RUNX2 co-operates with EGR1 to regulate osteogenic differentiation through Htra1 enhancers

Runt-related transcription factor 2 (Runx2) has been shown to regulate osteoblast differentiation by directly or indirectly regulating numerous osteoblast-related genes. However, our understanding of the transcriptional mechanisms of RUNX2 is mainly restricted to its transactivation, while the mechanism underlying its inhibitory effect during osteoblast differentiation remains largely unknown. Here, we incorporated the anti-RUNX2 chromatin immunoprecipitation (ChIP) sequencing in MC3T3-E1 cells and RNA-sequencing of parietal bone from Runx2 heterozygous mutant mice, to identify the putative genes negatively regulated by RUNX2. We identified HtrA serine peptidase 1 (Htra1) as a target gene and found ten candidate Htra1 enhancers potentially regulated by RUNX2, among which seven were verified by dual-luciferase assays. Furthermore, we investigated the motifs in the vicinity of RUNX2-binding sites and identified early growth response 1 (EGR1) as a potential partner transcription factor (TF) potentially regulating Htra1 expression, which was subsequently confirmed by Re-ChIP assays. RUNX2 and EGR1 co-repressed Htra1 and increased the expression levels of other osteoblast marker genes, such as osterix, osteocalcin, and osteoprotegerin at the messenger RNA and protein level. Moreover, Alizarin red staining combined with alkaline phosphatase (ALP) staining showed decreased calcified nodules and ALP activity in the siRUNX2+siEGR1 group compared with siRUNX2 group. Our findings revealed the detailed mechanism of the inhibitory function of RUNX2 towards its downstream genes, along with its partner TFs, to promote osteoblast differentiation.