Nucleosome architecture in aging and nuclear receptor activation in the liver

描述(由申请者提供)：我的长期目标是成为一名独立的多学科研究员，应用实验和计算方法来研究代谢功能障碍潜在的年龄相关性染色质变化。我正在努力争取在一家领先的研究机构获得一个终身教职的职位，这样我就可以把我的时间花在基础研究和实验室学生和研究员的培训上。在我的博士工作期间，我利用功能基因组学方法研究了肝脏中的转录调控网络，发现有翼螺旋转录因子Foxa2在胆汁酸代谢中发挥着重要作用。在完成我的博士学位后，我决定追随科学，由于我享有创造力和经济自由，我能够从事我在凯斯特纳博士的实验室开发的项目。作为凯斯特纳实验室的博士后研究员，我在一只只在肝脏发生基因缺失的小鼠模型中发现了一种新的年龄相关性肥胖表型，强调了肝脏在发育中的重要性。 肥胖。老年小鼠肝脏中Foxa2的丢失导致过早衰老表型，因为Foxa2缺陷小鼠表现出食物摄入量减少、能量消耗减少和甘油三酯积累。FOXA2拮抗mTOR信号，导致LXR�的激活和脂肪生成的增加。这一发现激起了我对年龄相关新陈代谢变化的兴趣。为了成为一名富有成效的多学科研究人员，既要知道如何恰当地进行实验以生成数据，又要知道如何恰当地分析数据。我来到布罗德研究所的雷格夫实验室，是为了补充我的实验背景，并通过培训我的计算技能来分析大型数据集和基因组数据建模。我目前正在分析我在不同年龄小鼠的肝脏中进行的MNase-seq和RNA-seq实验的数据(目标1)。我们预计在秋季完成分析并提交论文。雷格夫博士支持我在她的实验室继续新陈代谢研究的决定。虽然我的主要导师雷格夫博士是一名计算生物学家，在表观遗传调控、基因组学方法和计算分析方面拥有丰富的经验，但我的联合顾问是一个多学科委员会，成员包括控制肝脏基因表达的转录和表观遗传网络专家韦克斯曼博士、肝脏老化和年龄相关染色质变化领域的专家季姆琴科博士、核受体和脂质代谢领域的领军人物摩尔博士，以及在表观遗传学和人类肥胖方面有经验的临床医生罗森博士。在他们的指导下，作为一名独立的研究者，我将建立一个必要的基础，以探索年龄依赖性染色质变化与代谢功能障碍之间的关系。表观遗传变化是肝脏和其他组织中年龄相关性损伤的关键介质，这表明可能有共同的机制负责。我推测，核小体结构随年龄的变化是衰老肝脏这一机制的关键部分。为了验证这一假设，我将在目标1中检测从三个不同年龄的小鼠分离的肝脏中全基因组核小体的定位和占有率。接下来，我将在目标2中检验组蛋白修饰随年龄的变化。整合不同年龄的核小体占有率、染色质状态和基因表达应该收敛于调节因子和年龄相关功能障碍的分子机制。对年龄相关代谢表型调节剂的无偏见搜索将得到候选方法的补充。目前关于核受体的芯片序列数据已经挑战了核受体依赖的基因调控的经典模型。然而，全基因组定位分析表明，LXR�在肝脏中的结合在很大程度上是配体依赖的，并且激动剂使LXR�占据较少的可及部位。这些观察让我假设，染色质结构在核受体结合和代谢基因调控中发挥的作用比之前认识的更大。由于受LxR�调控的不受控制的肝脏脂肪生成可以促进年龄相关性肥胖的发展，而且线虫中的DAF-12和LxR同系物在衰老过程中介导了对环境条件的反应，因此理解激活的完整机制以及低和高亲和力LxR�结合位点对基因调控的贡献(Aim 3)至关重要，这可以推断到其他核受体及其代谢靶点。在这个应用中，我假设染色质的结构随着年龄的增长而变化，并在核受体结合和代谢基因调控中发挥比以前认识到的更大的作用，并提出了一系列实验和分析来验证这些想法。最终目标是确定表观遗传变化如何导致转录因子结合调节基因调控的变化，这些基因调控与年龄相关的代谢表型有关。这一无偏见的研究的目的是寻找可以被操纵来治疗年龄依赖性代谢功能障碍的候选调节剂，并辅之以候选方法。在K01奖项的支持下，将表观遗传学方面的培训和大规模基因组数据的分析与我在代谢研究、衰老、转录调控和基因调控网络方面的背景相结合，使我唯一有资格探索依赖年龄的染色质变化和代谢失调之间的关系。

DESCRIPTION (provided by applicant): My long-term goal is to become an independent multidisciplinary investigator, applying both experimental and computational approaches to age-dependent chromatin changes underlying metabolic dysfunction. I am working towards obtaining a tenure-track faculty position at a leading research institution, so that I can devote m time to basic research and training of students and fellows in my laboratory. During the course of my doctoral work, I utilized functional genomic approaches to investigate transcriptional regulatory networks in the liver and discovered that winged-helix transcription factor Foxa2 plays an important role in bile acid metabolism. After completing my doctoral degree I decided to follow the science and, due to creative and financial freedom I enjoyed, was able to pursue projects that I developed in Dr. Kaestner's laboratory. As a postdoctoral fellow in the Kaestner lab, I discovered a novel age-onset obesity phenotype in a mouse model where genetic deletion occurs only in the liver, underscoring the importance of the role the liver plays in development of obesity. Loss of Foxa2 in the liver of older mice results in a premature aging phenotype, as Foxa2-deficient mice exhibit reduced food intake, decreased energy expenditure, and triglyceride accumulation. Foxa2 antagonizes mTOR signaling, leading to activation of LXR� and increased lipogenesis. This finding piqued my interest in age-dependent metabolic changes. In order to become a productive multidisciplinary researcher, it is essential to know both how to perform experiments properly to generate the data and how to analyze the data appropriately. I came to Regev lab at the Broad Institute to complement my experimental background and build upon my computational skills with training in analysis of large data sets and modeling of genomic data. I am currently analyzing the data from MNase-seq and RNA-seq experiments I