Thesis: Paula Doria Borrell

Directors: Vicente Pérez García

Title: The role of BAP1 in early human placentation: Implications for trophoblast differentiation and preeclampsia

Abstract: The human placenta is a highly specialized, transient organ essential for fetal growth and maternal adaptation throughout pregnancy. Acting as the primary interface between maternal and fetal tissues, it mediates the exchange of nutrients, oxygen, hormones, and waste products, while also exerting crucial endocrine and immunological functions.
Placentation originates from the trophectoderm of the blastocyst, where proliferative cytotrophoblasts differentiate into two key lineages: extravillous trophoblasts, which invade the maternal endometrium and remodel spiral arteries, and syncytiotrophoblasts, which form the direct maternal–fetal interface. Disruptions in these processes underlie major pregnancy complications, with preeclampsia being a leading cause of maternal and perinatal morbidity and mortality. Understanding the molecular mechanisms governing early placentation is therefore critical for improving the diagnosis and treatment of pregnancy disorders. Recent advances have established immortalized human trophoblast stem cell lines capable of differentiating into both lineages, as well as three-dimensional trophoblast organoids that recapitulate the cellular and functional properties of the human placenta, providing powerful new platforms for mechanistic studies.
BRCA1-associated protein 1 (BAP1) is a nuclear deubiquitinase and tumor suppressor with broad functions in cell cycle regulation, apoptosis, DNA damage repair, metabolism, ferroptosis, and gene expression. As the catalytic subunit of the Polycomb Repressive–Deubiquitinase (PR-DUB) complex, BAP1 removes ubiquitin from monoubiquitinated histone H2A at lysine 119 (H2AK119Ub), counteracting Polycomb Repressive Complex 1 (PRC1).
Functional studies demonstrate that BAP1 is essential for early placentation in mice, and its regulatory role within PR-DUB appears conserved in humans. Intriguingly, BAP1 levels must be downregulated during trophoblast invasion, a phenomenon reminiscent of the mechanisms by which BAP1 mutations promote tumor metastasis. Building on these findings, this thesis explores the role of BAP1 in early human placental development, with the goal of understanding the molecular pathways controlled by BAP1 and how their disruption can contribute to pregnancy complications.
To address these questions, we employed immortalized human trophoblast stem cell (hTSC) lines and primary villous cytotrophoblasts isolated from first-trimester placentas, cultured in both two-dimensional and three-dimensional systems. In parallel, placental tissues from two independent clinical cohorts, together with publicly available datasets, were analysed. A combination of
molecular, functional, transcriptomic, proteomic, and epigenetic approaches was applied to assess the effects of BAP1 knockdown and overexpression on trophoblast biology.
The results of this thesis identifies BAP1 as a pivotal regulator of trophoblast stemness and epithelial identity, with a conserved role between human and mouse trophoblast cells. In hTSC and organoid models, sustained expression of BAP1 preserves the undifferentiated state by reinforcing cell–cell adhesion, whereas its downregulation activates transcriptional programs driving differentiation toward both lineages. Loss of BAP1 does not disrupt the core stem-cell gene regulatory network but increases proliferation, while BAP1 overexpression reduces proliferation and promotes adhesion-related pathways. These findings confirm the evolutionary conservation of BAP1’s dual role in stabilizing epithelial features and restraining proliferation in stem cells, while ensuring proper differentiation upon downregulation—providing a mechanistic framework for understanding trophoblast dysfunction in early-onset preeclampsia (EO-PE) placentas.
Integrated transcriptomic and proteomic analyses revealed specific pathways altered by BAP1 dysregulation, including glycoprotein metabolism and MAPK, Hippo, and JAK–STAT signaling. An organoid-derived gene signature generated from BAP1 overexpression clearly segregated EO-PE placentas into distinct clusters but failed to separate placentas from intrauterine growth restriction (IUGR) or gestational diabetes mellitus (GDM). This specificity indicates that BAP1-driven transcriptional changes are aligned with EO-PE pathophysiology rather than general placental dysfunction. Supporting this, tissue-based studies showed upregulation of BAP1 and its PR-DUB partners—particularly ASXL3 and ASXL2—in EO-PE but not in late-onset preeclampsia (LO-PE) or IUGR, suggesting that aberrant PR-DUB activity is specifically associated with the more severe, early-onset form of the disease.
This work uncovers how a well-known tumor suppressor orchestrates early placental development. BAP1’s role in maintaining epithelial identity and its regulated downregulation during invasive specification mirrors phenotypes observed in BAP1-deficient tumors. BAP1-overexpressing trophoblasts recapitulates key features of EO-PE, including disrupted STB and EVT formation, enhanced adhesion, and reduced proliferation, providing a physiologically relevant in vitro model.