The role of KLF4 in the development of human primordial germ cell

Primordial germ cells (PGCs) are the founder cells that develop into mature gametes. PGCs emerge during weeks 2-3 of human embryo development. Pluripotency genes are reactivated during PGCs specification, including Kruppel-like factor KLF4, but its precise role in PGC development is unknown. Here, we investigated the role of KLF4 in PGC development using our in vitro model for human PGC-like cells (hPGCLCs). We show that depletion of KLF4 using acute protein degradation strategies reduced the efficiency of hPGCLC specification. We conducted transcriptome analysis of KLF4-depleted hPGCLCs, and observed downregulation of genes involved in pattern specification, stem cell differentiation, and epigenetic modifications. Notably, KLF4 depletion led to the derepression of some somatic genes related to endodermal and neuronal differentiation. In PGCLCs, the KLF4 Cut&Run results showed significant co-enrichment with SP and STAT motifs, prominently localized to promoter regions. This represents a distinct binding pattern compared to that observed in naive embryonic stem cells. Our study reveals that KLF4 contributes to the differentiation and maintenance of human PGCLCs by comprehensively regulating both gene activation and repression. Overall design: To elucidate the role of KLF4 in the differentiation of human PGCLCs, we established an inducible knockdown system using the auxin-degron method in WIS2 NANOS3–tdTomato reporter cell lines. Cells isolated by cell sorting using PGCLC-specific markers underwent gene expression profiling via RNA-seq. Additionally, we performed CUT&RUN using KLF4 antibodies on normal PGCLCs to analyze the KLF4 binding profile across the genome.