performed in livers isolated from mice of different ages (Aim 1). We are expecting to finish the analysis and submit the paper in the fall. Dr. Regev has supported my decision to continue metabolic research in her laboratory. While my primary mentor Dr. Regev is a computational biologist with extensive experience in epigenetic regulation, genomics methods and computational analysis, I am co-advised by a multidisciplinary committee that includes Dr. Waxman, an expert in transcriptional and epigenetic networks that govern hepatic gene expression, Dr. Timchenko, an expert in the field of aging liver and age-associated chromatin changes, Dr. Moore, a leader in the field of nuclear receptors and lipid metabolism, and Dr. Rosen, a clinician with experience in epigenetics and human obesity. With their guidance, I will build a foundation necessary to pursue the relationship between age-dependent chromatin changes and metabolic dysfunction as an independent investigator. Epigenetic changes act as crucial mediators of age-dependent impairments in the liver and other tissues, suggesting a common mechanism could be responsible. I hypothesize that nucleosome architecture changes with aging are a key part of this mechanism in the aging liver. To test this hypothesis, I will examine genome-wide nucleosome positioning and occupancy in livers isolated from mice of three different ages in Aim 1. Next, I will examine changes in histone modifications with age in Aim 2. Integrating nucleosome occupancy, chromatin state, and gene expression at different ages should converge on regulators and a molecular mechanism for age-associated dysfunction. The unbiased search for regulators of age-dependent metabolic phenotypes will be complemented by a candidate approach. Current ChIP-Seq data for nuclear receptors have challenged the classical model of nuclear-receptor-dependent gene regulation. However, the genome-wide location analysis shows that LXR� binding in the liver is largely ligand-dependent and that the agonist enables LXR� to occupy less accessible sites. These observations led me to hypothesize that chromatin architecture plays a larger role in nuclear receptor binding and metabolic gene regulation than previously appreciated. Since uncontrolled hepatic lipogenesis, regulated by LXR�, can contribute to development of age-dependent obesity and DAF-12, LXR homolog in C.elegans, mediates responses to environmental conditions during aging, it is crucial to understand the complete mechanism of activation and the contribution of low and high affinity LXR� binding sites to gene regulation (Aim 3), which can be extrapolated to other nuclear receptors and their metabolic targets. In this application, I hypothesize that chromatin architecture changes with aging and plays a larger role in nuclear receptor binding and metabolic gene regulation than previously appreciated and propose a series of experiments and analyses to test these ideas. The ultimate goal is to determine how epigenetic changes lead to changes in transcription factor binding modulating gene regulation responsible for age-dependent metabolic phenotypes. The object of this unbiased search, which is complemented by a candidate approach, is to find candidate regulators that can be manipulated to treat age-dependent metabolic dysfunction. Integrating the training in epigenetics and analysis of large-scale genomic data, supported by this K01 award, with my background in metabolic research, aging, transcriptional regulation, and gene regulatory networks makes me uniquely qualified to pursue the relationship between age-dependent chromatin changes and metabolic dysregulation.