Regulation of yeast cellular aging through chromatin and novel pathways

描述(由申请人提供)：萌芽酵母的复制老化，酿酒酵母，已经成为衰老研究的一个非常有用的模型，为衰老提供了基本的遗传学和分子洞察力。染色质生物学的研究也得益于酵母模型，因为染色质的许多分子机制在真核生物中高度保守。这项建议的目标是建立两种筛选方法，这两种方法建立在我和其他人之前的工作基础上，以更好地理解表观遗传学和染色质调节通路如何参与衰老过程。这些筛选的结果有望为衰老研究确定许多新的靶点和染色质调节剂。表观遗传变化，包括组蛋白翻译后修饰，是关键的调控机制，涉及包括衰老在内的所有发育过程。然而，大多数研究和我们以前在这方面的研究只集中在少数已知的sirtuins的组蛋白修饰靶点上。发现sirtuins和其他染色质介导的衰老调节因子的新的组蛋白靶点是非常有意义的。我建议利用现有的组蛋白突变体库来建立一种基于成熟的旧细胞分选方法的新的筛选方法，并筛选能够改变酵母复制寿命的组蛋白突变(目标1)。可用的组蛋白突变体文库是由Jef Boeke的团队开发的。它包含组蛋白H3和H4的系统突变，每个残基都有多个版本的突变。更重要的是，这个文库以综合形式的突变为特色，这是用于衰老研究的关键因素。细胞表面标记和分选方法是由Leonard Guarente的实验室开发的，并在我之前的工作中用于分离108个数量的旧酵母细胞。提出的新的筛选方法建立在这种方法的基础上，并从组蛋白突变菌株池中丰富了旧细胞部分中的长寿命细胞。这种方法允许高通量筛选延长或缩短寿命的突变，而无需对所有突变菌株进行繁琐的寿命测试。筛查的命中结果将通过传统的寿命分析进行验证。酵母缺失文库是酵母菌株的集合，每个酵母菌株都有一个单一的基因缺失，对于定义基因功能，理解多个细胞通路的结构和逻辑，以及确定几种有希望的药物的靶点和作用模式，都是非常有价值的资源。肯尼迪和凯伯莱因的团队最近利用这一集合来识别新的衰老调节因子和途径。然而，开发像Sir2这样更有利于寿命的调控因子已经变得极其困难，因为许多损害关键途径的基因的破坏自然会导致寿命缩短，而且它们可能不直接参与衰老调节。这些长寿命的缺失突变体往往令人困惑，因为许多突变体参与了重要的生物学过程。因此，评估每个基因过度表达的衰老和生物学效应，并与缺失文库的结果进行比较，在识别像sirtuins这样的促进长寿的基因方面变得更加相关和有效。我建议构建一个酵母菌株集合，允许使用高通量方法系统地过度表达酵母基因，然后使用这个集合来发现支持衰老和年龄相关疾病生物学的新机制(目标2)。我将利用现成的酵母基因质粒库FLEX基因(包含5500多个酵母基因)和高效的网关克隆系统来生成具有适当标记的酵母质粒文库，用于整合和选择。大部分工作，包括质粒准备、网关克隆、酵母转化和验证，都将使用我的导师最近购买的最先进的液体处理机器人来执行。一个400个染色质相关基因的初步筛选被提议在这个项目的时间范围内完成特定的目标2。我希望找出直接参与染色质生物学或直接以染色质为靶标的新型衰老调节剂。 与公共健康相关：染色质是真核生物基因组的生理模板，遗传信息在这里存储、复制和表达。作为细胞遗传和表观遗传控制的中心，染色质在多个水平上受到众多途径的广泛调控。该项目将通过系统筛选来研究这些途径及其染色质靶点，识别新的衰老调节剂和靶点，并提供治疗潜力以促进健康寿命。

DESCRIPTION (provided by applicant): Replicative aging of budding yeast, Saccharomyces cerevisiae, has been a remarkably useful model for aging studies, providing fundamental genetic and molecular insights into aging. Studies of chromatin biology have also benefited from the yeast model, since many molecular mechanisms of chromatin are highly conserved among eukaryotes. The goal of this proposal is to establish two screening approaches that are built on previous works of mine and others for a better understanding on how epigenetics and chromatin regulatory pathways are involved in the process of aging. The results of these screenings are expected to identify many new targets and chromatin regulators for aging studies. Epigenetic changes, including histone post-translational modifications, are critical regulatory mechanisms, involved in all developmental processes including aging. However, most studies, and our previous research in this area have focused on only a few known histone modification targets of Sirtuins. It is of tremendous interest to discover new histone targets of Sirtuins and other chromatin-mediated aging regulators. I propose to utilize an existing systematic histone mutant library to establish a novel screening approach based on the well-established old cell sorting method, and to screen for histone mutants that alter replicative lifespan of yeast (aim 1). The available histone mutant library was developed by Jef Boeke's group. It contains systematic mutations for histone H3 and H4, with multiple versions of mutations for each residue. More importantly this library features mutations in an integrated form, which is a critical factor for use in aging studies. The cell surface labeling and sorting method was development by Leonard Guarente's lab and was used in my previous work to isolate old yeast cell in 108 quantities. The proposed novel screening approach builds on this method and enriches for long-lived cells in the old cell fraction from a pool of histone mutant strains. This approach allows high throughput screening for mutations that extend or shorten lifespan without performing the tedious lifespan assay for all mutant strains. Hits from the screening will be validated by conventional lifespan assay. The yeast deletion library, a collection of yeast strains, each harboring a single gene deletion, has been a tremendously valuable resource for defining gene function, understanding the structure and logic of multiple cellular pathways, many of which are conserved from yeast to humans, and for identifying the target and mode of action for several promising drugs. This collection was recently harnessed by Kennedy and Kaeberlein's groups to identify novel aging regulators and pathways. However, it has become exceedingly difficult to explorer more pro-lifespan regulators, like Sir2, because disruption of many genes that impair critical pathways naturally result in shorter lifespan and they may not be directly involved in aging regulation. Those long-lived deletion mutants are often confusing because many are involved in important biological processes. Therefore evaluate aging and biological effects of over-expressing each gene and compare to the results from the deletion library become more relevant and efficient in identifying genes that promote longevity, like Sirtuins. I propose to construct a collection of yeast strains allowing for systematic over-expression of yeast genes using high-throughput methods, and then to use this collection to discover novel mechanisms underpinning the biology of aging and age-related diseases (aim 2). I will take advantage of a readily available yeast gene plasmid collection FLEXgene, which contains more than 5,500 yeast genes, and the highly-efficient gateway cloning system to generate a library of yeast plasmids with an appropriate marker for integration and selection. Most of the work, including plasmid preparation, gateway cloning, yeast transformation and verification will be performed with a state-of-the-art liquid handling robot recently acquired by my mentor. An initial screen of 400 chromatin related genes is proposed to complete in specific aim 2 during this project timeframe. I expect to identify novel aging regulators that are either directly involved in chromatin biology or have direct targets in chromatin. PUBLIC HEALTH RELEVANCE: Chromatin is the physiological template of the eukaryotic genome, where genetic information is stored, replicated, and expressed. Being at the center of genetic and epigenetic control in the cell, chromatin is subject to an extensive network of regulations through numerous pathways at multiple levels. This project will examine these pathways and their chromatin target through systematic screens, identify novel aging regulators and targets, and provide therapeutic potentials to promote healthy lifespan.