DNMT1-mediated DNA methylation inhibits TP53BP2 transcription
First, we analyzed the genomic sequence of the TP53BP2 promoter to
evaluate the effect of epigenetic regulation on TP53BP2 expression via
the UCSC database. As expected, the TP53BP2 promoter contained a high
percentage of GC bases in the CpG islands (Figure S4A ). The
MethPrimer program identified a single CpG island measuring 1240 base
pairs in length. This island spans from position -599 to +641 relative
to the transcription start site. It has a CG content of 50% and a CpG
ratio of 0.6. This CpG island is located at the distal end of the
5’-flanking region of TP53BP2 (Figure S4B ), which may regulate
TP53BP2 levels through methylation. Several fragments of the TP53BP2
5’-flanking region were subsequently inserted into the firefly
luciferase vector pGL3, and the results of the luciferase activity assay
revealed that the -599–35 fragment, which spans most of the CpG
dinucleotides of the TP53BP2 promoter, presented the highest promoter
activity (Figure 5A). Consistent with this result , a luciferase
assay revealed increased transcription activity of TP53BP2 in both
HTR8/SVneo and JEG-3 cells after the cells were transfected with a
luciferase reporter of pGL3 harboring the fragment (-599/-35)
(Figure 5B ), indicating that this region (-599–35) serves as
the core regulatory region for TP53BP2. To examine whether DNA
methylation directly represses TP53BP2 promoter activity, we cloned the
TP53BP2 proximal promoter region from -599–35. The cloned inserts
were then methylated via the methylases Sss I (M.Sss I), Hha I (M.Hha
I), and Hpa II (M.Hha II). Sss I was adopted to methylate all 51
CpG sites within the sequence 5’-CpG-3’, Hha I methylated only 9 CpG
sites within the sequence 5’-GCGC-3’, and Hpa II methylated 3 CpG sites
within the sequence 5’-CCGG-3’. Proper methylation of the fragments was confirmed by digestion with the restriction
enzymes McrBC (methylation-specific restriction enzyme), Hha I, and Hpa II (methylation-sensitive restriction enzyme) (Figure 5C). By
transfecting a luciferase reporter vector into trophoblasts and
performing a luciferase assay, we observed that treatment with
three kinds of methylases reduced TP53BP2 promoter activity. Notably, Sss I methylase had the most significant inhibitory effect
(Figure 5D ). Next, we detected differences in TP53BP2 DNA
methylation levels via methylation-specific PCR (MSP). As shown inFigure 5E, F , global DNA methylation levels were
decreased in placentas from PE pregnancies and trophoblasts under hypoxia. Bisulfite sequencing PCR (BSP) further revealed a
remarkable decrease in DNA methylation levels within the (-599/-35)
region of the TP53BP2 promoter in HTR8/SVneo cells under hypoxia
(Figure 5G ). These results revealed that DNA
hypomethylation modulates the transcriptional activation of TP53BP2 in
trophoblasts in the placenta of PE pregnancies.
To determine the key enzymes involved in DNA methylation, HTR8/SVneo
cells were treated with DC_05 (DNMT1 inhibitor), Theaflavin-3,
3’-digallate (TFD, DNMT3a inhibitor) or Nanomycin A (NA, DNMT3b
inhibitor) under hypoxia. As shown in Figure 5H, DC_05
treatment, but not TFD or NA treatment, caused a significant decrease in
the DNA methylation level of the TP53BP2 promoter in HTR8/SVneo cells
under hypoxia. To further confirm the role of DNMT1 in regulating
TP53BP2 DNA methylation, we constructed HTR8/SVneo cells with DNMT1
knockdown (Figure S5 ). The results revealed that the knockdown
of DNMT1 decreased the DNA methylation level of the TP53BP2 promoter
(Figure 5I ) and increased the transcriptional activity and
protein levels of TP53BP2 (Figure 5J, K ). Taken together, these
results suggest that DNMT1-mediated DNA methylation strongly inhibits
TP53BP2 expression.