The transcriptional regulator Runx2 (runt-related transcription factor 2) has essential but distinct roles in osteoblasts and chondrocytes in skeletal development. However, Runx2-mediated regulatory mechanisms underlying the distinctive programming of osteoblasts and chondrocytes are not well understood. Here, we perform an integrative analysis to investigate Runx2-DNA binding and chromatin accessibility ex vivo using neonatal osteoblasts and chondrocytes. We find that Runx2 engages with cell-type-distinct chromatin-accessible regions, potentially interacting with different combinations of transcriptional regulators, forming cell-type-specific hotspots, and potentiating chromatin accessibility. Genetic analysis and direct cellular reprogramming studies suggest that Runx2 is essential for establishment of chromatin accessibility in osteoblasts. Functional enhancer studies identify an Sp7 distal enhancer driven by Runx2-dependent binding and osteoblast-specific chromatin accessibility, contributing to normal osteoblast differentiation. Our findings provide a framework for understanding the regulatory landscape encompassing Runx2-mediated and cell-type-distinct enhancer networks that underlie the specification of osteoblasts.

Osteoblast differentiation is a tightly regulated process in which key transcription factors (TFs) and their target genes constitute gene regulatory networks (GRNs) under the control of osteogenic signaling pathways. Among these TFs, Sp7 works as an osteoblast determinant critical for osteoblast differentiation. Following the identification of Sp7 and a large number of its functional studies, recent genome-scale analyses have made a major contribution to the identification of a “non-canonical” mode of Sp7 action as well as “canonical” ones. The analyses have not only confirmed known Sp7 targets but have also uncovered its additional targets and upstream factors. In addition, biochemical analyses have demonstrated that Sp7 actions are regulated by chemical modifications and protein–protein interaction with other transcriptional regulators. Sp7 is also involved in chondrocyte differentiation and osteocyte biology as well as postnatal bone metabolism. The critical role of SP7 in the skeleton is supported by its relevance to human skeletal diseases. This review aims to overview the Sp7 actions in skeletal development and maintenance, particularly focusing on recent advances in our understanding of how Sp7 functions in the skeleton under physiological and pathological conditions.

Skeletal development is tightly coordinated by chondrocytes and osteoblasts, which are derived from skeletal progenitors, and distinct cell-type gene regulatory programs underlie the specification and differentiation of cells. Runt-related transcription factor 2 (Runx2) is essential to chondrocyte hypertrophy and osteoblast differentiation. Genetic studies have revealed the biological functions of Runx2 and its involvement in skeletal genetic diseases. Meanwhile, molecular biology has provided a framework for our understanding of RUNX2-mediated transactivation at a limited number of cis-regulatory elements. Furthermore, studies using next-generation sequencing (NGS) have provided information on RUNX2-mediated gene regulation at the genome level and novel insights into the multiple layers of gene regulatory mechanisms, including the modes of action of RUNX2, chromatin accessibility, the concept of pioneer factors and phase separation, and three-dimensional chromatin organization. In this review, I summarize the emerging RUNX2-mediated regulatory mechanism from a multi-layer perspective and discuss future perspectives for applications in the treatment of skeletal diseases.

Tenomodulin (Tnmd) is a type II transmembrane protein characteristically expressed in dense connective tissues such as tendons and ligaments. Its expression in the periodontal ligament (PDL) has also been demonstrated, though the timing and function remain unclear. We investigated the expression of Tnmd during murine tooth eruption and explored its biological functions in vitro . Tnmd expression was related to the time of eruption when occlusal force was transferred to the teeth and surrounding tissues. Tnmd overexpression enhanced cell adhesion in NIH3T3 and human PDL cells. In addition, Tnmd -knockout fibroblasts showed decreased cell adhesion. In the extracellular portions of Tnmd, the BRICHOS domain or CS region was found to be responsible for Tnmd-mediated enhancement of cell adhesion. These results suggest that Tnmd acts on the maturation or maintenance of the PDL by positively regulating cell adhesion via its BRICHOS domain.

Although the skeleton is essential for locomotion, endocrine functions, and hematopoiesis, the molecular mechanisms of human skeletal development remain to be elucidated. Here, we introduce an integrative method to model human skeletal development by combining in vitro sclerotome induction from human pluripotent stem cells and in vivo endochondral bone formation by implanting the sclerotome beneath the renal capsules of immunodeficient mice. Histological and scRNA-seq analyses reveal that the induced bones recapitulate endochondral ossification and are composed of human skeletal cells and mouse circulatory cells. The skeletal cell types and their trajectories are similar to those of human embryos. Single-cell multiome analysis reveals dynamic changes in chromatin accessibility associated with multiple transcription factors constituting cell-type-specific gene-regulatory networks (GRNs). We further identify ZEB2, which may regulate the GRNs in human osteogenesis. Collectively, these results identify components of GRNs in human skeletal development and provide a valuable model for its investigation.