Genetic Analysis of H2B Ubiquitylation in Yeast

描述(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。在发芽酵母中，组蛋白H_2B(H_2Bub)的单价基化与转录激活有关，并调节一种新的转组蛋白途径，该途径导致组蛋白H3在赖氨酸4(H3K4)和79(H3K79)上甲基化。拟议研究的广泛目标是了解H2 Bub是如何调控的，以及组蛋白修饰如何影响转录和静止期，这是一种由营养剥夺引起的专门的静止状态。这些目标将通过三个具体目标来实现。在特定的目标1中，将使用遗传和分子方法确定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协同调节Pol II启动和延伸过程中的染色质稳定性。在特定目标2中，将通过重点研究E3连接酶Bre1在E2 Rad6激活中的作用来研究调节H2 Bub的因素。在具体目标3中，将利用分子和遗传学方法确定调节细胞进入稳定期时H_2Bub去泛素化和细胞重新进入生长期时H_2B快速泛素化的因素。将使用分子和细胞生物学方法研究H_2Bub在静止期的作用，以确定H_2Bub是否调节静止细胞的形成。转录异常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。H_2B泛素化的研究通过H_2Bub在H3K4的转组蛋白甲基化中的作用与癌症相关。人类SET结构域蛋白MLL1是一种在急性白血病中频繁重排的H3K4组蛋白甲基转移酶，因此对酵母的研究可以深入了解H3K4异常甲基化在肿瘤细胞中的作用。此外，对H_2Bub在细胞静止中的作用的研究可以提供对衰老和神经退行性疾病的新的理解。

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.