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.
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.
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