Transcriptional autoregulation of the bone related CBFA1/RUNX2 gene

The runt related transcription factor CBFA1 (AML3/PEBP2 alpha A/RUNX2) regu lates expression of several bone- and cartilage-related genes and is requir ed for bone formation in vivo. The gene regulatory mechanisms that control activation and repression of CBFA1 gene transcription during osteoblast dif ferentiation and skeletal development are essential for proper execution of the osteogenic pro gram. We have therefore defined functional contribution s of 5' regulatory sequences conserved in rat, mouse and human CBFA1 genes to transcription. Deletion analysis reveals that 0.6 kB of the bone-related rat or mouse CBFA1 promoter (P1, MASNS protein isoform) is sufficient to c onfer transcriptional activation, and that there are multiple promoter doma ins which positively and negatively regulate transcription. Progressive del etion of promoter segments between nt -351 and -92 causes a striking 30- to 100-fold combined decrease in promoter activity. Additionally, 5' UTR sequ ences repress reporter gene transcription 2- to 3-fold. Our data demonstrat e that CBFA1 is a principal DNA binding protein interacting with the 5' reg ion of the CBFA1 gene in osseous cells, that there are at least three CBFA1 recognition motifs in the rat CBFA1 promoter, and that there are three tan demly repeated CBFA1 sites within the 5' UTR. We find that forced expressio n of CBFA1 protein downregulates CBFA1 promoter activity and that a single CBFA1 site is sufficient for transcriptional autosuppression. Thus, our dat a indicate that the CBFA1 gene is autoregulated in part by negative feedbac k on its own promoter to stringently control CBFA1 gene expression and func tion during bone formation. J. Cell. Physiol. 184:341-350, 2000. (C) 2000 W iley-Liss, Inc.