Does Dietary Restriction Alter Stem Cell Function Through An Epigenetic Mechanism?

在所有已被证明可以延长寿命的干预措施中，饮食限制（DR）是研究最多的 和普遍的衰老干预。由于DR也延迟了大多数年龄相关性疾病的发作和进展， 疾病以及维持健康，人们普遍认为DR通过延迟/延缓 衰老然而，我们仍然不了解DR抗衰老作用的机制。 DR在很大程度上被忽视的一个方面是，在生命早期实施DR可以延长寿命 在他们的生活中，他们的生活是自由的。这提供了令人信服的 证据表明DR通过一种机制起作用，该机制涉及在DR发生后不久出现的分子信号。 实施DR，并在动物的整个生命周期内对其产生影响，即使在DR停止后，例如，一个 表观遗传机制，如DNA甲基化。在功能上，DNA甲基化（5mC）可以调节染色质 状态和影响转录因子和其他DNA结合蛋白进入DNA和调节 基因表达，从而改变细胞和组织的功能。包括我们实验室在内的几个小组， 已经表明，DR改变了小鼠组织中的DNA甲基化，逆转了DNA中的许多变化， 甲基化，以及诱导不随年龄变化的DNA甲基化的变化。 最近，我们发现短期DR可诱导肠粘膜DNA甲基化的改变， Nts 1基因。DNA甲基化的变化与Nts 1表达的增加密切相关， 当DR小鼠被随意喂养几个月时，这种情况持续存在。因为上皮细胞 肠粘膜每4 - 5天持续更新一次，我们观察到的DNA甲基化变化， 肠粘膜最可能产生于肠干细胞。因此，我们假设DR引起的变化 在肠道干细胞中特定基因组位点的DNA甲基化，导致分子记忆， 可能导致肠道干细胞功能中重要基因表达的改变。在 在本项目中，我们将测量DNA甲基化（5mC）和羟甲基化（5hmC）的变化， 使用我们小组开发的一种新的检测方法在肠道干细胞中进行DR，这使我们能够准确地测量 在基因组中约3000万个特定位点处的5mC和5hmC的单碱基分辨率变化。在R21中 格兰特，我们将在以下具体目标中检验我们的假设。 目的1：确定DR对基因调控区DNA甲基化和羟甲基化的影响 从三组小鼠的肠道中分离的干细胞的基因组：喂食AL的小鼠，喂食DR的小鼠， 个月，DR小鼠喂食DR 4个月，然后喂食AL 6个月。 目的2：识别可能由DNA甲基化/羟甲基化变化引起的转录变化 并在肠道干细胞功能中发挥作用。

Of all the interventions that have been shown to increase lifespan, dietary restriction (DR) is the most studied and universal aging intervention. Because DR also delays the onset and progression of most age-related diseases as well as maintaining healthspan, it is well accepted that DR increases lifespan by delaying/retarding aging. However, we still do not understand the mechanism(s) responsible for the anti-aging effect of DR. An important aspect of DR that has been largely overlooked is that when implemented early in life DR can increase lifespan of rodents even when rodents are fed ad libitum (AL) the remainder of their life. This provides compelling evidence that DR acts through a mechanism that involves a molecular signal(s) that arises shortly after the implementation of DR and has an impact on the animal over its lifespan, even after DR is discontinued, e.g., an epigenetic mechanism, such as DNA methylation. Functionally, DNA methylation (5mC) can regulate chromatin status and affect the ability of transcription factors and other DNA binding proteins to access DNA and regulate gene expression, thereby altering the function of cells and tissues. Several groups, including our laboratories, have shown that DR alters DNA methylation in tissues of mice, reversing many of the changes in DNA methylation that occur with age as well as inducing changes in DNA methylation that do not change with age. Recently we found that short-term DR induces changes in DNA methylation in intestinal mucosa in the promoter of the Nts 1 gene. The changes in DNA methylation are closely associated with increased expression of Nts 1, which persist when the DR mice are then fed ad libitum for several months. Because the epithelial cells in the intestinal mucosa are continuously renewed every 4 to 5 days, the changes in DNA methylation we observed in intestinal mucosa most likely arise in intestinal stem cells. Therefore, we hypothesize that DR induces changes in DNA methylation in intestinal stem cells at specific genomic sites that results in a molecular memory, which potentially leads to alterations in the expression of genes that are important in intestinal stem cell function. In this project, we will measure changes in DNA methylation (5mC) and hydroxymethylation (5hmC) induced by DR in intestinal stem cells using a novel assay developed by our group, which allows us to measure accurately at single base resolution changes in both 5mC and 5hmC at ~ 30 million specific sites in the genome. In this R21 grant, we will test our hypothesis in the following specific aims. Aim 1: Determine the effect of DR on DNA methylation and hydroxymethylation across gene regulatory regions of the genome of stem cells isolated from the intestines of three groups of mice: mice fed AL, mice fed DR for 4 months, and DR mice fed DR for 4 months and then fed AL for 6 months. Aim 2: Identify changes in transcription that could arise from changes in DNA methylation/hydroxymethylation and play a role in intestinal stem cell function.