A Genetic System Able to Screen for Active DNA Demethylation

DESCRIPTION (provided by applicant): With the increasing interest in epigenetic modification of DNA there is a need for research reagents to help study the phenomenon. Of especial interest would be an enzyme able to demethylate DNA that might provide a direct way to measure the extent of methylation in eukaryotic DNA. Such an enzyme is expected to be present in newly fertilized oocytes as an extensive active demethylation of embryonic DNA takes place immediately following fertilization and in the absence of DNA replication. However, so far attempts to isolate and characterize such an enzyme have failed. The literature report of an enzyme able to catalyze DNA demethylation has not been successfully reproduced. Several mechanisms can be conceived for DNA demethylation such as direct removal by a demethylase or a multistep process begun by a 5-methylcytosine DNA glycosylase. Initial studies will focus on searching for an enzyme able to directly demethylate DNA. In Phase II an in vivo system, devised during Phase I, will be used to screen or select for active demethylation. The selective system will use a methylation-sensitive restriction enzyme to kill cells unable to demethylate DNA. The screen will use a Type II restriction enzyme to induce an SOS response, visible by a blue phenotype, when demethylation takes place. Additionally, an in vitro transcription-translation system will be used that could detect active demethylation. Finally, a bioinformatics approach will be used to find candidate genes for the demethylase or a component of it in the human and mouse genomes. In Phase II these selection systems will be applied to eukaryotic cDNA libraries that are candidates to contain demethylases. The proposed research will have an impact on DNA modification, epigenetic memory and cancer biology. In addition, knowledge of the mechanism of demethylation could be crucial in converting fully differentiated adult stem cells into totipotent stem cells.