Combined EP300 and CREBBP Expression Levels Regulate Human Trophoblast Differentiation

Extravillous trophoblast (EVT) differentiation is essential for placental development and successful pregnancy, yet the molecular mechanisms regulating this process remain incompletely understood. CREB-binding protein (CREBBP) and E1A-binding protein p300 (EP300) are closely related lysine acetyltransferases that function as transcriptional co-activators. Previous studies reported that EP300 depletion impairs EVT differentiation whereas CREBBP depletion has little or no effect. We found that CREBBP mRNA expression was approximately one-third of EP300 expression in trophoblast stem cells (TSCs) and differentiated trophoblast lineages. To investigate whether differential CREBBP and EP300 expression explains this asymmetry in EVT differentiation initiation, we combined siRNA-mediated knockdown, CRISPR-generated CREBBP-deficient TSCs, pharmacological inhibition, and lentiviral Crebbp overexpression during EVT differentiation. While CREBBP depletion alone produced minimal phenotypic effects, CREBBP-deficient cells displayed increased sensitivity to the CREBBP/EP300 inhibitor A-485, with inhibitory effects appearing at lower A-485 concentrations in cells with reduced CREBBP level. Moreover, increased CREBBP expression partially rescued the EP300-depletion phenotype, including EVT-like morphology and selected differentiation marker expression. Together, these findings demonstrate that CREBBP contributes to EVT differentiation, but its role is obscured by its lower endogenous expression. We propose that the combined contributions of CREBBP and EP300, rather than EP300-specific activity alone, influence human trophoblast differentiation efficiency.