Regulation of yeast cellular aging through chromatin and novel pathways

摘要 芽殖酵母（Saccharomyces cerevisiae）的复制老化一直是一个非常有用的模型， 衰老研究，提供衰老的基本遗传和分子见解。染色质生物学研究 也受益于酵母模型，因为染色质的许多分子机制是高度相关的。 在真核生物中是保守的。该提案的目标是建立两种筛选方法， 为了更好地理解表观遗传学和染色质调节 参与了衰老过程。这些筛查的结果预计将确定许多 衰老研究的新靶点和染色质调节剂。 表观遗传学变化，包括组蛋白翻译后修饰，是关键的调控 机制，参与包括衰老在内的所有发育过程。然而，大多数研究和我们的 该领域的先前研究仅集中于Sirtuins的少数已知组蛋白修饰靶。具有 发现Sirtuins和其他染色质介导的衰老调节剂的新组蛋白靶点的巨大兴趣。 我建议利用现有的系统化组蛋白突变体库，建立一种新的筛选方法， 对成熟的旧细胞分选方法，并筛选组蛋白突变体，改变复制寿命 酵母（目的1）。可用的组蛋白突变体文库由Jef Boeke的小组开发。它包含 组蛋白H3和H4的系统性突变，每个残基具有多个突变版本。更 重要的是，该文库以整合形式的突变为特征，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伦纳德·瓜伦特的实验室开发的，并在我的实验室中使用 以前的工作，分离旧酵母细胞在108数量。提出的新的筛选方法建立在这一点上 方法，并从组蛋白突变株库中富集老细胞级分中的长寿细胞。这 该方法允许高通量筛选延长或缩短寿命的突变，而不需要进行 对所有突变株进行冗长的寿命测定。筛选的命中将通过常规寿命验证 比色法 酵母缺失文库是酵母菌株的集合，每个酵母菌株都含有单个基因缺失， 这是一个非常宝贵的资源，用于定义基因功能，理解多个基因的结构和逻辑， 细胞途径，其中许多是保守的从酵母到人类，并确定目标和模式 几种有前途的药物的作用。这些收藏品最近被肯尼迪和凯伯莱因的 小组确定新的老化调节剂和途径。然而，这已经变得非常困难， 探索更多的促寿命调节因子，如Sir 2，因为许多基因的破坏会损害关键途径， 自然导致寿命缩短，它们可能不直接参与衰老调节。那些长寿的 缺失突变体常常令人困惑，因为许多缺失突变体参与重要的生物学过程。因此 评估过表达每个基因的衰老和生物学效应，并与来自 缺失文库在鉴定促进长寿的基因（如Sirtuins）方面变得更加相关和有效。我 建议构建酵母菌株的集合，允许使用 高通量的方法，然后使用这个集合来发现支持 衰老生物学和与年龄有关的疾病（目标2）。我将利用一个现成的酵母基因 质粒收集FLEX基因，其中包含5,500多个酵母基因，以及高效的网关 克隆系统以产生具有用于整合和选择的适当标记的酵母质粒文库。 大部分的工作，包括质粒制备，网关克隆，酵母转化和验证将 我的导师最近买了一台最先进的液体处理机器人初始屏幕为400 染色质相关基因拟在本项目时间框架内完成具体目标2。我期望 鉴定直接参与染色质生物学或具有直接靶点的新的衰老调节剂， 染色质

Abstract 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.