Figure 5. DNA methylation represses TP53BP2 transcription via DNMT1. (A) The promoter activity of TP53BP2 was evaluated via a dual-luciferase reporter assay. Different fragments of the TP53BP2 promoter (-35/+1, -599/+1, -1018/+1, -1530/+1, -599/+641, and -2000/+1) were transfected into HEK293T cells with a Renilla luciferase vector (internal control), and the results are presented as firefly luciferase activity normalized to Renilla luciferase activity. (B) Dual-luciferase reporter assay analysis of the luciferase activities of the TP53BP2 promoter (-599/-35) in HTR8/SVneo and JEG-3 cells under hypoxia. (C) Methylation of the TP53BP2 promoter in HEK293T cells. Following methylation with SssI, HhaI, or HpaII methylases, TP53BP2 promoter fragments were digested with McrBC (a methylation-specific restriction enzyme), HpaII or HhaI (a methylation-sensitive restriction enzyme) to confirm the methylation status of the TP53BP2 promoter construct. (D) The activity of the TP53BP2 proximal promoter methylated with SssI, HhaI, or HpaII methylases was assessed via a luciferase reporter assay in HEK293T cells transfected with luciferase reporter constructs. (E, F) DNA methylation levels of the TP53BP2 promoter were evaluated via methylation-specific PCR (MSP) in placentas and in HTR8/SVneo and JEG-3 cells under hypoxia. U: unmethylated; M: methylated. (G) DNA methylation levels of the TP53BP2 promoter were evaluated via bisulfite sequencing PCR (BSP) in HTR8/SVneo cells under hypoxia. White cycle, unmethylated CpG dinucleotides; black cycle, methylated CpG dinucleotides. The percentage of methylation on each CpG dinucleotide was calculated by the number of methylated clones in each CpG site divided by the total number of clones in the same CpG site and is shown in the right panel. (H) The DNA methylation level of the TP53BP2 promoter was evaluated via BSP in HTR8/SVneo cells treated with DC_05 (a DNMT1-specific inhibitor), Theaflavin-3, 3’-digallate (TFD, a DNMT3a-specific inhibitor) or Nanomycin A (NA, a DNMT3b-specific inhibitor) under hypoxia. (I) The DNA methylation level of the TP53BP2 promoter was evaluated via BSP in HTR8/SVneo cells transfected with sh-DNMT1 under hypoxia. (J) The transcriptional activity of TP53BP2 in HTR8/SVneo cells transfected with sh-DNMT1. (K) TP53BP2 expression was evaluated via western blotting in HTR8/SVneo cells transfected with sh-DNMT1 under hypoxia. The data are presented as the means ± SDs. *P <0.05,**P <0.01,***P <0.001.