Dhx36 modulates the G-quadruplex within the Alpl promoter to promote osteogenesis and ameliorate osteoporosis

Abstract G-quadruplexes (G4s) are non-canonical nucleic acid structures that play important roles in diverse biological processes and represent promising therapeutic targets. However, their role in osteogenic differentiation remains largely unexplored. Here, we show that G4 abundance decreases during osteogenic differentiation and that pharmacological stabilization of G4 structures impairs osteogenesis in both cellular and animal models. We further identify a G4 structure within the promoter of the key osteogenic gene alkaline phosphatase (Alpl) as a critical cis-regulatory element governing osteogenic differentiation. Mechanistically, the topology of the Alpl G4 is dynamically modulated by the G4-binding proteins Nucleolin and Dhx36. Nucleolin stabilizes the Alpl G4, thereby suppressing Alpl promoter activity, whereas Dhx36 modulates the Alpl G4, resulting in the activation of Alpl transcription. Moreover, adeno-associated virus-mediated Dhx36 gene therapy effectively restored bone mass in ovariectomy-induced osteoporotic mice. Collectively, these findings establish G4 structures as key regulators of osteogenic differentiation and highlight modulation of G4 dynamics through Dhx36 as a promising therapeutic strategy for osteoporosis.