Down-regulation of EHMT2 through irisin-mediated epigenetic modification promotes osteogenesis via promoting DLX3 transcription

Irisin, a cleavage peptide by fibronectin type III domain-containing protein 5 (FNDC5), has been recognized as a promotor of osteogenesis to alleviate osteoporosis (OP). However, the detailed regulatory mechanism of osteogenesis remains unclear. This study uncovered the mechanisms underlying bone formation. Osteogenic activity was evaluated by ALP and ARS staining. Expression of target molecules and osteogenic-related factors was assessed by RT-qPCR, Western blotting, and immunohistochemical staining. The binding of euchromatic histone lysine methyltransferase 2 (EHMT2) to distal-less homeobox 3 (DLX3) promoter was validated by ChIP and luciferase reporter assays. Protein interaction, ubiquitination, and small ubiquitin-like modifier (SUMO) modification were determined by Co-IP or Ni2+-NTA pull-down assay. In vivo bone formation was evaluated in a mouse model. Histological analysis was performed by HE and Masson staining. EHMT2 expression was decreased, while DLX3 expression was increased during osteogenic differentiation in vitro. Down-regulation of EHMT2 facilitated osteogenesis via enhancing DLX3 transcription and expression with assistance of H3K9me2. Moreover, EHMT2 was down-regulated by S-phase kinase-associated protein 2 (SKP2)-mediated ubiquitination, whereas protein inhibitor of activated STAT 4 (PIAS4)-mediated SUMOylation exerted an opposite role. Finally, irisin down-regulated EHMT2 via repressing SUMOylation and promoting ubiquitination of EHMT2, thus contributing to bone formation. Epigenetic modification-mediated down-regulation of EHMT2 by irisin leads to transcriptional activation of DLX3, thus promoting bone formation.