Chromatin Modifications by Histone Ubiquitination and Methylations

描述（由申请人提供）：涉及混合谱系白血病（MLL）基因的染色体易位经常发生在人急性髓系和淋巴系白血病中。我们鉴定了酿酒酵母的Set 1蛋白作为MLL同源物，并在我们称为COMPASS的复合物中对其进行纯化。Set 1/COMPASS能够使组蛋白H3在其K4（H3 K4）上甲基化。基于酵母研究，我们现在知道人MLL也存在于能够甲基化H3 K4的COMPASS样复合物中。在过去的十年中，我们实验室的酵母研究已经鉴定了参与组蛋白H3 K4甲基化的分子机制（COMPASS）和参与H3 K79甲基化的分子机制（Dot 1）。例如，我们证明了组蛋白H2 B通过Rad 6/Bre 1的单泛素化是通过COMPASS和Dot 1进行适当的H3 K4三甲基化所必需的。在酵母中鉴定的这些酶机制从酵母到人类是高度保守的。鉴于人类MLL和Dot 1参与白血病发病机制的事实，本研究的中心假设是，从酵母研究中获得的这方面的信息将对我们理解和治疗MLL易位为基础的白血病产生直接和有价值的影响。这些目标将通过三个具体目标实现。我们最近已经能够完全重组活性酵母COMPASS。因此，本申请的具体目标1将集中于定义复合物的每个亚基如何促进H3 K4甲基化过程;以及H2 BK 123单泛素化如何改变酶的催化性质。我们最近的分子研究表明，令人惊讶的少量H2 B单泛素化足以在酵母细胞中提供几乎完全水平的H3 K4三甲基化。鉴于我们最近开发了H2 B单泛素化的特异性多克隆抗体，本申请的具体目标2集中于通过采用遗传和生物化学筛选来鉴定独立于H3 K4甲基化的适当H2 B单泛素化所需的因子。我们在酵母中的研究表明，组蛋白H3 K79甲基化是一个参与转录调控的动态过程。然而，没有已知的H3 K79脱甲基酶。因此，本申请的具体目标3集中于使用分子筛选鉴定酵母S中的H3 K79脱甲基酶机制。酿酒酵母和完整的分子和生物化学表征这些因素。实现上述三个目标所获得的数据不仅将对我们了解组蛋白H2 BK 123通过单倍喹啉化和H3 K4/K79通过甲基化的调节产生根本性影响，而且将有助于全面了解这些因素在MLL易位型血液恶性肿瘤发病机制中所发挥的作用，以及这些途径如何用于MLL易位引起的白血病的靶向治疗。 公共卫生相关性：该更新申请的重点是定义在酵母酿酒酵母中实施和去除组蛋白H2 B单泛素化和H3 K4和H3 K79甲基化所涉及的分子机制和机制。在人类，这些标志物和机制与儿童白血病的发病机制有关。我们计划对酵母中这些因子的生化、分子和酶特性进行表征，并产生针对其活性的小分子抑制剂 希望它们可以用于基于易位的白血病的靶向治疗。

DESCRIPTION (provided by applicant): Chromosomal translocations involving the mixed lineage leukemia (MLL) gene occur frequently in human acute leukemias of myeloid and lymphoid lineages. We identified the Set1 protein of yeast Saccharomyces cerevisiae as a MLL homologue and purified it in a complex we call COMPASS. Set1/COMPASS is capable of methylating histone H3 on its K4 (H3K4). Based on the yeast studies, we now know that human MLL is also found in a COMPASS-like complex capable of methylating H3K4. The yeast studies in our laboratory during the past ten years have resulted in the identification of the molecular machineries involved in histone H3K4 methylation by COMPASS and H3K79 methylation by Dot1. For example, we demonstrated that histone H2B monoubiquitination by Rad6/Bre1 is required for proper H3K4 trimethylations by COMPASS and Dot1. These enzymatic machineries identified in yeast are highly conserved from yeast to human. Given the fact that human MLL and Dot1 are involved in the pathogenesis of leukemia, the central hypothesis of this study is that information obtained from studies in yeast in this regard will have a direct and valuable impact on our understanding and the treatment of MLL translocation-based leukemia. These objectives will be achieved through three specific aims. We have recently been able to fully reconstitute active yeast COMPASS. Therefore the Specific Aim 1 of this application will be focused on defining how each subunit of the complex contributes to the process of H3K4 methylation; and how H2BK123 monoubiquitination alters the catalytic properties of the enzyme. Our recent molecular studies demonstrated that a surprisingly small amount of H2B monoubiquitination is enough to provide almost a full level of H3K4 trimethylation in yeast cells. Given that we have recently developed H2B monoubiquitinated specific polyclonal antibodies, the Specific Aim 2 of this application is focused on identifying factors required for proper H2B monoubiquitination independently of H3K4 methylation by employing genetic and biochemical screens. Our studies in yeast have demonstrated that histone H3K79 methylation is a dynamic process involved in transcriptional regulation. However, there are no known H3K79 demethylases. Therefore, Specific Aim 3 of the application is focused on the use of molecular screens identifying H3K79 demethylase machinery in yeast S. cerevisiae and full molecular and biochemical characterization of these factors. Data obtained as the result of the implementation of the above proposed three aims will not only have a fundamental impact on our understanding of the regulation of histones H2BK123 by monoubiquitination and H3K4/K79 by methylations, but also will be instrumental in obtaining a comprehensive understanding of the roles these factors play in the pathogenesis of MLL translocation-based hematological malignancies, and how such pathways could be used for targeted therapeutics for leukemia caused by MLL translocations. PUBLIC HEALTH RELEVANCE: The focus of this renewal application is on defining the molecular machineries and mechanisms involved in the implementation and removal of histones H2B monoubiquitination and H3K4 and H3K79 methylations in yeast Saccharomyces cerevisiae. In human, these marks and the machineries are associated with the pathogenesis of childhood leukemia. We plan to characterize the biochemical, molecular, and enzymatic properties of these factors in yeast and to generate small molecule inhibitors for their activities with the hope that they can be used for targeted therapeutics for translocation-based leukemia.