A Strategy for Accurate Quantification of 5-Methylcytosine and 5-Hydroxymethylcytosine at CpG Sites Within Gene Promoter.
5-Methylcytosine (5mC) can be converted to 5-hydroxymethylcytosine (5hmC) in mammalian DNA by the ten-eleven translocation (TET) enzymes. Traditional bisulfite-based DNA methylation analysis techniques have been widely used in the detection of 5mC. However, they can not discriminate 5hmC from 5mC, leading to overestimate 5mC levels. We here introduce a strategy, combination of selective oxidation and bisulfite pyrosequencing (BS-Pyroseq), for quantification of both 5mC and 5hmC at CpG sites within the promoters of CDH1, DAPK, RARβ and RUNX3 genes in a panel of cell lines and clinical samples. As expected, oxidative bisulfite pyrosequencing (oxBS-Pyroseq) assay decreased overall or site-specific methylation levels of three of these genes in most cell lines as compared with BS-Pyroseq assay. Similarly, decreased overall or site-specific methylation levels of DAPK, RARβ and RUNX3 genes in laryngeal, gastric and thyroid cancer and their matched normal tissues, respectively, were also found by a comparison between these two techniques, particularly in cancerous tissues. In addition, by using this combined strategy and hydroxymethylcytosine DNA immunoprecipitation (hMeDIP) assay, we demonstrated that TET1 up-regulated DAPK expression through promoter demethylation. Collectively, this strategy is easy to establish and accurately discriminates and quantifies 5mC and 5hmC at CpG sites within selected gene promoters.
Yiping
Y
Qui
Qi
Q
Yang
Fang
F
Sui
Rong
R
Lu
Siwen
S
Dang
Meiju
M
Ji
Nongyue
N
He
Bingyin
B
Shi
Peng
P
Hou
D044503:5-Methylcytosine / Q000032:analysis* / Q000031:analogs & derivatives; D018899:CpG Islands; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis; D019175:DNA Methylation; D005260:Female; D006801:Humans; D010084:Oxidation-Reduction; D011401:Promoter Regions, Genetic; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000191:economics / Q000379:methods*; D014407:Tumor Cells, Cultured
C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine