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.