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髓系和淋巴系的人类急性白血病中。我们鉴定了酿酒酵母的Set1蛋白是MLL同源物，并在我们称为COMPASS的复合物中纯化了它。Set1/COMPASS能够在其K4 （H3K4）上甲基化组蛋白H3。基于酵母的研究，我们现在知道人类MLL也存在于能够甲基化H3K4的compass样复合体中。在过去十年的酵母研究中，我们通过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Rad6/Bre1对组蛋白H2B单泛素化是COMPASS和Dot1对H3K4三甲基化的必要条件。这些酵母菌的酶促机制从酵母菌到人类都高度保守。鉴于人类MLL和Dot1参与白血病的发病机制，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对MLL易位性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COMPASS。因此，本应用程序的具体目标1将集中于定义该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能。我们最近的分子研究表明，令人惊讶的是，少量的H2B单泛素化足以在酵母细胞中提供几乎完全水平的H3K4三甲基化。鉴于我们最近开发了H2B单泛素化特异性多克隆抗体，本申请的特异性目标2主要是通过采用遗传和生化筛选来确定独立于H3K4甲基化的适当H2B单泛素化所需的因子。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，没有已知的H3K79去甲基化酶。因此，本申请的Specific Aim 3侧重于利用分子筛选技术鉴定酿酒酵母中H3K79去甲基酶机制，并对这些因素进行完整的分子和生化表征。实现上述三个目标所获得的数据不仅将对我们理解组蛋白H2BK123通过单泛素化和H3K4/K79通过甲基化的调控产生根本性影响，而且将有助于全面了解这些因素在MLL易位型血液系统恶性肿瘤发病机制中的作用。以及如何将这些途径用于靶向治疗由MLL易位引起的白血病。

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.