Hypomethylation of PRDM1 is associated with recurrent pregnancy loss

Abstract Recurrent pregnancy loss (RPL) rates have continued to rise during the last few decades, yet the underlying mechanisms remain poorly understood. An emerging area of interest is the mediation of gene expression by DNA methylation during early pregnancy. Here, genome‐wide DNA methylation from placental villi was profiled in both RPL patients and controls. Subsequently, differentially expressed genes were analysed for changes in gene expression. Many significant differentially methylated regions (DMRs) were identified near genes dysregulated in RPL including PRDM1. Differentially expressed genes were enriched in immune response pathways indicating that abnormal immune regulation contributes to RPL. Integrated analysis of DNA methylome and transcriptome demonstrated that the expression level of PRDM1 is fine‐tuned by DNA methylation. Specifically, hypomethylation near the transcription start site of PRDM1 can recruit other transcription factors, like FOXA1 and GATA2, leading to up‐regulation of gene expression and resulting in changes to trophoblast cell apoptosis and migration. These phenotypic differences may be involved in RPL. Overall, our study provides new insights into PRDM1‐dependent regulatory effects during RPL and suggests both a mechanistic link between changes in PRDM1 expression, as well as a role for PRDM1 methylation as a potential biomarker for RPL diagnosis.


| INTRODUC TI ON
Recurrent pregnancy loss (RPL), defined as more than two consecutive miscarriages, is viewed as a distinct disorder. 1 It is estimated that 5% of women experience two clinical miscarriages and approximately 1% experience three or more losses. 2 The aetiology of RPL is complicated, with known causal factors of RPL including genetic factors, anatomic abnormalities, autoimmune and endocrine factors. 3 However, the aetiology of approximately half of RPL cases still remains unknown. [3][4][5] During pregnancies, extravillous trophoblast cells invade the decidualized endometrium and remodel uterine spiral arteries to increase maternal blood flow to the placenta villi. 6 The transitions from undifferentiated progenitors to differentiated trophoblast cells require dynamic epigenetic regulation, 6 suggesting a role for DNA methylation as a potential mechanism regulating trophoblast differentiation. Despite numerous studies demonstrated that aberrant DNA methylation of genes is closely correlated with pregnancy loss, it remains unclear the roles of DNA methylome and transcriptome perturbations in RPL patients. 7 In this study, we aimed to investigate the DNA methylome and transcriptome perturbations in RPL patients.

| DNA isolation and bisulphite conversion
1μg DNA was used for bisulphite conversion with the EZ DNA methylation kit. (detailed in the Supplementary Materials).

| Genome-wide methylation profiling
Methylation data were processed using the R ChAMP package. (detailed in the Supplementary Materials).

| Luciferase reporter assay
See Supplementary Materials.

| Cell cycle, cell apoptosis and cell migration
See Supplementary Materials.

| Statistical analysis
See Supplementary Materials.

| Differential DNA methylation pattern between recurrent pregnancy losses (RPLs) and control
We performed Illumina EPIC Bead Chip to investigate the differential DNA methylation between normal controls (n = 4) and RPL patients (n = 2). The overall methylation pattern was significantly different between cases and controls (Supplementary Material, Figure S1A, B). Evaluating genome-wide DNA methylation level by β value, both site-specific hyper-and hypomethylation was observed ( Figure 1A,B). We totally observed 147 711 significant DMPs  Table S3). An example of hypo DMR is displayed in Figure 1E. The distribution patterns of hyperand hypo-methylated DMRs were similar ( Figure 1F). Functional annotation of genes near hypo-methylated DMRs demonstrates enrichment for development signalling pathways ( Figure 1G).
Meanwhile, the hyper-methylated DMRs were enriched in pathways related to embryo development ( Figure 1H), suggesting DNA methylation during development is a fine-tuning mechanism.

