Active remodeling of the chromatin landscape directs extravillous trophoblast cell lineage development

Abstract

The extravillous trophoblast (EVT) cell lineage is a key feature of placentation and critical for spiral artery remodeling and successful pregnancy. Our knowledge of transcriptional regulation driving EVT cell development is limited. Here, we mapped the transcriptome and epigenome landscape as well as chromatin interactions of human trophoblast stem (TS) cells and their transition into the differentiated EVT cell lineage. Chromatin accessibility in intergenic regions was more extensive in EVT cells than in TS cells in the stem state, which is consistent with increased enhancer-driven gene regulation. Using reference epigenome annotation, we noted that 18% of the chromatin landscape in EVT cells was uncharted. We linked regulatory regions to their cognate target genes and characterized the three-dimensional organization of the TS cell functional genome by high throughput chromosome conformation capture. Integration of chromatin accessibility, long-range chromatin interactions, transcriptomic, and transcription factor binding motif enrichment enabled identification of transcription factors and regulatory mechanisms associated with EVT cell lineage development. Subsequent analyses elucidated functional roles for TFAP2C, SNAI1, and EPAS1 in the regulation of EVT cell lineage development. EPAS1 was identified as an upstream regulator of key EVT cell transcription factors, including ASCL2 and SNAI1 and together with its target genes, was linked to diseases of pregnancy, including idiopathic recurrent pregnancy loss and low birth weight. Collectively, we have revealed activation of a dynamic regulatory network that provides a framework for understanding EVT cell specification in trophoblast cell lineage development and human placentation.

Single-cell assessment of trophoblast stem cell-based organoids as human placenta-modeling platforms

“…Future models incorporating decidual cell (uterine gland epithelia, blood vessel cells, and immune cells) co-cultures may address this limitation, however the integration of such complex models pose significant challenges. Regardless, gene regulatory analyses confirm the importance of SOX4, ASCL2 and SNAI1 TFs involved in extravillous differentiation in vivo and in vitro [53][54][55] while identifying additional new TF targets (i.e., TCF7L2, BATF3, XRCC4, MXD4). Alternatively, computational modeling resolved two routes of TSC maturation towards the SCTp state in hTSC-TOrgs: one conventional CTB to SCTp route defined by fusogenic gene enrichment, and an alternative direct TSC to SCTp route defined by endocrine gene enrichment.…”

Core Conserved Transcriptional Regulatory Networks Define the Invasive Trophoblast Cell Lineage

“…Candidate TFs driving gene regulation in invasive trophoblast cells were identified through their expression in invasive trophoblast cells and through the presence of corresponding TF DNA binding motifs associated with invasive trophoblast cell specific genes. The most striking TF families linked to the invasive trophoblast cell lineage exhibit conservation in human EVT cells [39] and have been previously implicated in trophoblast cell biology [70,71]. Most interestingly, many of the invasive trophoblast cell relevant TFs are implicated in early phases of trophoblast cell lineage development or the differentiation of other trophoblast cell lineages.…”

“…To determine if transcripts that have multiple associated peaks in rat invasive trophoblast cells also possess multiple associated peaks in human EVT cells, we incorporated open regions (ATAC-seq peaks) identified in EVT cells into our analysis [39]. First, we associated the EVT cell open regions to genes.…”

“…We found that of the 439 rat peaks with TFAP2C motifs, 208 (47.38%) overlapped with TFAP2C peaks in differentiated mouse TS cells, which was significant (p-value=0.009). Additionally, all 11 TF families enriched in the rat peaks were enriched in EVT cell ATAC-seq peaks [39] (Table S3). These comparisons provide evidence for the validity of the computationally based binding site predictions.…”

“…In this report, we interrogated the chromatin landscape of invasive trophoblast cells isolated from the uterine-placental interface of the rat using single-nucleus assay for transposase-accessible chromatin-sequencing (snATAC-seq). These datasets were integrated with scRNA-seq datasets from rat and human invasive trophoblast/EVT cells [20], as well as ATAC-seq from EVT cells [39], to identify conserved gene regulatory networks controlling the invasive trophoblast cell lineage.…”