Dlx5 Regulates 3D Chromatin Architecture To Enhance Osteogenesis

This study explores the molecular mechanism underlying regulation of osteogenic differentiation of mesenchymal stem cells by Dlx5 from a novel perspective of chromatin 3D conformation regulation. In vitro, DLX5-overexpressing BMSCs were established. CUT&Tag and Hi-C sequencing were used to identify DLX5 and CTCF binding sites and map chromatin interactions. Validation studies with luciferase reporter assays and binding site mutations confirmed the functional relevance of DLX5/CTCF interactions. Osteogenic differentiation was evaluated through alkaline phosphatase activity, Alizarin Red S staining, RT-qPCR, and Western blotting. In vivo, adenovirus-mediated DLX5 overexpression was applied in mice, with bone formation assessed via micro-CT imaging and histological analysis. CUT&Tag and Hi-C analysis revealed significant dynamic changes in chromatin architecture during osteogenesis, demonstrating that DLX5 modulates chromatin looping through CTCF to regulate Runx1 expression. Functional assays, both in vitro and in vivo, showed that DLX5 overexpression significantly enhanced osteogenic differentiation, as indicated by elevated ALP activity and increased expression of osteogenic proteins. In contrast, CTCF silencing disrupted chromatin interactions and impaired osteogenesis. This study uncovers a novel regulatory axis in which DLX5 modulate 3D chromatin conformation through CTCF, facilitating the osteogenic differentiation of BMSCs. By identifying the DLX5/CTCF/Runx1 axis as a critical regulatory mechanism, the research provides a transformative perspective on the role of 3D chromatin dynamics in osteogenesis. These findings not only enhance our understanding of the epigenetic regulation of bone formation but also offer valuable theoretical insights and identify promising therapeutic targets for bone regeneration.