Abstract 396: TET oxidase activity accumulated on methyl-CpG sites extensively upregulates methylated genes through DNA demethylation

Abstract

Abstract Ten-Eleven Translocation (TET) proteins play an important role in regulating DNA methylation fidelity and their inactivation contributes to the DNA hypermethylation phenotype in cancer; they are Fe²+- and 2-oxoglutarate-dependent dioxygenases that successively oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) and these oxidized methylcytosines are important intermediates in the process of 5mC demethylation. TET proteins convert 5mC to 5hmC in vitro, but it is difficult to explore the process of DNA demethylation and their effects on endogenous hypermethylated genes in cells. In this study, we tried to develop a system that accumulates TET oxidase activity at hypermethylated promoters. To accomplish this, we constructed a fusion protein-producing vector consisting of methyl-CpG binding domain (MBD) and TET1 catalytic domain (TET1 CD) and asked whether this fusion protein (MBD-TET1 CD) could lead to DNA demethylation and gene reactivation. We first prepared DNA constructs that contain wild-type (wt) TET1 CD as well as catalytically inactive TET1 CD mutant (mut) with or without MBD from MBD2 protein and then transfected each construct into human embryonic kidney cell line 293T. We isolated stably transfected 293T cell lines and analyzed the expression of genes transcriptionally silenced by DNA hypermethylation using RT-PCR. Surprisingly, only plasmids containing TET1 CDwt reactivated silenced MAL, SOX17, TRH, MT1M, and MLH1 genes in a MBD dependent manner. To clarify the mechanisms of the antisilencing activities in clones stably expressed MBD-TET1 CDwt, we examined the CpG islands in MAL, TRH, and MT1M promoters by genomic sequencing after bisulfite modifications. Almost all CpG sites were methylated in parental cell line 293T, a TET1 CDwt clone, a TET1 CDmut clone, and a MBD-TET1 CDmut clone. On the other hand, MBD-TET1 CDwt clones showed an extensive demethylation at CpG islands of all three genes. In addition, because bisulfite sequencing does not distinguish between 5mC and 5hmC, we converted 5hmC to glucosylated form and discriminated between 5mC and 5hmC using restriction enzymes MspI and HpaII. Quantitative real time PCR analysis clearly demonstrated that MBD-TET1 CDwt promoted demethylation at CpG sites with some 5hmC bases. We further examined the changes in global gene expression in stable cell lines containing different TET constructs by microarray analyses. All top 50 genes upregulated by MBD-TET1 CDwt-stably expressing cell lines contained CpG islands and interestingly, 43 of top 50 genes were also upregulated after treatment with DNA demethylating agent, 5-azacytidine. These results suggest that specific recruitment of TET oxidase activity on hypermethylated gene promoters induces DNA demethylation and promotes reactivation of epigenetically silenced genes. Citation Format: Shinichi Fukushige, Yasuhiko Mizuguchi, Akira Horii. TET oxidase activity accumulated on methyl-CpG sites extensively upregulates methylated genes through DNA demethylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 396. doi:10.1158/1538-7445.AM2014-396