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 和普遍的老龄化干预。因为DR也延缓了大多数与年龄相关的 除了保持健康寿命外，人们普遍认为DR通过延迟/延缓来延长寿命 衰老。然而，我们仍然不清楚(S)博士抗衰老作用的重要机制 灾难恢复在很大程度上被忽视的一个方面是，在生命早期实施灾难恢复可以延长寿命 即使在啮齿动物的余生中随意喂食(AL)，也是如此。这提供了令人信服的 DR通过涉及分子信号的机制起作用的证据(S)，该信号在 DR的实施，并对动物的生命周期产生影响，即使在DR停止之后，例如 表观遗传机制，如DNA甲基化。从功能上讲，DNA甲基化(5mC)可以调节染色质 转录因子和其他DNA结合蛋白进入DNA并调节的状态和影响能力 基因表达，从而改变细胞和组织的功能。包括我们的实验室在内的几个组织， 已经表明DR改变了小鼠组织中的DNA甲基化，逆转了DNA的许多变化 随着年龄而发生的甲基化，以及引起不随年龄变化的DNA甲基化的变化。 最近我们发现短期DR在启动子中肠黏膜DNA甲基化发生改变 NTS-1基因。DNA甲基化的改变与NTS1表达的增加密切相关， 当DR小鼠被随意喂食几个月后，这种情况仍然存在。因为上皮组织中的细胞 肠粘膜每4至5天持续更新一次，我们观察到DNA甲基化的变化 肠粘膜最有可能起源于肠道干细胞。因此，我们假设DR会引起变化 在肠道干细胞中特定基因组位置的DNA甲基化，导致分子记忆，这 这可能会导致肠道干细胞功能中重要的基因表达发生变化。在……里面 在这个项目中，我们将测量DNA甲基化(5mC)和羟甲基化(5hmC)的变化 使用我们团队开发的一种新的方法在肠道干细胞中进行DR，该方法使我们能够准确地测量 在单碱基分辨率下，基因组中约3000万个特定位置的5mC和5hmC的分辨率都发生了变化。在此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DNA甲基化和跨基因调控区羟甲基化的影响 从三组小鼠的肠道分离的干细胞基因组的比较：饲喂AL的小鼠，饲喂DR的小鼠 3个月，DR小鼠饲喂DR 4个月，然后饲喂AL 6个月。 目的2：确定DNA甲基化/羟甲基化变化可能引起的转录变化 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.