Development of DNA demethylation technology in HEK293T cells

DNA demethylating agents are epigenetic drugs for the therapy of myeloid leukemias. They not only induce DNA demethylation but also have significant cytostatic and cytotoxic effects, however, the relationships between these characteristics have not been established yet due to the lack of method to induce only DNA demethylation. Here we show that a fusion protein comprising the methyl-CpG binding domain (MBD) and the catalytic domain of Ten-eleven translocation protein 1 (TET1-CD) globally demethylates and upregulates a number of methylated genes. Gene expression microarray analyses using human embryonic kidney cell line 293T indicate that cells expressing wild-type (wt) TET1 catalytic domain with MBD (MBD-TET1-CDwt) upregulated more genes than ones expressing TET1-CDwt without MBD or catalytically inactive TET1-CD mutant (mut) with MBD (MBD-TET1-CDmut) and their upregulated genes frequently contained CpG islands (CGIs) within ± 1,000 bp of the transcription start site (TSS). Interestingly, 88% of genes upregulated 5-fold or more by MBD-TET1-CDwt were also reactivated after treatment with DNA demethylating agent, 5-azacytidine, suggesting that gene reactivation by both methods is primarily based on DNA demethylation. HEK293T cells were transfected with pcDNA6/myc-His vector, pcDNA6-TET1-CDwt, pcDNA-MBD-TET1-CDwt, or pcDNA6-MBD-TET1-CDmut and two independent stable transformants expressing vector, TET1-CDwt, MBD-TET1-CDwt, or MBD-TET1-CDmut were constructed respectively. RNAs from these eight stable cell lines were prepared and analyzed by Agilent Human Gene Expression 4x44K v2 Microarray.