Quantitative analysis of global 5-methyl- and 5-hydroxymethylcytosine in TET1 expressed HEK293T cells

Abstract DNA methylation at the 5-position of cytosine bases (5-methylcytosine, 5 mC) in genomic DNA is representative epigenetic modification and is involved in many cellular processes, including gene expression and embryonic development. The hydroxylation of 5 mC provide 5-hydroxymethylcytosine (5 hmC), the so-called sixth base rediscovered recently in mammalian cells, is also considered to act as an epigenetic regulator. We report herein the immunochemical assessment of 5 hmC achieved by an enzyme-linked immunosorbent assay (ELISA) using our linker technology. The keys to this assay are 1) the immobilization of genomic DNA with the bifunctional linker molecule, and 2) quantitative analysis by using guaranteed standard samples containing defined amounts of 5 hmC. We succeeded in the sensitive and quantitative detection of 5 hmC as well as 5 mC in HEK293T cells transfected with TET1, and also monitored the effect of ascorbate on the TET1 catalyzed conversion of 5 mC to 5 hmC. Our linker technology enables the rapid and stable immobilization of genomic samples and thus contributes to the realization of a reproducible 5 hmC evaluation method.