TWIST inactivation reduces CBFA1/RUNX2 expression and DNA binding to the osteocalcin promoter in osteoblasts

Abstract

The Saethre–Chotzen (SC) syndrome is characterized by increased osteogenesis and premature fusion of cranial sutures, resulting from mutations in TWIST, a basic helix–loop–helix transcription factor. The molecular target genes for Twist in osteoblasts are however unknown. We report here that TWIST haploinsufficiency in mutant osteoblasts reduces mRNA and protein levels for CBFA1/RUNX2, a specific osteoblast transcription factor, during both osteoblast cell growth and in vitro osteogenesis. Moreover, this is associated with altered expression of major osteoblast-specific genes. Electrophoretic mobility shift assay (EMSA) showed reduced-binding ability of Cbfa1 to its target OSE2 element in the osteocalcin promoter in mutant osteoblasts. By contrast, TWIST inactivation does not hamper Cbfa1 binding on a similar upstream element present in the α1(I) collagen promoter in mutant osteoblasts. This provides the first evidence that TWIST inactivation alters CBFA1/RUNX2 expression and Cbfa1 binding ability to the osteocalcin promoter, indicating that CBFA1/RUNX2 is a target gene for TWIST in human osteoblasts.