Regulation of DNA Methylation by a Major Folate Enzyme

描述(申请人提供)：叶酸衍生物参与对细胞内过程至关重要的单碳转移反应。叶酸代谢障碍可导致多种异源疾病，如癌症和糖尿病并发症。叶酸状态的变化通常与DNA甲基化的变化有关，DNA甲基化是一种有丝分裂遗传的修饰，可以导致基因表达的显著变化，并进一步促进疾病。10-甲酰基四氢叶酸脱氢酶(FDH)是最丰富的叶酸酶之一，它催化10-甲酰基四氢叶酸在NADP的依赖下转化为四氢叶酸和二氧化碳。有两个观察结果促使我们提出了这项研究建议：(I)外劳直接影响细胞内S-腺苷蛋氨酸的水平，腺苷甲硫氨酸是DNA甲基转移酶的主要底物，(Ii)外佣需要用4‘-磷酸丙氨酸假体基团修饰才能具有催化活性。我们假设，FDH通过控制叶酸库中碳基的可用性来调节DNA甲基化。我们进一步建议，催化活性的磷酸化外佣是完成这一调节功能所必需的。我们将通过以下具体目标来检验和探索这些假说：(1)确定FDH表达对基因组DNA甲基化的影响。(2)研究FDH对细胞内叶酸库和DNA甲基化的影响是否需要对其进行磷酸化。(3)应用基因芯片技术评价外佣对基因表达的影响。细胞培养技术、酶活性分析、免疫化学方法、分子生物学、siRNA蛋白质沉默、测量细胞内叶酸库的分析、高压液相色谱和微阵列技术都将被用于实现所描述的目标。这个项目将提供对叶酸库如何影响DNA甲基化状态的洞察。这一特殊过程可能与表观遗传性疾病状态的发生有关，特别是在与叶酸缺乏和糖尿病相关的病理中。了解FDH的新陈代谢可能提供能够调整细胞甲基化状态的药物疗法。 公共卫生相关性：与维生素B9(叶酸)相关的蛋白质的基因突变与糖尿病并发症有关。这个项目将描述与叶酸相关的代谢过程，这可能为预防或治疗糖尿病疾病提供洞察力。

DESCRIPTION (provided by applicant): Folate derivatives participate in single-carbon transfer reactions that are vital to intracellular processes. Disruptions of folate metabolism can lead to numerous heterologous diseases, such as cancer and diabetic complications. Changes in folate status are often associated with alterations in DNA methylation, a mitotically heritable modification that can lead to significant changes in gene expression and further promote disease. One of the most abundant folate enzymes, 10-formyltetrahydrofolate dehydrogenase (FDH), catalyzes the NADP-dependent conversion of 10-formyltetrahydrofolate to tetrahydrofolate and carbon dioxide. Two observations have prompted this research proposal: (i) that FDH has a direct effect on intracellular levels of S- adenosyl methionine, the major substrate for DNA methyltransferases, and (ii) that FDH requires modification with a 4'-phosphopantetheine prosthetic group for its catalytic activity. We hypothesize that FDH regulates DNA methylation by controlling availability of carbon groups in the folate pool. We further suggest that catalytically active, phosphopantetheinylated FDH is required to fulfill this regulatory function. We will test and explore these hypotheses through the following specific aims: (1) Determine the effect of FDH expression on genomic DNA methylation. (2) Investigate whether phosphopantetheinylation of FDH is required for its effects on intracellular folate pools and DNA methylation. (3) Evaluate the effect of FDH on gene expression using a microarray approach. Cell culture techniques, enzyme activity assays, immunochemical methods, molecular biology, protein silencing by siRNA, assays to measure intracellular folate pools, high-pressure liquid chromatography, and microarray technologies will all be used to achieve the described aims. This project will offer insight into how folate pools affect DNA methylation status. This particular process may have a relationship to the occurrence of epigenetic disease states, particularly in pathologies associated with folate deficiency and diabetes. Knowledge of FDH metabolism may provide pharmacological therapies capable of adjusting cellular methylation status. PUBLIC HEALTH RELEVANCE: Genetic mutations of proteins related to the vitamin B9 (folate), are associated with diabetic complications. This project will characterize a metabolic process related to folic acid that may provide insight into the prevention or treatment of diabetic disease.