G9a and DNMT1 cooperatively suppress the binding of E2F1 to TP53BP2
To elucidate the potential relationship between DNMT1 and G9a in regulating TP53BP2 expression, DNMT1 or G9a was overexpressed in HTR8/SVneo cells via Ad-DNMT1 and/or Ad-G9a transfection (Figure S8). Overexpression of DNMT1 or G9a decreased E2F1 binding to the TP53BP2 promoter, whereas co-overexpression of both DNMT1 and G9a further attenuated this binding (Figure 8A). Moreover, knockdown of both G9a and DNMT1 significantly decreased TP53BP2 DNA methylation levels and H3K9me2 enrichment at the TP53BP2 promoter in HTR8/SVneo cells under hypoxia (Figure 8B, C ), ultimately resulting in significant upregulation of TP53BP2 transcription and protein expression (Figure 8D, E ). Similar results were also observed in HTR8/SVneo cells treated with DC_05 or/and BIX under hypoxia (Figure S9 ). Notably, the injection of AAV-shG9a or/and AAV-shDNMT1 into the placenta of PE rats significantly increased placental damage, as evidenced by HE staining (Figure 8F ). Additionally, PE rats injected with AAV-shG9a or/and AAV-shDNMT1 presented significant increases in blood pressure and urinary protein levels (Figure 8G, H ), suggesting a synergistic inhibitory effect of G9a and DNMT1 on E2F1 binding to the TP53BP2 promoter in placental dysfunction. Next, we investigated the interactions among E2F1, DNMT1 and G9a in trophoblasts. Co-IP assays revealed that DNMT1 physically interacted with G9a and E2F1 in HTR8/SVneo cells under hypoxia, whereas there was almost no interaction between G9a andE2F1 (Figure 8I ). Immunofluorescence staining revealed that both DNMT1 and G9a were colocalized with E2F1 in the nuclei of HTR8/SVneo cells (Figure 8J). Considering the multiple functional domains of DNMT1, we constructed a series of truncated constructs of DNMT1, and plasmids encoding different GST-tagged DNMT1 fragments (GST-Control, GST-WT, GST-1-446, GST-431-703, GST-643-835, GST-836-1060, and GST-1061-1632) were cotransfected with plasmids encoding Myc-tagged G9a (Myc-G9a) or Flag-tagged E2F1 (Flag-E2F1) into HEK293 cells (Figure 8K ). Co-IP assays revealed that the 1–446 region of DNMT1 interacted with G9a, whereas the 1061–1632 region of DNMT1 interacted with E2F1 (Figure 8L ). Most importantly, a qRT‒PCR assay revealed a marked increase in TP53BP2 expression in HTR8/SVneo cells transfected with the Δ1-446 mutation, whereas transfection with the Δ1061-1632 mutation decreased its expression (Figure 8M ). Furthermore, we constructed plasmids with a deleted region of DNMT1 that interacts with G9a (Δ1-446) or E2F1 (Δ1061-1632) and transfected these mutants into HTR8/SVneo cells. Interestingly, deletion of the 1-446 region of DNMT1 markedly increased the enrichment of E2F1 at the TP53BP2 promoter in HTR8/SVneo cells under hypoxia while reducing the enrichment of DNMT1 at the TP53BP2 promoter. On the other hand, deletion of the 1061–1632 region of DNMT1 promoted E2F1 binding at the TP53BP2 promoter and enhanced H3K9me2 enrichment (Figure 8N). Collectively, these data indicate that the interaction between G9a and DNMT1 can suppress E2F1-mediated activation of TP53BP2 in trophoblasts .