DNMT1 inhibited TP53BP2 expression via inversely modulating E2F1
TP53BP2 has been reported to be a direct target of E2F1. Here, both qRT‒PCR and western blot assays revealed a significant increase in E2F1 levels in placentas from PE pregnancies compared with those from non-PE pregnancies (Figure 6A ). This finding was further confirmed by immunofluorescence staining of trophoblasts from the placentas of PE pregnancies (Figure 6B ). Next, we examined the impact of E2F1 on TP53BP2 expression by overexpressing or knocking down E2F1 in HTR8/SVneo and JEG-3 cells (Figure S6 ). E2F1 overexpression and E2F1 knockdown increased and inhibited TP53BP2 expression, respectively, in HTR8/SVneo cells under hypoxia (Figure 6C ). Furthermore, a chromatin immunoprecipitation (ChIP) assay using an anti-E2F1 antibody revealed significant enrichment of E2F1 at the TP53BP2 promoter under hypoxia (Figure 6D ). Next, via the use of the JASPAR database to compute putative transcription factor binding elements encompassing the hypomethylated CpG site in the TP53BP2 promoter, we identified three putative E2F1 binding sites (-33/-22, -99/-88 and -368/-357 )in the TP53BP2 promoter (Figure 6E ). A ChIP assay revealed remarkable binding of E2F1 with the TP53BP2 promoter at the -33/-22, -99/-88 and -368/-357 sites (Figure 6F). In addition, a luciferase reporter assay revealed a significant decrease in the activity of the TP53BP2 promoter after mutation of the -33/-22 site (Mut1), -99/-88 site (Mut2) and -368/-357 site (Mut3) (Figure 6G ). As methyltransferases can regulate target gene expression by directly interacting with transcription factors33(), we th en adopted the Co-IP method to investigate the relationship between DNMT1 and E2F1. Consistent with a previous report, DNMT1 physically interacts with E2F1 in trophoblasts under hypoxia (Figure 6H ). Moreover, a ChIP assay further revealed that knockdown of DNMT1 increased the enrichment of E2F1 in the TP53BP2 promoter (Figure 6I). This result demonstrated that DNMT1 mediated the binding between E2F1 and the TP53BP2 promoter. Collectively, these data suggest that DNMT1 suppresses E2F1 binding to the TP53BP2 promoter, resulting in reduced autophagy in trophoblasts under hypoxia.