hCINAP regulates the differentiation of embryonic stem cells by regulating NEDD4 liquid-liquid phase-separation-mediated YAP1 activation

YAP1 functions in lineage differentiation of pluripotent embryonic stem cells (ESCs); however, the detailed mechanisms underlying the regulation of YAP1 activity during ESC differentiation remain elusive. Here, we report that hCINAP serves as a negative regulator of YAP1 during ESC fate decisions. The expression of mCINAP, the murine homolog of hCINAP, is downregulated during the differentiation process of murine ESC (mESC) ectoderm lineage, leading to liquid-liquid phase separation (LLPS) of NEDD4 and activation of YAP1. Mechanistically, hCINAP interacts with and prevents NEDD4 from forming cytoplasmic condensates that compartmentalize YAP1 and its kinase NLK, facilitating YAP1 phosphorylation at Ser128 and promoting YAP1 activation. mCINAP depletion leads to the formation of NEDD4 condensates and YAP1 activation, which impedes endoderm differentiation of mESCs. Our study shows that hCINAP is a vital regulator of YAP1 activity and is essential for stem cell fate decisions, which provides mechanistic insight into early embryogenesis. [Display omitted] •Deubiquitinase OTUB1 stabilizes hCINAP to regulate YAP1 activity•hCINAP interacts with NEDD4 and prevents NEDD4 from forming cytoplasmic condensates•NEDD4 LLPS promotes YAP1 activity through upregulating its phosphorylation at Ser128•hCINAP and NEDD4 synergistically regulate lineage differentiation of ESCs Zhuge et al. demonstrate that hCINAP acts as a negative regulator of YAP1 by modulating NEDD4 liquid-liquid phase separation and identify the role of the OTUB1-hCINAP-NEDD4-YAP1 axis in regulating the differentiation of ESCs.