Discussion
PE is a condition that typically occurs after the 20th week of
pregnancy and is characterized by high blood pressure and often the
presence of protein in the urine
(proteinuria)35() . Moreover, clinical
evidence indicates that leaving the placenta from the mother can
alleviate symptoms in patients with
PE36(). As a conserved process in
eukaryotes that maintains cellular
homeostasis5(), autophagy can be activated
in PE cases and trophoblasts under
hypoxia37(). Thus, it has been identified
as a target for therapeutic intervention in PE, which supports our findings that excessive autophagy occurs in placentas from PE
pregnancies and in trophoblasts under hypoxia. However, the specific
mechanism of placental dysfunction caused by excessive autophagy should
be further explored. Here, we demonstrated that TP53BP2 could induce
trophoblast autophagy in placentas from early-onset PE
pregnancies. Mechanistically, we found that DNMT1-mediated DNA
hypomethylation and G9a-mediated H3K9me2 suppressed the binding of E2F1
to the TP53BP2 promoter, eventually inhibiting TP53BP2
expression and autophagy in trophoblasts during PE pregnancies.
Moreover, TP53BP2 was found to be a predictive biomarker associated with
the clinicopathological characteristics of early-onset PE and
a promising target for the treatment of early-onset PE.
As a p53-binding protein, TP53BP2 can promote p53-mediated
apoptosis12(). Recent studies have shown
that TP53BP2 is involved in the regulation of diverse diseases through
interactions with different molecules. For example, TP53BP2 was found to
mitigate acute kidney injury induced by ischemia‒reperfusion by
promoting LC3B-II conversion and facilitating p62 degradation
38(). Similarly, Wang et al discovered that TP53BP2 could attenuate HBV replication in hepatocytes
through binding to HSF1, thereby inhibiting the transactivation of Atg7
in a p53-independent manner39(). TP53BP2
can also inhibit RAS-induced senescence by preventing the formation of
the ATG16-ATG5-ATG12 complex40(). In this
study, we not only detected high expression of TP53BP2 in
placentas from PE pregnancies and trophoblasts under hypoxia
but also demonstrated that TP53BP2 knockdown alleviated the
pathological process of PE via the inhibition of autophagy in
trophoblasts. Moreover , TP53BP2 induced autophagy by promoting the
release of Beclin-1 from the Beclin-1-Bcl-2 complex in trophoblasts.
However, more investigations are needed to elucidate the detailed
mechanism involved.
Epigenetic regulation of gene expression involves several mechanisms,
including DNA methylation, histone modification, and the biogenesis and
action of noncoding RNAs40(). They
regulate gene expression by modulating the accessibility of
transcription factors and other regulatory proteins to DNA. Pregnancy
involves dynamic genetic and epigenetic modifications that are essential
for the development and health of both the mother and the fetus. These
modifications encompass a range of processes that influence gene
expression, chromatin structure, and cellular function throughout
gestation 41(). DNA methylation is a
fundamental epigenetic mechanism that usually occurs at cytosine‒guanine
dinucleotide (CpG) sites and is typically mediated by DNMTs (DNMT1,
DNMT3a and DNMT3b)42(). Studies have
reported that DNA methylation regulates diverse biological processes,
including chromatin structure remodeling, gene transcription, genome
imprinting, and chromosome
stability43-45(). In addition, the
processes of pregnancy, including fertilization, embryo implantation,
and placental development during early human early embryo development,
are intricately intertwined with DNA
methylation45(). Therefore, it is
reasonable to hypothesize that PE could be linked to abnormal DNA
methylation patterns in crucial genes. As expected, the TP53BP2 promoter
contains a high percentage of GC bases in the form of CpG islands, and
the TP53BP2 DNA methylation level is markedly decreased in PE. Moreover,
the present study revealed that the suppression of DNMT1 binding betweenE2F1 and the TP53BP2 promoter contributes to the inhibition of
TP53BP2 transcription in trophoblasts. In turn, the inhibition of DNMT1
expression significantly increases TP53BP2 expression by decreasing its
methylation, suggesting that the upregulation of TP53BP2 is mediated in
a DNA methylation-dependent manner.
More importantly, as a transcription activator, after dimerization with
partner proteins, E2F1 binds to DNA via the E2 recognition site of
5’-TTTC[CG]CGC-3’ in the promoter region of various
genes46(). E2F1 is overexpressed in
cancers such as lung cancer, colorectal cancer, and bladder cancer, as
well as sporadic Burkitt’s
lymphomas4748(,).
Consistent with these reports, E2F1 is highly expressed in
placentas from PE pregnancies and trophoblasts under hypoxia.
A previous study reported that E2F1 regulates cell growth and increases
cell size through the activation of mTORC1, a major regulator of protein
synthesis and autophagy49(). In addition,
in the context of obesity, high levels of E2F1 in adipose tissue can
induce the expression of autophagy-related genes and activate
autophagy50(). Consistent with this
observation, our results indicated that high levels of E2F1
significantly upregulated TP53BP2 expression and autophagy in
trophoblasts under hypoxia. Moreover, our work identified three putative
E2F1 binding sites in the TP53BP2 promoter.
In addition, by observing the role of histone methylation in the
pathogenesis of PE , we demonstrated that, under hypoxia, the enrichment
of H3K9me2 at the TP53BP2 promoter was noticeably reduced in HTR8/SVneo
cells, accompanied by the activation of TP53BP2. Our results further
indicated that the reduction in H3K9me2 enrichment was attributed to the
downregulation of G9a and reduced recruitment of G9a to the TP53BP2
promoter in HTR8/SVneo cells under hypoxia. Notably, DNA methylation and
histone modification can often cooperatively regulate gene expression by
modifying chromatin structure51(). In
this study, we found that cooperative inhibition of DNMT1 and G9a led to
TP53BP2 activation, transcription and trophoblast autophagy in PE.
Multiple mechanisms can explain the change in TP53BP2 transcription.
First, DNA methylation and histone modification share the same methyl
donor. Second, the physical interaction between DNMT1 and G9a suggests a
potential mechanism for coordinating DNA methylation and histone
modification of chromatin regions in vivo during
replication52(). Moreover, H3K9me2 can
act as a docking site for chromatin-modifying proteins and
heterochromatin proteins. This interaction subsequently recruits DNMT1
and enhances its activity, resulting in DNA
hypermethylation53(). In addition,
decreased DNMT1 and G9a promote E2F1 binding to the TP53BP2 promoter,
leading to the upregulation of TP53BP2 in trophoblasts. This may occur
through the inhibition of the formation of compact and active chromatin
structures, thereby making the region accessible to E2F1. This
observation was supported by our previous finding that DNA methylation
and H3K9ac are involved in the reduction of miR-195-3p in Hcy-induced
atherosclerosis54(). Intriguingly, G9a
restrains the enrichment of E2F1 in the TP53BP2 promoter via direct
interaction with amino acids 1–446 of DNMT1 but not E2F1.
In conclusion, the interplay between H3K9me2 and DNA hypomethylation
cooperatively suppresses TP53BP2 transcription by inhibiting the binding
of E2F1 to the TP53BP2 promoter, leading to excessive trophoblast
autophagy and consequently the development of PE. Therefore, TP53BP2 may
be a new independent biomarker for the diagnosis or prognostic
prediction of early-onset PE (Fig 9 ).