Comparing gene expression effects of Hnrnpf knockout in mESCs while they are pluripotent vs. after they have been differentiating for 4 days due to LIF removal [RNA-seq]

The ERK (extracellular signal-regulated kinase) signaling pathway plays a crucial role in maintaining the self-renewal and regulating the differentiation of mouse embryonic stem cells (ESCs). However, the underlying mechanisms by which ERK exerts these effects remain incompletely understood. In this study, we identified heterogenous nuclear ribonucleoprotein F (HNRNPF) as a novel ERK2-interacting protein and phosphorylation substrate. We demonstrated that ERK2 phosphorylates HNRNPF on Ser346 and Tyr356. Hnrnpf knockout (KO) ESCs manifested impaired self-renewal, due to downregulation of CDK1 and CCNB1. Notably, phosphorylation of HNRNPF rescued CDK1 and CCNB1 expression, leading to restoring self-renewal capacity of Hnrnpf KO ESCs. Additionally, loss of HNRNPF promoted ESC differentiation toward mesoderm and trophectoderm lineages, a phenotype attributed to reduced EED expression and diminished H3K27me3 levels. Phosphorylation of HNRNPF restored EED expression and H3K27me3 levels, thereby mitigating differentiation bias in Hnrnpf KO ESCs. Collectively, our findings uncover a critical role for HNRNPF as a downstream effector of ERK2 signaling in regulating ESC self-renewal and differentiation.