Chromatin Modifications by Histone Ubiquitination and Methylations

描述(申请人提供)：涉及混合血统白血病(MLL)基因的染色体易位在人类髓系和淋巴系急性白血病中频繁发生。我们鉴定了酿酒酵母的Set1蛋白为MLL同源物，并在一个我们称为COMPASS的复合体中对其进行了纯化。SET1/COMPASS能够甲基化K4(H3K4)上的组蛋白H3。根据酵母研究，我们现在知道，人类MLL也发现在能够甲基化H3K4的指南针状复合体中。在过去的十年中，我们实验室的酵母菌研究已经用COMPASS和Dot1鉴定了组蛋白H3K4甲基化和H3K79甲基化的分子机制。例如，我们证明了组蛋白H2B由Rad6/Bre1单泛素化是COMPASS和Dot1正确的H3K4三甲基化所必需的。在酵母中发现的这些酶机制从酵母到人类都高度保守。鉴于人类MLL和Dot1参与了白血病的发病机制，本研究的中心假设是从酵母中获得的这方面的信息将对我们对MLL易位白血病的理解和治疗产生直接而有价值的影响。这些目标将通过三个具体目标来实现。我们最近已经能够完全重建活性酵母指南针。因此，本申请的具体目标1将集中在定义复合体的每个亚基如何促进H3K4甲基化过程；以及H2BK123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，令人惊讶的少量的H2B单泛素化足以在酵母细胞中提供几乎全部水平的H3K4三甲基化。鉴于我们最近开发了H2B单素化的特异性多克隆抗体，本申请的具体目标2集中在通过使用遗传和生化筛选来识别独立于H3K4甲基化的适当的H2B单素化所需的因素。我们在酵母中的研究表明，组蛋白H3K79甲基化是一个参与转录调控的动态过程。然而，目前还没有已知的H3K79去甲基酶。因此，本申请的具体目标3集中在使用分子筛选鉴定酿酒酵母H3K79去甲基酶机制，并对这些因素进行全分子和生化表征。上述三个目标的实现所获得的数据不仅将对我们理解组蛋白H2 BK123通过单素化和H3K4/K79通过甲基化进行调控具有根本性的影响，而且将有助于全面了解这些因素在MLL易位引起的血液系统恶性肿瘤的发病机制中所起的作用，以及如何将这些途径用于MLL易位引起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.