Studies on cell conversion methods by site-specific DNA demethylation

Research Project

Project/Area Number	24510284
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Multi-year Fund
Section	一般
Research Field	System genome science
Research Institution	National Institute of Health Sciences
Principal Investigator	KUBOSAKI Atsutaka 国立医薬品食品衛生研究所, 衛生微生物部, 主任研究官 (30425673)
Research Collaborator	TOMARU Yasunobu FURUHATA Erina SUZUKI Takahiro JAY Shin CHRISTOPHE Simon ANDO Yoshinori HASEGAWA Ryota HAYASHIZAKI Yoshihide SUZUKI Harukazu YOSHINARI Tomoya
Project Period (FY)	2012-04-01 – 2015-03-31
Project Status	Completed (Fiscal Year 2014)
Budget Amount *help	¥5,590,000 (Direct Cost: ¥4,300,000、Indirect Cost: ¥1,290,000) Fiscal Year 2014: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000) Fiscal Year 2013: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000) Fiscal Year 2012: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
Keywords	エピゲノム制御 / ゲノム / 発現制御 / DNA脱メチル化
Outline of Final Research Achievements	Hydroxymethylcytosine (hmC), one of several reported cytosine modifications, was recently thought to play an important role in DNA demethylation. To obtain the fundamental knowledge of hmC, methylcytosine dioxygenase was overexpressed in HEK293T cells and 5-hydroxymethyl deoxycytidines were analyzed by liquid chromatography-tandem mass spectrometry. I also used an original episomal vector-based method called MoCEV to monitor the fate of hmC beyond DNA replication in HEK293T cells. The MoCEV system containing fully hydroxymethylated-cytosine fragments revealed a significant modification towards methylcytosine after several rounds of DNA replication. Since the unmodified MoCEV did not undergo any DNA methylation during cell division, the results strongly suggest that somatic cells undergo hmC to methylcytosine specifically at the CpG sites during cell division. These data may help to induce site-specific DNA demethylation for cell conversion.