Abstract

Cbfa1 is a critical regulator of cell differentiation expressed only in the osteochondrogenic lineage. To define the molecular basis of this cell-specific expression we analyzed the murine Cbfa1 promoter. Here we show that the first 976 bp of this promoter are specifically active in osteoblastic cells. Within this region DNase I footprinting delineated a 40-bp area (CE1) protected differently by nuclear extracts from osteoblastic cells and from non-osteoblastic cells. When multimerized, CE1 conferred an osteoblast-specific activity to a heterologous promoter in DNA transfection experiments; this enhancing ability was conserved between mouse, rat, and human CE1 present in the respective Cbfa1 promoters. CE1 site-specific mutagenesis determined that it binds NF1- and AP1-like activities. Further analyses revealed that the NF1 site acts as a repressor in non-osteoblastic cells due to the binding of NF1-A, a NF1 isoform not expressed in osteoblastic cells. In contrast, the AP1 site mediates an osteoblast-specific activation caused by the preferential binding of FosB to CE1 in osteoblastic cells. In summary, this study identified an osteoblast-specific enhancer in the Cbfa1 promoter whose activity is achieved by the combination of an inhibitory and an activatory mechanism.

Highlights

  • Osteoblasts are cells of mesenchymal origin required to form the skeleton during development and to maintain bone mass thereafter

  • Cbfa1 element 1 (CE1) conferred an osteoblast-specific activity to a heterologous promoter in DNA transfection experiments; this enhancing ability was conserved between mouse, rat, and human CE1 present in the respective Cbfa1 promoters

  • We narrowed down this cell specificity to a 40-bp region, CE1, located at Ϫ415 compared with the Cbfa1 start site of transcription and show that CE1 acts as an osteoblast-specific enhancer in DNA transfection experiments

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Summary

Introduction

Osteoblasts are cells of mesenchymal origin required to form the skeleton during development and to maintain bone mass thereafter. The results of the footprinting analysis of the Ϫ976/ Ϫ89 region of the mouse Cbfa1 promoter indicate that CE1, a 40-bp area lying between Ϫ415 and Ϫ375 (Fig. 2D), is the only region binding distinct factors in osteoblastic and non-osteoblastic cells.

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