The role of DNA methylation in human trophoblast differentiation

Abstract

The placenta is a vital fetal exchange organ connecting mother and baby. Specialised placental epithelial cells, called trophoblasts, are essential for adequate placental function. Trophoblasts transform the maternal vasculature to allow efficient blood flow to the placenta and facilitate adequate nutrient uptake. Placental development is in part regulated by epigenetic mechanisms. However, our understanding of how DNA methylation contributes to human trophoblast differentiation is limited. To better understand how genome-wide methylation differences affect trophoblast differentiation, reduced representation bisulfite sequencing (RRBS) was conducted on four matched sets of trophoblasts; side-population trophoblasts (a candidate human trophoblast stem cell population), cytotrophoblasts (an intermediate progenitor population), and extravillous trophoblasts (EVT, a terminally differentiated population) each isolated from the same first trimester placenta. Each trophoblast population had a distinct methylome. In line with their close differentiation relationship, the methylation profile of side-population trophoblasts was most similar to cytotrophoblasts, whilst EVT had the most distinct methylome. In comparison to mature trophoblast populations, side-population trophoblasts exhibited differential methylation of genes and miRNAs involved in cell cycle regulation, differentiation, and regulation of pluripotency. A combined methylomic and transcriptomic approach was taken to better understand cytotrophoblast differentiation to EVT. This revealed methylation of 41 genes involved in epithelial to mesenchymal transition and metastatic cancer pathways, which likely contributes to the acquisition of an invasive EVT phenotype. However, the methylation status of a gene did not always predict gene expression. Therefore, while CpG methylation plays a role in trophoblast differentiation, it is likely not the only regulatory mechanism involved in this process.

Keywords: DNA methylation; Placenta; cytotrophoblasts; extravillous trophoblasts; trophoblast.

Figure 1.

(a) Schematic diagram of a first trimester human placental villus showing the three major mature trophoblast populations. Extravillous trophoblasts are shown invading into the decidua and remodelling a maternal spiral artery (b) Flow diagram showing the lineage differentiation pathway of trophectoderm derived trophoblast populations.

Figure 2.

(a) Cluster dendogram showing hierarchical clustering based on global methylation for side-population trophoblasts (SP), cytotrophoblasts (CTB) and extravillous trophoblasts (EVT) from the four placentae used in this work (denoted by numbers 1–4). (b) Venn diagram of differentially methylated CpG sites between SP and CTB, SP and EVT or CTB and EVT.

Figure 3.

(a) Pie chart showing the mean distribution of CpGs in promoter, exon, intron and intergenic regions across all 12 trophoblast samples. b-d) Pie charts showing the percentage of differentially methylated CpG located in promoter, exon, intron and intergenic regions when (b) side-population trophoblasts (SP) are compared to cytotrophoblasts (CTB), (c) SP are compared to extravillous trophoblasts (EVT), or (d) CTB are compared to EVT.

Figure 4.

(a) Bar graph of the percentage methylation at 16 CpG across 6 genes that were significantly differentially methylation when analysed using RRBS and chosen for further validation using Sequenom methylation analysis in side-population trophoblasts, cytotrophoblasts and extravillous trophoblasts. Each number correlates to the CpG in the Sequenom amplicon presented in Figure 4 plots B-G. (b-g) Dot plots showing percentage methylation of CpGs in GATA2 (b), CPZ (c), CARD9 (d), GALNT6 (e), LOC100289580 (f), and C22orf31 (g) in SP, CTB, and EVT when analysed by Sequenom MassARRAY EpiTYPER Analysis. * indicates an adjusted p value ≤0.05 and ** indicates an adjusted p value ≤0.01, *** indicates an adjusted p value ≤0.001, **** indicates an adjusted p value ≤0.0001. Error bars SEM.

Figure 5.

Bar graphs showing the normalised miRNA expression in side-population trophoblasts (SP), cytotrophoblasts (CTB) or extravillous trophoblasts (EVT) for (a) miR-let-7b, (b) miR-637 and (c) miR4487. ** indicates an adjusted p value ≤0.01. Error bars are SEM.

Figure 6.

Bar graphs showing (a) the normalised mRNA fold change in extravillous trophoblasts (EVT) compared to cytotrophoblasts (CTB) of the top 20 significantly different genes associated with EMT or invasive cancer that were hypomethylated at CpG islands in promoters regions of EVT compared to CTB (b) the normalised mRNA fold change in CTB compared to EVT of the top 20 significantly different genes that were hypermethylated at CpG island in promoters regions of CTB compared to EVT.