Chromatin Modifications by Histone Ubiquitination and Methylations

Project: Research project

Project Details

Description

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.
StatusFinished
Effective start/end date11/1/148/31/16

Funding

  • National Institute of General Medical Sciences (5R01GM069905-14)

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