CDK8 regulates definitive endoderm commitment through cAMP-lysosomal homeostasis and neuroectodermal suppression

Abstract Definitive endoderm (DE) specification is essential for gastrointestinal and respiratory organogenesis, yet upstream regulators that integrate signaling pathways with metabolic and epigenetic cues remain poorly understood. Here, we identified cyclin-dependent kinase 8 (CDK8) as an important regulator of human DE commitment. CDK8 deficiency significantly impaired DE formation and subsequent hepatic differentiation via EPAC-dependent cAMP hyperactivation and lysosomal acidification impairment. Multi-omics analyses revealed that CDK8 repressed neuroectoderm and cAMP pathway genes while activating endodermal and lysosomal programs, with associated changes in promoter-proximal H3K27me3 deposition. We further found that CDK8 and OTX2 coordinately regulated a FOSL2 / HNF4A / FOXH1 network in which FOXH1 serves as a downstream effector node, maintaining EPAC-dependent cAMP homeostasis and lysosomal acidification, thereby suppressing neuroectodermal gene expression while promoting endodermal commitment. Moreover, OTX2 could compensate for CDK8 loss in regulating this process. In summary, our study identified CDK8 as an upstream regulator that coordinates signaling, metabolic, and epigenetic cues to promote endodermal specification while restraining neuroectodermal mis-specification, providing new mechanistic insights into human endoderm development.