BHLHE40 Cooperates with GATA2/3 to Control Human Syncytiotrophoblast Lineage Differentiation

Abstract Syncytiotrophoblasts (STBs) constitute one of the core components of the placenta, responsible for synthesizing pregnancy‐sustaining hormones such as human chorionic gonadotropin (HCG). Deficient syncytialization of cytotrophoblasts affects the hormonal secretion and placental development, contributing to pregnancy‐associated disorders, including spontaneous miscarriage. To date, the molecular mechanisms, particularly the role of transcription factors (TFs), in STB lineage specification remain incompletely understood. Through targeting direct regulators of a STB lineage‐specific marker, CGB (encoding chorionic gonadotropin‐β), by DNA pull‐down coupled with mass spectrometry, basic helix‐loop‐helix family member 40 (BHLHE40) has been identified as a key regulator in human STB differentiation. BHLHE40 expression is increased during STB differentiation but reduced in villous samples from women with miscarriages. CRISPR/Cas9‐mediated knockout of BHLHE40 in human trophoblast stem cells (TSCs) prevents STB differentiation in vitro, impairing gene expression critical for hormone synthesis and cell syncytialization. Mechanistically, BHLHE40 interacts directly with GATA2 and GATA3 to facilitate their chromatin occupancy, thereby activating transcriptional programs essential for STB differentiation. These findings uncover a BHLHE40‐GATA2/3 regulatory network governing human trophoblast lineage commitment, providing insights into placental development and potential therapeutic targets for pregnancy disorders.