With the recent discovery of 5-hydroxymethylcytosine in mammalian DNA and the TET enzyme family responsible for its generation, it is clear that the mammalian DNA epigenome is much more complex than had been thought. A lot of questions remain to be answered about the in vivo roles and the in vitro properties of the TET enzymes. In this fast-track application, we propose studying the TET-like enzymes in the single-cell protist Naegleria gruberi. They are much smaller in size than the human TET enzymes and are much easier to produce and engineer. A panel of biochemical assays will be carried out to thoroughly characterize the in vitro properties of the active TET-like enzymes in N. gruberi.
We aim at providing these enzymes as reagents to the research market. Since 5-methylcytosine (5mC) is an important epigenetic marker in mammalian genomic DNA, these enzymes should enable many creative experiments, e.g., direct sequencing of the epigenome, because they functionalize the chemically inert 5mC. To enhance their versatility, we propose using enzyme engineering to look for mutants with altered or improved properties. For this purpose, we will determine the 3D structure of at least one active TET-like enzymes. We plan to apply the wild-type enzymes or derived mutants in several epigenomic applications and determine the methylome/hydroxymethylome changes during the differentiation process of N. gruberi, which may shed lights on the similar processes in the more complex biological systems. In summary, our studies will bring a number of novel enzymatic reagents to the research market and generate scientific insights on epigenetic regulation involving 5hmC.

Public Health Relevance

Dynamic DNA epigenomes provide a basis for the phenotypic variation of a single genome. Many pathological processes, including cancer, are associated with aberrant landscapes of the epigenome. By studying a unique family of 5-methylcytosine oxidases, this proposal aims at providing novel reagents for better characterization of the DNA epigenomes and further understanding of their dynamic transitions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Small Business Innovation Research Grants (SBIR) - Phase II (R44)
Project #
4R44GM105132-02
Application #
8815475
Study Section
Special Emphasis Panel (ZRG1-IMST-G (10))
Program Officer
Maas, Stefan
Project Start
2013-09-01
Project End
2016-02-29
Budget Start
2014-03-15
Budget End
2015-02-28
Support Year
2
Fiscal Year
2014
Total Cost
$461,343
Indirect Cost
Name
New England Biolabs, Inc.
Department
Type
DUNS #
066605403
City
Ipswich
State
MA
Country
United States
Zip Code
01938
Horton, John R; Wang, Hua; Mabuchi, Megumu Yamada et al. (2014) Modification-dependent restriction endonuclease, MspJI, flips 5-methylcytosine out of the DNA helix. Nucleic Acids Res 42:12092-101
Hashimoto, Hideharu; Pais, June E; Zhang, Xing et al. (2014) Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA. Nature 506:391-5