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.

Public Health Relevance

The focus of this renewal application is on defining the molecular machineries and mechanisms involved in the implementation and removal of histones H2B monoubiquitination and H3K4 and H3K79 methylations in yeast Saccharomyces cerevisiae. In human, these marks and the machineries are associated with the pathogenesis of childhood leukemia. We plan to characterize the biochemical, molecular, and enzymatic properties of these factors in yeast and to generate small molecule inhibitors for their activities with the hope that they can be used for targeted therapeutics for translocation-based leukemia.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM069905-11
Application #
8521314
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Carter, Anthony D
Project Start
2003-12-01
Project End
2016-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
11
Fiscal Year
2013
Total Cost
$426,272
Indirect Cost
$163,141
Name
Stowers Institute for Medical Research
Department
Type
DUNS #
614653652
City
Kansas City
State
MO
Country
United States
Zip Code
64110
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Mohan, Man; Herz, Hans-Martin; Shilatifard, Ali (2012) SnapShot: Histone lysine methylase complexes. Cell 149:498-498.e1
Schulze, Julia M; Hentrich, Thomas; Nakanishi, Shima et al. (2011) Splitting the task: Ubp8 and Ubp10 deubiquitinate different cellular pools of H2BK123. Genes Dev 25:2242-7
Smith, Edwin; Lin, Chengqi; Shilatifard, Ali (2011) The super elongation complex (SEC) and MLL in development and disease. Genes Dev 25:661-72
Yu, Yao; Srinivasan, Madhusudhan; Nakanishi, Shima et al. (2011) A conserved patch near the C terminus of histone H4 is required for genome stability in budding yeast. Mol Cell Biol 31:2311-25
Takahashi, Yoh-hei; Westfield, Gerwin H; Oleskie, Austin N et al. (2011) Structural analysis of the core COMPASS family of histone H3K4 methylases from yeast to human. Proc Natl Acad Sci U S A 108:20526-31
Takahashi, Yoh-Hei; Schulze, Julia M; Jackson, Jessica et al. (2011) Dot1 and histone H3K79 methylation in natural telomeric and HM silencing. Mol Cell 42:118-26
Mohan, Man; Lin, Chengqi; Guest, Erin et al. (2010) Licensed to elongate: a molecular mechanism for MLL-based leukaemogenesis. Nat Rev Cancer 10:721-8
Eissenberg, Joel C; Shilatifard, Ali (2010) Histone H3 lysine 4 (H3K4) methylation in development and differentiation. Dev Biol 339:240-9

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