Figure 8. DNMT1 and G9a cooperatively regulate TP53BP2 expression in PE. (A) Enrichment of E2F1 at the promoter region of TP53BP2 was analyzed via ChIP in HTR8/SVneo cells transfected with Ad-DNMT1 and/or Ad-G9a under hypoxia. (B) BSP analysis was performed to determine the TP53BP2 DNA methylation level in HTR8/SVneo cells transfected with sh-DNMT1 and/or sh-G9a under hypoxia.(C) H3K9me2 enrichment at the promoter region of TP53BP2 was analyzed via ChIP in HTR8/SVneo cells transfected with sh-DNMT1 and/or sh-G9a under hypoxia. (D) The promoter transcription activity of TP53BP2 was analyzed via a dual-luciferase reporter assay in HTR8/SVneo cells transfected with sh-DNMT1 and/or sh-G9a under hypoxia.(E) Western blotting was performed to determine TP53BP2 expression in HTR8/SVneo cells transfected with sh-DNMT1 and/or sh-G9a under hypoxia. (F) Placental pathological changes in PE rats injected with AAV-shG9a and/or AAV-shDNMT1 were evaluated via HE staining. Scale bars=500 μm. (G) Noninvasive tail-cuff blood pressure measurement system used to detect the systolic blood pressure and diastolic blood pressure of PE rats injected with AAV-shG9a and/or AAV-shDNMT1. (H) Total urine protein levels in PE rats injected with AAV-shG9a and/or AAV-shDNMT1. (I) Co-IP assay followed by immunoblotting showing the interactions between G9a and DNMT1 or E2F1 in HTR8/SVneo cells under hypoxia. (J) Immunofluorescence staining was used to measure the colocalization of DNMT1 (red) and E2F1 (green) or G9a (green) in HTR8/SVneo cells under hypoxia. Nuclei were stained with DAPI. Scale bar=20 μm. (K) Schematic diagram depicting the structure of DNMT1 and truncation mutants of the GST-tagged DNMT1 fragments (GST-Control, GST-WT, GST-1-446, GST-431-703, GST-643-835, GST-836-1060, and GST-1061-1632). (L) The interactions between DNMT1 and G9a or between DNMT1 and E2F1 were examined via Co-IP with an anti-Myc antibody in HEK293T cells cotransfected with plasmids encoding different GST-tagged DNMT1 fragments and plasmids encoding Myc-tagged G9a (Myc-G9a) or Flag-tagged E2F1 (Flag-E2F1), respectively.(M) The transcriptional activity of the TP53BP2 promoter in HTR8/SVneo cells transfected with the Δ1-446 mutation or the Δ1061-1632 mutation. (N) The enrichment of E2F1, DNMT1, G9a and H3K9me2 at the promoter region of TP53BP2 in HTR8/SVneo cells transfected with wild-type DNMT1 (WT), the G9a binding region deletion mutant DNMT1 (Δ1-446) or the E2F1 binding region deletion mutant DNMT1 (Δ1061-1632) was assessed via a ChIP assay under hypoxia. The data are presented as the means±SDs. Student’s t test (unpaired, two-tailed) was used to compare two independent groups, and two-way ANOVA was performed for comparisons of multiple groups.*P <0.05,**P <0.01,***P <0.001;#P <0.05.