Characterization of an osteoblast-specific enhancer element in the CBFA1 gene.
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.
Departments of Molecular and Human Genetics and Medicine, Baylor College of Medicine, Houston, Texas 77030, USA.
Adriana
A
Zambotti
Huda
H
Makhluf
Jianhe
J
Shen
Patricia
P
Ducy
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D005075:Biological Evolution; D002454:Cell Differentiation; D002460:Cell Line; D050659:Core Binding Factor Alpha 1 Subunit; D004742:Enhancer Elements, Genetic / Q000502:physiology*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D025542:Neurofibromin 1 / Q000378:metabolism; D010006:Osteoblasts / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics*
D050659:Core Binding Factor Alpha 1 Subunit; D009363:Neoplasm Proteins; D025542:Neurofibromin 1; D018808:Transcription Factor AP-1; D014157:Transcription Factors