Single-cell ATAC-seq analysis of human embryoid bodies reveals crucial transcription factor networks involved in early germ layer specification

Primary germ layer formation is a crucial event that follows fertilization and is accompanied by significant epigenetic changes. However, the precise mechanism of cell fate specification remains inadequately defined. In this study, we employed human embryoid body as an in vitro model to mimic early development and conducted single-cell ATAC sequencing to delineate lineage-specific chromatin regulatory elements and their associated transcription factor (TF) networks. By assessing the consistency of TF gene activity and binding in each cell, we classified the three germ layers and performed pseudotime analysis, leading to the identification of “early committed cells” at the onset of each germ layer. Subsequently, we established TF regulatory networks by integrating the inferred loop signals (InferLoop) of binding site and target gene promoters, pinpointing the key TFs that govern the early committed cells in each germ layer. Finally, through gene knockdown and phenotypic analysis, we confirmed their essential roles in early cell fate determination. Our findings demonstrate that scATAC-seq can delineate transitional cell populations and identify a series of key TFs crucial in early germ layer determination. Furthermore, our methodological framework presents a pathway for future explorations in this intricate field of biology and provides valuable resources for mechanistic study. Collectively, this work provides novel insights to the intricate process of cell differentiation and germ layer specification, potentially contributing to significant advancements in the field of developmental biology research.