Regulation of yeast cellular aging through chromatin and novel pathways

摘要 萌芽酵母的复制老化是一个非常有用的模型 衰老研究，提供对衰老的基本遗传学和分子洞察力。染色质生物学的研究 也受益于酵母模型，因为染色质的许多分子机制是高度 在真核生物中保守的。该提案的目标是建立两种筛选方法 关于我和其他人以前的工作，以更好地理解表观遗传学和染色质调控 衰老过程中涉及多种途径。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。 表观遗传变化，包括组蛋白翻译后修饰，是关键的调控因素 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 以往在这方面的研究主要集中在几个已知的sirtuins组蛋白修饰靶点上。它是 对发现sirtuins和其他染色质介导的老化调节剂的新的组蛋白靶点非常感兴趣。 我建议利用现有的组蛋白突变体文库来建立一种新的筛选方法，该方法基于 在成熟的旧细胞分选方法上，并筛选改变复制寿命的组蛋白突变体 酵母菌(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，每个残基有多个版本的突变。更多 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，并在我的 之前的工作是分离108个数量的老酵母细胞。拟议的新筛选方法建立在此基础上 方法，并在组蛋白突变菌株池中的旧细胞组分中丰富长寿命细胞。这 该方法允许高通量筛查延长或缩短寿命的突变，而无需执行 对所有突变菌株进行繁琐的寿命测试。筛查的命中率将根据常规寿命进行验证 化验。 酵母删除文库是酵母菌株的集合，每个酵母菌株都含有一个基因缺失， 为定义基因功能、理解多个基因的结构和逻辑提供了极有价值的资源 细胞途径，其中许多是保守的，从酵母到人类，并用于识别目标和模式 对几种有希望的药物采取行动。这些藏品最近被肯尼迪和凯伯莱因利用 小组，以确定新的衰老调节器和途径。然而，要做到这一点已经变得异常困难 探索者更倾向于延年益寿，比如Sir2，因为破坏了许多破坏关键途径的基因 自然会导致寿命缩短，而且它们可能不直接参与衰老调节。那些长寿的人 缺失突变体往往令人困惑，因为许多都参与了重要的生物过程。因此 评估过度表达每个基因的衰老和生物学效应，并与 缺失文库在识别像sirtuins这样促进长寿的基因方面变得更加相关和有效。我 建议使用以下方法构建允许酵母基因系统过度表达的酵母菌株集合 高吞吐量方法，然后使用此集合来发现支撑 老龄化生物学和老年病(目标2)。我将利用一种现成的酵母基因 含有5500多个酵母基因的FLEX基因和高效通道的质粒库 克隆系统，以产生具有适当标记的酵母质粒文库，用于整合和选择。 包括质粒制备、门户克隆、酵母转化和验证在内的大部分工作将 用我的导师最近买来的最先进的液体搬运机器人表演。初始屏幕为400 染色质相关基因建议在本项目时间框架内完成特定目标2。我希望是这样 确定直接参与染色质生物学或直接针对染色质的新型衰老调节因子 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.