演題詳細

一般口演 / Oral Session

【E】一般口演 33 (Oral Session 33) :MDS/MPN:Basic Research 1

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日程
2013年10月11日(金)
時間
14:25 - 15:25
会場
第14会場 / Room No.14 (札幌市教育文化会館 3F 研修室305)
座長・司会
桐戸 敬太 (Keita Kirito):1
1:Department of Hematology, University of Yamanashi, Japan
 
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Determination of 5-hydroxymethylcytosine at single base resolution using next generation sequencer

演題番号 : OS-1-169

松井 啓隆 (Hirotaka Matsui):1、金井 昭教 (Akinori Kanai):1、稲葉 俊哉 (Toshiya Inaba):1

1:Research Institute for Radiation Biology and Medicine, Hiroshima University

 

TET2 and IDH1/2 genes, whose gene products play roles in converting 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), shows loss-of-function mutations in some myeloid diseases. However, the study of 5hmC has been hindered by the lack of a method that determines the proportion and the position of 5hmC at single-base resolution. Traditional bisulfite sequencing cannot distinguish 5hmC from 5mC as both of them are read as C. Meanwhile it is recently reported that Tet-assisted bisulfite sequencing (TAB-seq) successfully detects 5hmC at the whole genome level. Here we established a system that enables us to analyze the 5hmC level focusing on CpG rich gene promoters and CpG islands at relatively low cost by combining reduced representative bisulfite sequencing (RRBS) and TAB-seq methods. In our system, CpG-rich DNA fragments of 150-250bp are first selectively purified from genomic DNA. 5hmC sites in these fragments are then glucosylated to 5gmC by βglucosyltransferase, while 5mC sites are oxidized to 5-carboxycytosine (caC) by Tet enzyme, followed by the bisulfite conversion. After these treatments, unmodified C and 5caC are sequenced as T, while only 5gmC is read as C. In combination with traditional bisulfite sequencing, we are now able to estimate the proportions of C/5mC/5hmC in more than a half million CpG sites. We found that 5hmC distributes from 0 to 32.4% (Mean 0.55%) in promoters in hematopoietic cells. Promoters containing lower CpG numbers tend to have higher 5hmC levels. The changes in 5hmC in primary MDS/AML patients will be discussed in this presentation.

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