| DNA methylation affected Gene Expressions in recurrent pregnancy losses (RPLs)
In order to assess whether DNA methylation changes affect the gene To further explore the DNA methylation responsible for the regulation of PRDM1, we carried out the dual-luciferase reporter assay (Figure 2A). The reporter vector with either the methylated or the unmethylated insert was co-transfected with an internal control (Renilla vector) into the human trophoblast cell line HTR-8/SVneo, JEG-3 and 293T cell line, respectively. Luciferase activities of unmethylated PRDM1 DMR were significantly (P < .001) higher than that with the methylated insert in these three cell lines (Figure 2A).

| PRDM1 DMR regulated trophoblast cells apoptosis and migration by recruiting GATA2 and FOXA1
To determine whether PRDM1 have effects on cell functions, we overexpressed PRDM1 in HTR-8/SVneo and JEG-3 cell lines. As shown in Figure 2B-E, apoptosis levels were significantly increased in the HTR-8/SVneo (P < 0.01) and JEG-3 cell lines (P < 0.01) after overexpressed PRDM1. Additionally, the high expression level of PRDM1 delayed cell progression from S to M cell cycle transition in JEG-3 cell line (P < 0.05) ( Figure 2F). By transwell cell migration assays, we found that the number of cells migrating through transwell pores was significantly increased after PRDM1 overexpression in HTR-8/SVneo cell ( Figure 2G).
In order to test whether there were other factors binding to the hypo-methylated region of PRDM1, we used the online tool called PROMO to predict the putative transcription factor binding sites in DNA sequences based on TRANSFAC database. 10,11 We validated these binding sites by ChIP-qPCR, and the results showed that

| D ISCUSS I ON
In the present study, we set out to identify changes in placental villi from recurrent pregnancy loss, when compared to healthy normal term placenta. PRDM1 was identified to be both significantly differentially methylated and expressed. To validate the functional effects of these changes, knockdown studies were performed in the placental cell lines, JEG-3 and HRT8. These findings demonstrate that epigenetic regulation of PRDM1 is a potential aetiological factor for recurrent pregnancy loss. PRDM1, a transcription regulator of cell fate in the embryo, is required for primordial germ cell specification and reprogramming of intestinal enterocytes. 12 In our study, compared with controls, PRDM1 was found to be expressed significantly higher in placental villi of RPL patients. All together, these results suggested that not only PRDM1 deficiency, but also excess would both cause adverse effects on embryo development. Furthermore, we found that the DMR near transcription start site of PRDM1 was hypo-methylated.
Hypomethylation in regulatory region will open the chromatin region and expose DNA sequence to transcription factors, which will recruit cofactors and affect the gene transcription. 13 In this study, we found that hypomethylation of PRDM1 could recruit GATA2 and FOXA1 binding. GATA2, a transcription factor in GATA family, was reported to regulating gene regulatory network during self-renew and differentiation of the trophoblast cells. 14 Specifically, others have shown that knockout Gata2/Gata3 led to embryonic lethality probably through impairing trophoblast development. 15 Additionally, we identified major differences in immune genes in the transcriptomic analysis. Similar to previous findings that suggest disruption of immune tolerance will affect normal pregnancy, transcriptomic data for RPL patients were significantly changed in immune genes. Specifically, disturbance of immune balance induced pro-inflammatory with highly expressed chemokines (CXCL8, CCL2) and adhesion molecules (ICAM1) in RPL 16 was observed in our data.
In summary, we propose a model ( Figure 2K) that hypomethylation at the PRDM1 promoter induces the binding of GATA2 and FOXA1, leading to an induction of PRDM1 expression. This increase in expression results in increased migration and apoptosis in trophoblast cells, leading to RPL. Of importance, this suggests that the methylation level of PRDM1 may be a promising target for intervention in RPL as well as a promising biomarker for early diagnosis of RPL.

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts of interest associated with this study.

AUTH O R S ' CO NTR I B UTI O N S
XW, CL and YX directed the study, obtained financial support and were responsible for study design. GD, MY, QX and ZH performed experiments. MY and YQ analysed data. GD, MY and YQ wrote the manuscript. XH, LH, YF, YZ, RW, SX, XH, GF and SL provided reagents and processed tissue samples. All authors read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.