulation and, as the key distributor of most ingested nutrients due to its anatomical proximity to the gut, is an ideal candidate for testing epigenetic changes from overnutrition. We propose to test the hypothesis that the development of obesity in adulthood alters gene expression and energy metabolism in the liver at least partially through changes in DNA methylation. Aim 1 is perform a quantitative genome-wide analysis of the hepatic DNA methylome following exposure to a high fat diet, which will be accomplished using Methyl-Sensitive Cut Counting (MSCC). Aim 2 is to perform a quantitative genome-wide analysis of the hepatic transcriptome following exposure to a high-fat diet, which will be accomplished using genome-wide 5' SOLID-SAGE. Aim 3 is to determine mechanistic relationships between DNA methylation and gene expression, which will be accomplished using state of the art computational approaches to identify genes that are both differentially methylated and expressed, and then selected genes will be validated with qRT-PCR and single gene methylation analysis. Finally mechanistic relationships between transcription factor recruitment and transcriptional rate will be determined by chromatin immunoprecipitation and nuclear run-on. This project has the potential to provide groundbreaking information concerning the epigenetics of obesity. PUBLIC HEALTH RELEVANCE: Obesity and its related disorders, metabolic syndrome and cardiovascular disease, often have an underlying genetic component that only becomes apparent after exposure to overnutrition and/or sedentary lifestyle. This suggests that the environment plays an important role in the development of obesity, and biologically this may manifest as an epigenetic phenomenon; i.e., genetics affected at a level other than the sequence of the DNA. We propose to test the hypothesis that the development of obesity in adulthood alters gene expression and energy metabolism in the liver at least partially through changes in DNA methylation. Both DNA methylation and gene expression will be measured after a high fat diet at the level of the entire genome and at high resolution. Using state-of the art bioinformatic approaches and sophisticated molecular assays, we will then identify and validate individual genes that have changes in both methylation and expression after a high fat diet, and will determine how DNA methylation alters the recruitment of transcription factors and the rate of transcription. This project has the potential to provide groundbreaking information concerning how the environment (in this case a high fat diet) affects the genetics of obesity.