Testing the role of chromatin in healthspan

描述（由申请人提供）：自从发现组蛋白脱乙酰酶SIRT 1以来，已经清楚的是，改变染色质的结构对寿命具有有益的影响。考虑到有许多蛋白质被赋予染色质重塑活性（组蛋白乙酰基转移酶（HAT）、经典组蛋白脱乙酰基酶（HDAC）、组蛋白甲基转移酶、组蛋白脱甲基酶等），它们中的至少一些可能不仅在寿命上而且在健康寿命上具有有益或有害的影响。不幸的是，敲除这些蛋白质中的任何一种都会导致胚胎死亡。为了克服这种负面结果，我们决定调整染色质的结构，以询问这种改变是否可以用于健康寿命的益处。为了实现这一目标，我们产生了一个转基因小鼠表达的CH3结构域的p300（CH3p300）。选择该结构域是因为它被大量转录因子以及HDAC 1结合，并且作为显性负性发挥作用。这些动物出生时正常，在幼年或老年（20月龄）时均无显著的表型变化。然而，当用高脂肪饮食攻击时，与野生型同窝仔相比，CH3p300动物积累的脂肪少30%。我们假设CH3p300的表达通过调节代谢转录组减少了与高脂饮食相关的有害结果。使用我们的创新小鼠模型，我们提出解决以下目标：具体目标1：确定CH3p300转基因小鼠是否显示与抵抗高脂肪诱导的肥胖和肝脂肪变性相关的染色质特征。我们将使用CH3p300和喂食正常食物或高脂肪饮食的野生型小鼠的肝脏组织的ChIP测序来绘制染色质修饰或"标记"的全局变化，并使用选定靶标的ChIP-qPCR来验证这些数据。具体目标2：确定全局染色质标记对基因转录的影响。我们将从CH3p300和喂食高脂肪饮食的野生型小鼠的肝脏中产生基因表达谱。将这些数据与目标1中生成的ChIP-Seq数据进行比较。这些研究对衰老领域的影响：如果我们的假设得到证实，从我们的实验中获得的知识可能有助于设计未来改善老年人健康状况的新治疗方法，因为HAT，HDAC和甲基转移酶抑制剂已经在临床试验中用于多种疾病。 公共卫生相关性：维持健康直到老年是人类的目标，但实际上没有动物模型来研究它。我们已经产生了表达p300组蛋白乙酰转移酶CH3结构域的转基因小鼠。当用高脂肪饮食攻击时，转基因小鼠比野生型同窝出生的小鼠积累更少的体重和更少的体脂。本提案的目的是确定CH3p300表达引起的染色质改变是否在高脂饮食诱导的代谢应激下维持健康的寿命。

DESCRIPTION (provided by applicant): Since the discovery of the histone deacetylases SIRT1, it has become clear that modifying the structure of chromatin has a beneficial effect in lifespan. Considering that there are many proteins endowed with chromatin remodeling activity (histone aceyltransferases (HATs), classical histone deacetylases (HDACs), histone methyltransferases, histone demethylases, and others), it is likely that at least some of them have either beneficial or detrimental effects not only in lifespan but also in healthspan. Unfortunately, knocking down any of these proteins is embryonic lethality. To overcome this negative outcome we decided to tweak the structure of chromatin to ask whether such alterations could be used for healthspan benefits. To achieve this goal we generated a transgenic mouse that expresses the CH3 domain of the p300 (CH3p300). This domain was chosen since it is bound by a large number of transcription factors as well by HDAC1, and functions as a dominant negative. The animals are born normal and do not have significant phenotypic changes either at young age or in old age (20 month old). However, when challenged with a high fat diet, the CH3p300 animals accumulate 30% less fat compared to wild-type littermates. We hypothesize that that expression of CH3p300 diminishes deleterious outcomes associated with a high fat diet by regulating the metabolic transcriptome. Using our innovative mouse model we propose to address the following aims: Specific Aim 1: To determine whether the CH3p300 transgenic mice display chromatin signatures associated with resistance to high fat-induced obesity and hepatic steatosis. We will map global changes in chromatin modifications or "marks" using ChIP-sequencing of liver tissue from CH3p300 and wild-type mice that are fed a normal chow or a high fat diet, and verify these data using ChIP-qPCR of selected targets. Specific Aim2: To determine the impact of the global chromatin marks on gene transcription. We will generate gene expression profiles of livers from CH3p300 and wild-type mice fed a high fat diet. These data will be compared to the ChIP-Seq data generated in Aim 1. Impact of the studies proposed to the field of Aging: If our hypothesis is confirmed, the knowledge obtained from our experiments may help design in the future new therapeutic approaches for improving healthspan in older adults as HAT, HDAC, and methyltransferase inhibitors are already in clinical trials for several diseases. PUBLIC HEALTH RELEVANCE: Maintaining healthspan till old age is a goal of mankind, but there are virtually no animal models to study it. We have generated transgenic mice that express the CH3 domain of the p300 histone acetyltransferase. When challenged with a high fat diet, the transgenic mice accumulate less weight and less body fat than wild type littermates. The goal of this proposal is to determine whether chromatin alterations resulting from expression of CH3p300 maintain a healthy lifespan in spite of the metabolic stress induced by a high fat diet.