Genetic Analysis of H2B Ubiquitylation in Yeast

描述（由申请人提供）：染色质结构由翻译后组蛋白修饰、组蛋白变异、核小体重塑和核小体交换调节。组蛋白修饰与广泛的生物过程有关，并通过作为调节因子的结合位点或通过调节染色质折叠状态来促进或抑制蛋白质- dna相互作用。H2B （H2Bub1）的单泛素化与活性转录密切相关，特别是与转录延伸有关。在从酵母到人类的生物体中，H2Bub1通过泛素化机制与延长RNA聚合酶II （Pol II）的关联以及转录延伸因子的介导而共转录建立。与H2Bub1功能相关的关键问题是它是否与转录以外的其他生物过程有关，以及它在染色质中的存在如何影响其细胞作用。我们假设H2Bub1在许多不同的细胞过程中调节多蛋白复合物的组装或活性，因为它促进了稳定的染色质环境。我们发现H2Bub1存在于酵母的复制起点，它在DNA复制叉的进展中起作用，这是一种与转录伸长机制相似的现象。在Specific Aim 1中，我们将使用遗传和分子方法来确定在起源处调节H2Bub1的因素，并通过检查htb-K123R突变对复制依赖性核小体动力学的影响来确定H2Bub1在复制中的作用。组蛋白修饰的调控和功能主要是在生长细胞的背景下研究的。然而，大多数真核细胞在其生命周期的很大一部分处于非生长或静止状态，其特征是转录的普遍抑制。酵母静止期（SP）已成为研究细胞静止在时间顺序寿命（CLS）背景下的良好模型。我们的研究揭示了一种特殊的组蛋白修饰模式是SP静止细胞的特征。这种模式包括H2Bub1及其下游标记H3K79me2的选择性丢失，以及与转录起始（H3K4me3）和延伸（H3K36me3, H3K79me3）相关的标记的保留。在Specific Aim 2中，我们将使用遗传、分子和基因组方法来研究这些修饰在静止发育和细胞从静止状态释放中的功能意义。我们还将探讨CLS缩短或延长的突变体是否具有组蛋白修饰模式的紊乱，以及H2B的去泛素化是否决定了分化为静止和非静止细胞的时间。总之，这两个目标中概述的研究应该为染色质在基本细胞过程中的作用提供新的机制见解，并揭示染色质结构的改变如何潜在地影响人类疾病。

DESCRIPTION (provided by applicant): Chromatin structure is regulated by post-translational histone modifications, histone variants, nucleosome remodeling, and nucleosome exchange. Histone modifications have been connected to a wide range of biological processes, and function to promote or inhibit protein-DNA interactions by serving as binding sites for regulatory factors or by regulating the state of chromatin folding. The monoubiquitylation of H2B (H2Bub1) has been tightly linked to active transcription, and in particular to transcription elongation. In organisms from yeast to humans, H2Bub1 is established co-transcriptionally through the association of the ubiquitylation machinery with elongating RNA Polymerase II (Pol II) and the mediation of transcription elongation factors. The key issues relating to the function of H2Bub1 are whether it is linked to other biological processes besides transcription, and how its presence in chromatin affects its cellular roles. We hypothesize that H2Bub1 regulates the assembly or activity of multi-protein complexes in a number of different cellular processes because it promotes a stable chromatin environment. We have found that H2Bub1 is present at yeast origins of replication and that it plays a role in progression of the DNA replication fork, a mechanistically similar phenomenon to transcription elongation. In Specific Aim 1, we will use genetic and molecular approaches to identify the factors that regulate H2Bub1 at origins and define the role of H2Bub1 in replication by examining the effect of an htb-K123R mutation on replication-dependent nucleosome dynamics. The regulation and function of histone modifications have been studied primarily in the context of growing cells. However, most eukaryotic cells spend a large fraction of their life cycle in a non-growing or quiescent state tha is characterized by a general repression of transcription. Yeast stationary phase (SP) has emerged as an excellent model for the study of cellular quiescence in the context of chronological lifespan (CLS). Our studies have revealed that a specific pattern of histone modifications characterize SP quiescent cells. This pattern includes the selective loss of H2Bub1 and its downstream mark of H3K79me2, and the retention of marks associated with transcription initiation (H3K4me3) and elongation (H3K36me3, H3K79me3). In Specific Aim 2, we will use genetic, molecular, and genomic approaches to investigate the functional significance of these modifications in the development of quiescence and in release of cells from the quiescent state. We will also explore if mutants with shortened or lengthened CLS have perturbed patterns of histone modifications, and if the deubiquitylation of H2B determines the timing of differentiation into quiescent and nonquiescent cells. Together, the studies outlined in the two Aims should provide new mechanistic insights into the role of chromatin in basic cellular processes and reveal how alterations in chromatin structure potentially impact human disease. PUBLIC HEALTH RELEVANCE: H2B ubiquitylation is a histone modification with significant relevance to human health, particularly cancer. A tumor suppressor encodes a key regulator of H2B ubiquitylation, and several downstream histone modifications that are mediated by H2B ubiquitylation are linked to the inappropriate expression of developmentally important genes in leukemia. Understanding the fundamental roles of H2B in chromatin structure and function could lead to the development of new therapies in cancer. Cellular quiescence is a key mechanism for cell and tissue regeneration and has emerged as important factor in the survival of both normal adult and cancer stem cells. Defining the underlying epigenetic mechanisms that contribute to cellular quiescence could eventually lead to the development of new treatments to stimulate cellular regeneration in response to injury or to eliminate cancer stem cells.