Roles of Histone Ubiquitination in Yeast

酵母中泛素化H2B （uH2B）的鉴定及其缺失相关表型的发现将被用来了解这种组蛋白修饰在染色体结构和功能中的作用。总体目标是确定依赖于uH2B的生物过程，并了解这种修饰是如何在体内调节的。第一个目标是确定uH2B是否为染色质成分，因为uH2B的亚细胞定位可能决定其生物学功能。一般染色质关联将在富集染色质相关蛋白的制剂中进行测量，并在核小体纯化后，使用一种新的免疫检测方法。采用染色质免疫沉淀（ChIP）方法检测uH2B与特定基因的优先染色质关联。组蛋白翻译后修饰对染色质结构的调节已成为真核生物调节染色体功能的关键机制。尽管大量的研究已经调查了组蛋白乙酰化如何影响转录调控，但其他组蛋白修饰的生物学作用在很大程度上仍然未知。uH2B存在于酵母中，是最佳细胞生长和减数分裂所必需的，这一发现为研究这种修饰对遗传易感生物的影响提供了一个独特的机会。由于组蛋白泛素化是一个进化保守的过程，期望这些研究将对我们理解这种修饰如何影响真核生物染色体上发生的多种过程做出重大贡献。

The identification of ubiquitinated H2B (uH2B) in the yeast Saccharomyces cerevisiae and the discovery of phenotypes associated with its absence will be exploited to understand the role of this histone modification in chromosome structure and function. The overall objectives are to define the biological processes that depend on uH2B and to learn how this modification is regulated in vivo. The first goal is to determine whether uH2B is a chromatin constituent, since the subcellular localization of uH2B may determine its biological functions. General chromatin association will be measured in preparations that enrich for chromatin-associated proteins and after purification of nucleosomes, using a novel method of immunological detection. Preferential chromatin association of uH2B with specific genes will be assayed by the method of chromatin immunoprecipitation (ChIP). The modulation of chromatin structure by the posttranslational modification of histones has emerged as a key mechanism to regulate chromosome function in eukaryotes. Although a wealth of studies have investigated how transcriptional regulation is affected by histone acetylation, the biological roles of other histone modifications remain largely unknown. The discovery that uH2B is present in yeast and required for optimal cell growth and meiosis has provided a unique opportunity to study the impact of this modification in a genetically tractable organism. Since histone ubiquitination is an evolutionarily conserved process, it is expected that these studies will make significant contributions to our understanding of how this modification influences the multiple processes that occur on eukaryotic chromosomes.