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.