Selective Distal Enhancer Control of the Mmp13 Gene Identified through Clustered Regularly Interspaced Short...

Matrix metalloproteinase 13 (Mmp13, collagenase-3) plays an essential role in bone metabolism and mineral homeostasis. It is regulated by numerous factors, including BMP-2, parathyroid hormone, and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), through transcription factors such as Runt-related transcription factor 2 (RUNX2), CCAAT/enhancer-binding protein β (C/EBPβ), OSX, and vitamin D receptor (VDR). During osteoblast maturation, the basal expression of Mmp13 and its sensitivity to 1,25(OH)2D3 are strikingly increased. In this report, ChIP-sequencing analysis in mouse preosteoblasts revealed that the Mmp13 gene was probably regulated by three major enhancers located -10, -20, and -30 kb upstream of the gene promoter, occupied by activated VDR and prebound C/EBPβ and RUNX2, respectively. Initially, bacterial artificial chromosome clone recombineering and traditional mutagenesis defined binding sites for VDR and RUNX2. We then employed a CRISPR/Cas9 gene editing approach to delete the -10 and -30 kb Mmp13 enhancers, a region proximal to the promoter, and VDR or RUNX2. VDR-mediated up-regulation of Mmp13 transcription was completely abrogated upon removal of the -10 kb enhancer, resulting in a 1,25(OH)2D3-directed repression of Mmp13. Deletion of either the -30 kb enhancer or RUNX2 resulted in a complete loss of basal transcript activity and a ChIP-identified destabilization of the chromatin enhancer environment and factor binding. Whereas enhancer deletions only affected Mmp13 expression, the RUNX2 deletion led to changes in gene expression, a reduction in cellular proliferation, and an inability to differentiate. We conclude that the Mmp13 gene is regulated via at least three specific distal enhancers that display independent activities yet are able to integrate response from multiple signaling pathways in a model of activation and suppression.