PU.1 (SPI1) is a crucial transcription factor in hematopoiesis, yet its role in human endothelial-to-hematopoietic transition (EHT) remains unclear. By comparing the human in vivo and in vitro EHT transcriptomes, we observed similarities in the transcriptional profiles of SPI1, suggesting its regulatory role in EHT. Knocking down SPI1 in in vitro-generated hemogenic endothelial cells (HECs) led to a decrease in the generation of hematopoietic progenitor cells (HPCs) and their differentiation potential. Through multi-omic analysis, we identified KLF1 and LYL1 - transcription factors specific to erythroid/myeloid and lymphoid cells, respectively - as downstream targets of SPI1. Overexpressing KLF1 and LYL1 partially rescues the SPI1 knockdown-induced reduction in HPC formation. Specifically, KLF1 overexpression restores myeloid lineage potential, while LYL1 overexpression re-establishes lymphoid lineage potential. We also observed a SPI1-LYL1 axis in the regulatory network of in vivo EHT. Taken together, our findings shed new light on the role of SPI1 in regulating erythroid, lymphoid, and myeloid cell lineage commitment during EHT, potentially contributing to the heterogeneity of hematopoietic stem cells (HSCs).
scRNA_seq_analysis.R: Custom script for single-cell data analysis. Input for single-cell data analysis can be found here
RNA&ATAC-seq_analysis.R: Custom script for RNA&ATAC-seq data analysis. Input for RNA&ATAC-seq data analysis can be found here