Ectopic expression of DNMT3L in human trophoblast stem cells restores features of the placental methylome [ChIP-seq]

The placental DNA methylation landscape is unique, with widespread partially methylated domains (PMDs) and hundreds of placental-specific imprinted domains. Furthermore, the placental 'methylome' has been the focus of extensive study for links with pregnancy complications. Human trophoblast stem cells (hTSCs) offer exciting potential for functional epigenetic studies; however, whether the hTSC epigenome recapitulates primary cytotrophoblast, remains poorly described. In this study, we demonstrate that hTSCs exhibit an atypical methylome, with DNA methylation present over transcribed gene bodies but a complete loss of placental PMDs. Using single-cell RNA-seq from human embryogenesis, hTSCs display a notable absence of DNMT3L expression. Ectopic expression of DNMT3L in hTSCs restored placental PMDs. DNMT3L-expressing hTSCs showed comparable stemness but failed to syncytialise in organoid culture, associated with hypermethylation of STB transcription factor motifs. These findings reveal that DNMT3L is essential in establishing the human placental methylome and that DNMT3L downregulation is necessary for successful trophoblast differentiation. Overall design: To understand determinants of the DNA methylation landscape in hTSCs, we cultured blastocyst- (BTS11) and 1st trimester cytotrophoblast- (CT29) derived hTSCs in 2D conditions (Okae et al., 2018), 2D in organoid media, 3D as organoids (Sheridan et al., 2021), and as organoids cultured in hypoxic conditions. We assayed histone modifications using ChIP_seq to evalue the localisation of DNA methylation in hTSCs. Please note that the following published datasets were re-analyzed as replicates (as detailed in the re-analyzed_sample_details.txt). The accessions of the public datasets used in the current study: Sample GSM accns Series SRX accns Biosample accns ================================================ GSM3633664 GSE127288 SRX5443225 SAMN11029824 GSM3633665 GSE127288 SRX5443226 SAMN11029822 GSM3633672 GSE127288 SRX5443233 SAMN11029813 GSM3633673 GSE127288 SRX5443234 SAMN11029848 GSM3633676 GSE127288 SRX5443237 SAMN11029842 GSM3633677 GSE127288 SRX5443238 SAMN11029846 GSM5862947 GSE196221 SRX14060225 SAMN25690350 GSM5862950 GSE196221 SRX14060228 SAMN25690347 GSM5862951 GSE196221 SRX14060229 SAMN25690346 GSM6043305 GSE200760 SRX14845721 SAMN27567865 GSM6043306 GSE200760 SRX14845722 SAMN27567864 GSM6043307 GSE200760 SRX14845723 SAMN27567863 GSM6043317 GSE200761 SRX14845739 SAMN27567878 GSM6043318 GSE200761 SRX14845740 SAMN27567877 GSM1436879 GSE59434 SRX652746 SAMN02911906 GSM1436880 GSE59434 SRX652747 SAMN02911907 GSM1436881 GSE59434 SRX652748 SAMN02911908 GSM1436882 GSE59434 SRX652749 SAMN02911909 GSM2232855 GSE84382 SRX1945295 SAMN05389843 GSM2232856 GSE84382 SRX1945296 SAMN05389844 GSM2232857 GSE84382 SRX1945297 SAMN05389845 GSM2232858 GSE84382 SRX1945298 SAMN05389846 GSM2232859 GSE84382 SRX1945299 SAMN05389847 GSM2232860 GSE84382 SRX1945300 SAMN05389848