Lysine methylation at chromatin and cellular responses to stress

项目摘要 细胞在波动的环境信号存在下的生存严重依赖于快速 基因表达的变化。染色质修饰酶是基因组的关键调节因子 在应激过程中重新编程，这些酶的异常调节或突变导致 扰乱了基因表达程序，并对细胞应激做出了不适当的反应。是这样的 后果有助于人类的病理过程，包括肿瘤发生和衰老 细胞。尽管染色质修饰物在这些途径中起着关键作用，但仍有大量的 关于组蛋白修饰的新位置及其对基因组的影响的知识差距 监管，特别是在环境中遇到不同压力的情况下。我们的 初步工作发现，酿酒酵母蛋白Set4，一个潜在的 人类蛋白MLL5的同源基因对细胞在氧化应激中的生存很重要，而且它是 一种活性的组蛋白甲基转移酶。我们工作的中心假设是，SET4是一种压力-- 受调控的甲基转移酶，激活已定义的基因表达程序以响应 应激通过其赖氨酸甲基化活性。提出了三个具体目标。在Aim I中，我们将 用生化和质量测定确定Set4在体外和细胞内的底物特异性 基于光谱学的方法。我们将测试Set4是否主要针对组蛋白，或者它是否 也有额外的非组蛋白甲基赖氨酸底物，有助于氧化应激 回应。在AIM II中，将使用分子和遗传分析来确定 调节Set4本身，以应对氧化和其他压力。设置4所依据的机制 对照基因的表达将通过RNA测序分析和芯片分析来阐明。 胁迫下一系列突变体的set4及其同源甲基标记的测序。AIM III将 通过以下方法测试Set4依赖应激反应需要Set4的PhD手指的假设 将其定位于染色质。生化和蛋白质组学分析将确定 Set4的PhD指的组蛋白或非组蛋白结合伴侣，以及靶向分子和 基因组实验将评估PHD手指在定位和活动中的作用 Set 4位于染色质。这些研究目的将为我们深入了解 新的表观遗传修饰物，我们预计将适用于其潜在的人类直系物MLL5， 它与干细胞的维持、肿瘤的发生和神经发育有关 精神错乱。此外，这项工作将揭示环境压力和环境压力之间的新联系 以染色质为基础的基因表达调节，这将是我们理解 异常应激信号对基因组的错误调控如何导致人类疾病。

Project Summary Cell survival in the presence of fluctuating environmental signals is critically dependent on rapid changes in gene expression. Chromatin-modifying enzymes are key regulators of genome reprogramming during stress, and aberrant regulation or mutation of these enzymes results in disrupted gene expression programs and inappropriate responses to cellular stress. Such consequences contribute to pathological processes including oncogenesis and aging of human cells. Despite critical roles for chromatin modifiers in these pathways, there are still substantial gaps in our knowledge regarding novel sites of histone modification and their effects on genome regulation, particularly in the presence of diverse stresses encountered in the environment. Our preliminary work has uncovered that the Saccharomyces cerevisiae protein Set4, a potential ortholog of the human protein MLL5, is important for cell survival in oxidative stress and that it is an active histone methyltransferase. The central hypothesis of our work is that Set4 is a stress- regulated methyltransferase that activates a defined gene expression program in response to stress through its lysine methylation activity. Three specific aims are proposed. In Aim I, we will define the substrate specificity of Set4 both in vitro and in cells using biochemical and mass spectrometry based approaches. We will test whether Set4 primarily targets histones, or if it also has additional, non-histone methyl-lysine substrates that contribute to the oxidative stress response. In Aim II, molecular and genetic analysis will be used to determine pathways that regulate Set4 itself in response to oxidative, and other, stresses. Mechanisms by which Set4 controls gene expression will be elucidated through RNA-sequencing analysis and chIP- sequencing of Set4 and its cognate methyl mark in a series of mutants under stress. Aim III will test the hypothesis that the PHD finger of Set4 is required for Set4-depdent stress responses by promoting its localization to chromatin. Biochemical and proteomic assays will determine the histone or non-histone binding partner of the PHD finger of Set4, and targeted molecular and genomic experiments will assess the role of the PHD finger in the localization and activity of Set4 at chromatin. These research aims will provide substantial insight in to the function of a novel epigenetic modifier that we expect to be applicable to its potential human ortholog MLL5, which has been implicated in stem cell maintenance, tumorigenesis and neurodevelopmental disorders. Furthermore, this work will uncover new links between environmental stress and chromatin-based regulation of gene expression, which will be critical to our understanding of how misregulation of the genome by aberrant stress signaling contributes to human disease.