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 ).