The Multifaceted Role of Nanog in Endoderm Differentiation: Unveiling the Nanog-Tbx3-Gata6 Regulatory Network in mESCs

Nanog plays a pivotal role in the second fate decision during preimplantation, yet its molecular mechanism remains incompletely understood. Here, we have discovered that Nanog forms a complex with Tbx3 to sustain the self-renewal of mESCs through the repression of endoderm genes Gata6, Gata4, and Sox17 via newly identified distal enhancers. Activation of these enhancers converts ESCs into XEN cells. When Nanog is lost, the repression by the PRC2 complex on Gata6 expression is lifted, leading to Tbx3-independent initiation of Gata6 expression. Additionally, we found that Tbx3 forms a complex with Gata6 to promote the expression of Gata6, Gata4, and Sox17, thereby facilitating endoderm differentiation initiated by the Nanog deletion. Our study also sheds light on Nanog's ability to regulate the binding of Setd2 on chromatin, thereby influencing the deposition of histone H3K36me3 and modulating RNA splicing efficiency, which ultimately regulates the expression of endoderm genes. In summary, our findings unveil the intricate and multi-layered regulatory network of Nanog in the process of endoderm differentiation in ESCs.