Luciferase assay

The 885-bp fragment from the Dnmt3a gene promotor region (including OCT1-binding motif) and the 696-bp fragment from −622 to +74 bp of the Kcna2 gene promotor and 5′-untranslated region were amplified by PCR from genomic DNA with the primers (Supplementary Table 3) to, respectively, construct the Dnmt3a and Kcna2 gene reporter plasmids. Vectors containing mutations at the −482, −457, −444 or −440 CpG site were achieved by deleting the corresponding C using the MutanBEST kit (Takara, Berkeley, CA) to achieve site-directed mutagenesis. The PCR products were ligated into the pGL3-Basic vector (containing firefly luciferase reporter gene, Promega, Madison, WI) using the SmaI and HindIII restriction sites. DNA sequencing was carried out to verify the sequences of recombinant clones. HEK-293T cells were cultured as described above. One day after the cells were plated on 12-well plate, the cells were transfected with 40 ng of the pRL-TK plasmid (as a control containing renilla luciferase reporter gene, Promega) alone or plus 1 μg of the pGL3-Basic vectors using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. The wells were divided into different groups as indicated. Two days after transfection, the cells were collected in passive lysis buffer. Approximately 40 μl of supernatant was used to measure the luciferase activity with the Dual-Luciferase Reporter Assay System (Promega). Independent transfection experiments were repeated for three times. The relative reporter activity was calculated after normalization of the firefly activity to renilla activity.