Genetic Analysis of H2B Ubiquitylation in Yeast

描述（由申请人提供）：组蛋白共价修饰与真核基因表达的调控密切相关。出芽酵母（Saccharomyces cerevisiae）中组蛋白H2B （H2Bub）的单泛素化与转录激活有关，并调节一种新的反式组蛋白途径，导致赖氨酸4 （H3K4）和赖氨酸79 （H3K79）上的组蛋白H3甲基化。所提出的研究的主要目标是了解H2Bub是如何被调节的，以及这种组蛋白修饰如何影响转录和固定期（一种由营养剥夺导致的特殊静止状态）。这些目标将在三个具体目标中实现。在Specific Aim 1中，将使用遗传和分子方法定义H2Bub在Pol II转录中的作用，以确定组蛋白修饰是否与组蛋白伴侣蛋白合作，在Pol II起始和延伸过程中调节染色质稳定性。在Specific Aim 2中，将重点研究E3连接酶Bre1在E2， Rad6激活中的作用，从而研究调节H2Bub的因素。在Specific Aim 3中，我们将采用分子和遗传学的方法来确定细胞进入静止期时H2Bub的去泛素化和细胞重新进入生长期时H2B的快速泛素化的调控因素。我们将利用分子生物学和细胞生物学的方法来研究H2Bub在静止期的作用，以确定H2Bub是否调节静止细胞的形成。转录畸变通常是由与组蛋白修饰状态相关的表观遗传变化引起的。这些变化反过来又与许多疾病状态有关。我们提出的关于H2B泛素化的研究通过H2Bub在H3K4反式组蛋白甲基化中的作用与癌症相关。人类SET结构域蛋白MLL1是一种H3K4组蛋白甲基转移酶，在急性白血病中经常重排，因此对酵母的研究可以深入了解肿瘤细胞中异常H3K4甲基化的作用。此外，研究H2Bub在细胞静止中的作用可以为衰老和神经退行性疾病提供新的认识。

DESCRIPTION (provided by applicant): Histone covalent modifications are tightly linked to the regulation of eukaryotic gene expression. Monoubiquitylation of histone H2B (H2Bub) in the budding yeast, Saccharomyces cerevisiae, is associated with transcriptional activation, and regulates a novel trans-histone pathway that leads to methylation of histone H3 on lysines 4 (H3K4) and 79 (H3K79). The broad goals of the proposed research are to understand how H2Bub is regulated and how this histone modification influences transcription and stationary phase, a specialized quiescent state that results from nutrient deprivation. These goals will be pursued in three specific aims. In Specific Aim 1, the role of H2Bub in Pol II transcription will be defined using genetic and molecular approaches to determine if the histone modification co-operates with histone chaperones to regulate chromatin stability during Pol II initiation and elongation. In Specific Aim 2, the factors that regulate H2Bub will be studied by focusing on the roles of the E3 ligase, Bre1, in activation of the E2, Rad6. In Specific Aim 3, molecular and genetic approaches will be employed to identify the factors that regulate the deubiquitylation of H2Bub when cells enter stationary phase and the rapid ubiquitylation of H2B when cells re-enter the growth phase. The role of H2Bub in stationary phase will be investigated using molecular and cell biological approaches to determine whether H2Bub regulates the formation of quiescent cells. Aberrations in transcription can frequently result from epigenetic changes that are linked to the modification state of histones. These changes, in turn, have been associated with a number of disease states. The proposed studies on H2B ubiquitylation have relevance to cancer through the role of H2Bub in the trans- histone methylation of H3K4. The human SET domain protein, MLL1, is an H3K4 histone methyltransferase that is frequently rearranged in acute leukemias, and thus studies in yeast could provide insight into the roles of aberrant H3K4 methylation in tumor cells. In addition, studies on the role of H2Bub in cellular quiescence could provide a new understanding of aging and neurodegenerative diseases.