Dot 1 Complex, Transcriptional Elongation Control and Human Cancer

Dot 1 复合物、转录延伸控制和人类癌症

• 批准号: 8204703
• 负责人: Ali Shilatifard
• 金额: $ 25.63万
• 依托单位: STOWERS INSTITUTE FOR MEDICAL RESEARCH
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8204703
• 关键词: Antibodies; Biochemical; Cell Line; Cells; Childhood Leukemia; Chimera organism; Complex; Data; Elongation Factor; Enzymes; Epigenetic Process; Gene Expression; Gene Expression Regulation; Gene Targeting; Genetic Transcription; Goals; Hematologic Neoplasms; Histone H3; Human; In Vitro; Learning; Leukemic Cell; Link; Lysine; Macromolecular Complexes; Malignant Neoplasms; Methods; Methylation; Modification; Molecular; Myeloid Progenitor Cells; Pathogenesis; Pathway interactions; Patients; Play; Property; Proteins; RNA Polymerase II; Reagent; Regulation; Reporting; Role; Staging; Transcriptional Regulation; base; cell immortalization; histone methyltransferase; inhibitor/antagonist; leukemia; member; novel; public health relevance; reconstitution; small molecule; therapeutic target

DESCRIPTION (provided by applicant): The goals of this proposal are full molecular and biochemical characterization of the human Dot1 complex and the human AFF4 complex, and molecular identification of how the components of their complexes contribute to the pathogenesis of hematological malignancies. Epigenetic modifications play a vital role in the regulation of transcription and gene expression. Therefore, it is not surprising that enzymes and molecular machineries engaged in epigenetic modifications have been linked to the pathogenesis of human malignances. Dot1, is a Histone Methyltransferase (HMTases) capable of methylating lysine 79 of histone H3 (H3K79) marking the elongation stage of transcription by RNA Polymerase II. Dot1 was recently reported to interact with AF10, one of the fusion partners of MLL involved in the pathogenesis of leukemia. Direct fusion of Dot1 to MLL also results in the immortalization of myeloid progenitor cells and this immortalization activity depends on the H3K79 HMTase function of Dot1. To learn more about the molecular role of Dot1 in leukemia pathogenesis, we have recently biochemically isolated a novel macromolecular complex containing the human Dot1. Therefore, Specific Aim 1 of this application is focused on the identification of the components of the human Dot1 complex using biochemical and mass spectrometric methods. Once fully identified, we plan to generate reagents such as antibodies and cell lines expressing tagged versions of the components of the complex to study how these subunits associate within the Dot1 complex with the ultimate goal of reconstituting a catalytically active Dot1 complex in vitro. Since H3K79 methylation by Dot1 contributes to the pathogenesis of leukemia, a full understanding of its molecular and enzymatic properties will allow us to better define how misregulation of its activity results in the pathogenesis of leukemia. With a possible role for the RNA polymerase II elongation complex in the MLL translocation- based leukemia, we have recently demonstrated that AFF4, and several of the known RNA polymerase II elongation factors are shared components of many of the MLL-chimeras. Therefore, the Specific Aim 2 is focused on the biochemical isolation and molecular characterization of the human AFF4 complex. In this aim of the application, we will identify the gene targets of AFF4 and the members of its complex in leukemic cell lines and in primary cells from patients suffering from MLL translocation-based leukemia. Studies proposed under Aim 2 of the proposal will be instrumental in defining the role of transcriptional elongation control in the regulation of gene expression and how the misregulation of such activity will result in the pathogenesis of childhood leukemia. Data obtained as the result of the implementation of the above proposed two aims will not only have a fundamental impact on our understanding on the regulation of the elongation stage of transcription by the Dot1 and AFF4 complexes, but also will be instrumental for an understanding of the role these factors play in the pathogenesis of MLL translocation-based hematological malignancies, and how such pathways could be used for targeted therapeutics of leukemia caused by MLL-translocations. PUBLIC HEALTH RELEVANCE: The focus of this application is on a group of proteins (MLL1, Dot1, and AFF4), which are involved in the pathogenesis of childhood leukemia. We plan to characterize the biochemical, molecular and enzymatic properties of these factors and generate small molecule inhibitor of their activities with the hope that they can be used for targeted therapeutics of translocation-based leukemia.

描述（由申请人提供）：该提案的目标是人Dot1复合物和人类Aff4复合物的完全分子和生化表征，以及对其复合物成分的分子鉴定如何有助于血液学恶性肿瘤的发病机理。表观遗传修饰在转录和基因表达的调节中起着至关重要的作用。因此，毫不奇怪，从事表观遗传修饰的酶和分子机器与人类恶性肿瘤的发病机理有关。 DOT1是一种组蛋白甲基转移酶（HMTase），能够甲基化组蛋白H3的甲基化赖氨酸79（H3K79），标志着RNA聚合酶II的转录延伸阶段。据报道，DOT1与AF10相互作用，AF10是参与白血病发病机理的MLL的融合伙伴之一。 DOT1与MLL的直接融合还导致髓样祖细胞的永生化，而这种永生化活性取决于DOT1的H3K79 HMTase功能。为了更多地了解DOT1在白血病发病机理中的分子作用，我们最近在生化分离了一种新型的含有人Dot1的大分子分子复合物。因此，使用生化和质谱方法，本应用的特定目的1集中于鉴定人dot1复合物的成分。一旦充分确定，我们计划生成诸如表达复合物组件的标记版本的抗体和细胞系等试剂，以研究这些亚基如何在DOT1复合物中关联到与重建催化活性Dot1复合物的最终目标。由于DOT1的H3K79甲基化有助于白血病的发病机理，因此对其分子和酶促性质的完全理解将使我们能够更好地定义其活性的正直如何导致白血病的发病机理。在基于MLL的易位性白血病中RNA聚合酶II伸长率复合物的可能作用，我们最近证明AFF4和几个已知的RNA聚合酶II伸长因子是许多MLL-Chimeras的共享组成部分。因此，特定的目标2集中在人AFF4复合物的生化分离和分子表征上。在应用程序的这一目标中，我们将确定AFF4的基因靶标及其在白血病细胞系中的复合物以及患有基于MLL易位性白血病的患者的主要细胞中的基因靶标。提案的目标2提出的研究将有助于定义转录伸长控制在基因表达调节中的作用，以及这种活性的正调将如何导致儿童白血病的发病机理。由于上述两个目标的实施而获得的数据不仅会对我们对DOT1和AFF4复合物对转录延伸阶段的调节的理解产生根本性的影响，而且还将对这些因素在MLL基于基于MLL的血液学恶性中的病原体中的作用而发挥作用，以及这些因素在对这些因素的病原体中的作用起着作用MLL转移。 公共卫生相关性：该应用的重点是参与儿童白血病发病机理的一组蛋白质（MLL1，DOT1和AFF4）。我们计划表征这些因素的生化，分子和酶促特性，并产生其活性的小分子抑制剂，希望它们可用于基于易位性白血病的靶向治疗剂。