Testing the role of chromatin in healthspan

描述(申请人提供)：自从发现组蛋白去乙酰基酶SIRT1以来，改变染色质的结构对寿命有好处已经变得很清楚了。考虑到有许多蛋白质被赋予染色质重塑活性(组蛋白乙酰转移酶(HATS)、经典的组蛋白去乙酰基酶(HDAC)、组蛋白甲基转移酶、组蛋白去甲基酶等)，很可能至少其中一些对寿命和健康都有有益或有害的影响。不幸的是，击倒这些蛋白质中的任何一种都会导致胚胎死亡。为了克服这一负面结果，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。为了实现这一目标，我们培育了一只表达p300(CH3p300)CH3结构域的转基因小鼠。之所以选择这个结构域，是因为它被大量的转录因子结合，也被HDAC1结合，并作为显性负值发挥作用。这些动物出生时都是正常的，无论是在年轻时还是在老年(20个月大)时都没有明显的表型变化。然而，当受到高脂肪饮食的挑战时，CH3p300动物积累的脂肪比野生型小鼠少30%。我们假设CH3p300的表达通过调节代谢转录组来减少与高脂肪饮食相关的有害后果。利用我们的创新小鼠模型，我们建议解决以下目标：具体目标1：确定CH3p300转基因小鼠是否表现出与抵抗高脂诱导的肥胖和肝脏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组织芯片测序来绘制染色质修饰或“标记”的全球变化图，并使用选定目标的芯片qPCR来验证这些数据。特异性AIM2：确定全球染色质标记对基因转录的影响。我们将生成CH3p300和喂高脂饮食的野生型小鼠肝脏的基因表达谱。这些数据将与AIM 1中生成的CHIP-SEQ数据进行比较。建议的研究对老龄化领域的影响：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在未来设计新的治疗方法来改善老年人的健康寿命，因为HAT、HDAC和甲基转移酶抑制剂已经在对几种疾病进行临床试验。 公共卫生相关性：保持健康寿命到老年是人类的目标，但几乎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。当受到高脂肪饮食的挑战时，转基因小鼠比野生型小鼠积累的体重和体脂更少。这项建议的目的是确定CH3p300的表达导致的染色质变化是否在高脂肪饮食诱导的代谢应激下保持健康的寿命。

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.