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. Overall design: To evaluate the differentiation potential of Hnrnpf KO ESCs, we performed RNA sequencing (RNA-seq) to profile global transcriptional changes in both differentiated and undifferentiated ESCs following HNRNPF deletion.

ERK（细胞外调节蛋白激酶，extracellular signal-regulated kinase）信号通路在维持小鼠胚胎干细胞（ESCs）的自我更新及调控其分化进程中发挥关键作用。然而，ERK介导上述效应的潜在分子机制仍未完全阐明。本研究鉴定出异质性细胞核核糖核蛋白F（HNRNPF）为一种新型ERK2相互作用蛋白与磷酸化底物。研究证实，ERK2可在HNRNPF的Ser346与Tyr356位点发生磷酸化修饰。Hnrnpf敲除（KO）的ESCs表现出自我更新能力受损，该现象源于CDK1与CCNB1的表达下调。值得注意的是，HNRNPF的磷酸化修饰可挽救CDK1与CCNB1的表达水平，进而恢复Hnrnpf KO ESCs的自我更新能力。此外，HNRNPF的缺失会促进ESCs向中胚层与滋养外胚层谱系分化，这一表型归因于EED表达水平降低以及组蛋白H3K27三甲基化（H3K27me3）水平下降。HNRNPF的磷酸化修饰可恢复EED的表达与H3K27me3水平，从而缓解Hnrnpf KO ESCs的分化偏向性。综上，本研究的发现揭示了HNRNPF作为ERK2信号通路下游效应分子，在调控ESCs自我更新与分化过程中的关键作用。

总体实验设计：为评估Hnrnpf KO ESCs的分化潜能，我们开展了RNA测序（RNA-seq）实验，以分析HNRNPF缺失后分化态与未分化态ESCs的全转录组表达谱变化。