Regulon active landscape reveals cell development and functional state changes of human primary osteoblasts in vivo

Background: While transcription factor (TF) regulation is known to play an important role in osteoblast development, differentiation, and bone metabolism, the molecular features of TFs in human osteoblasts at the single-cell resolution level have not yet been characterized. Here, we identified modules (regulons) of co-regulated genes by applying single-cell regulatory network inference and clustering to the single-cell RNA sequencing profiles of human osteoblasts. We also performed cell-specific network (CSN) analysis, reconstructed regulon activity-based osteoblast development trajectories, and validated the functions of important regulons both in vivo and in vitro. Results: We identified four cell clusters: preosteoblast-S1, preosteoblast-S2, intermediate osteoblasts, and mature osteoblasts. CSN analysis results and regulon activity-based osteoblast development trajectories revealed cell development and functional state changes of osteoblasts. CREMand FOSL2regulons were mainly active in preosteoblast-S1, FOXC2regulons were mainly active in intermediate osteoblast, and RUNX2and CREB3L1regulons were most active in mature osteoblasts. Conclusions: This is the first study to describe the unique features of human osteoblasts in vivo based on cellular regulon active landscapes. Functional state changes of CREM, FOSL2, FOXC2, RUNX2, and CREB3L1regulons regarding immunity, cell proliferation, and differentiation identified the important cell stages or subtypes that may be predominantly affected by bone metabolism disorders. These findings may lead to a deeper understanding of the mechanisms underlying bone metabolism and associated diseases.