Dot 1 Complex, Transcriptional Elongation Control and Human Cancer

Project: Research project

Project Details

Description

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 translocationbased
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.
StatusFinished
Effective start/end date12/2/1411/30/15

Funding

  • National Cancer Institute (7R01CA089455-15)

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