Figure 2.

NRF1 coordinates with DNA methylation to modulate ASZ1 expression. A) Asz1 core promoter contains 2 NRF1 binding sites. B) ChIP assays followed by real-time PCR analyses with a NRF1 antibody from various tissues on the Asz1 promoter containing predicated NRF1 binding sites. C, D) Gel shift assays of a GST-NRF1 fusion protein with synthesized FITC- or ³²P-labeled probe containing NRF1 binding sites 1 (C) and 2 (D), respectively. The GST-ASZ1 fusion protein was used as a negative control. E) Activities of luciferase driven by wild-type, mutated (with 1 or both NRF1 binding site deleted: e.g., pΔb1), or methylated promoter of Asz1 were measured upon cotransfection of a NRF1 transgene or an empty vector control (ctrl). The pGL3 is an additional vector control without Asz1 promoter. Relative firefly luciferase activity is normalized to the Renilla and represented as mean ± 1 sem from 6 biologic replicates. *P < 0.05, **P < 0.01, ***P < 0.001. F) Vector containing the Asz1 promoter (pASZ1 in E) was treated with or without CpG methyltransferase M.SssI (MpAsz1) and methylation status was confirmed by digestion of the methylation-sensitive restriction enzyme HpaII.