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

Abstract

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. CREM and FOSL2 regulons were mainly active in preosteoblast-S1, FOXC2 regulons were mainly active in intermediate osteoblast, and RUNX2 and CREB3L1 regulons 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 CREB3L1 regulons 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.

The genetically predicted causal relationship of inflammatory bowel disease with bone mineral density and osteoporosis: evidence from two-sample Mendelian randomization

“…In addition to its role in inflammation and autoimmune diseases, Taiwanese scholars found that CREM expression was increased in patients with rheumatoid arthritis and its variants were involved in the progression of the disease ( 39 ). Single-cell sequencing studies showed that CREM regulons were most active in preosteoblast-S1 ( 40 ), implying that CREM variants are engaged in osteogenesis and bone homeostasis.…”

The Regulatory Network of CREB3L1 and Its Roles in Physiological and Pathological Conditions

“…At the same time, ER stress marker X-Box binding protein 1 (Xbp1) and the chaperone protein Heat Shock Protein Family A (Hsp70) Member 5 (HSPA5/GRP78/BiP) were induced as well 11 . In human beings, compared with the preosteoblasts (including preosteoblast-S1 and preosteoblast-S2) and the intermediate osteoblasts, the active scores for CREB3L1 and RUNX2 regulons in mature osteoblasts are the highest, and the PHOSPHO1, SPNS2, and COL1A1 in the CREB3L1 regulon increase as osteoblasts mature 37 . Differential proteomics-based identification of ubiquitylation substrates (DiPIUS) revealed that CREB3L1 serves as the substrate of ubiquitin ligase F-Box and WD repeat domain containing 7 (Fbxw7).…”