CRISPR Screening Reveals a Novel Role for FOXH1 in Regulating Pluripotency of Porcine Embryonic Stem Cells

Porcine extended potential stem cells (pEPSCs), which exhibit both self-renewal and pluripotency, are promising for application in both agricultural biotechnology and regenerative medicine. However, the molecular mechanisms governing these two interconnected properties remain elusive. Here, two types of CRISPR-Cas9 screenings are conducted in pEPSCs. This fitness screening identified several genes essential for cell viability, including PRMT1, MYBL2, and NASP. Concurrently, FACS-based screenings revealed genes crucial for pluripotency, such as SOX2, ZFP42, and FOXH1. Notably, it is demonstrated that FOXH1 is required for maintaining pluripotency in pEPSCs, which complements the understanding of its role in mesendoderm specification. pEPSCs lacking FOXH1 exhibited a flatter and more dispersed clonal morphology, accompanied by downregulation of pluripotency genes and upregulation of lineage-specific genes. Additionally, FOXH1 knockdown significantly impaired blastocyst formation during early pig embryogenesis. Functionally, the dual role of FOXH1 in pluripotency maintenance and cell differentiation is validated: FOXH1 preserves pluripotency by enhancing chromatin accessibility at pluripotency gene loci, while also influencing lineage specification through H3K4me3 modification at developmental related genes. Thus, these findings uncover a novel role of FOXH1 involved in the core regulatory network that orchestrates gene expression programs to maintain the pluripotency state of pEPSCs and provide valuable insights into categorizing gene function.