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.
Zambotti
Adriana
A
Departments of Molecular and Human Genetics and Medicine, Baylor College of Medicine, Houston, Texas 77030, USA.
Makhluf
Huda
H
Shen
Jianhe
J
Ducy
Patricia
P
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