Cloning and functional characterization of the 5′-flanking region of the human bone morphogenetic protein-2 gene

Abstract

Bone morphogenetic protein-2 (BMP-2) is involved in bone formation, organogenesis or pattern formation during development. The expression of BMP-2 is regulated accurately and coordinately with that of other transforming growth factor-beta (TGF-beta) superfamily members. To elucidate the mechanism underlying the regulation of BMP-2 expression, a 6.7 kb SpeI-SalI fragment, from the P1 phage library, encompassing the 5'-flanking region of the human BMP-2 gene, was isolated and sequenced. Transcription start sites were mapped by the 5'-rapid amplification of cDNA ends (RACE) method. It has been found that the human BMP-2 gene contains, largely, two promoter regions surrounded by GC-rich sequences with several Sp1 consensus motifs. The proximal promoter possesses a single start site, whereas several start sites are clustered in the distal promoter region. Neither TATA nor CAAT consensus sequences are found in the proximity of the start sites for either promoter. Interestingly, in no case is the transcription-initiation site common between the human and mouse BMP-2 genes, although the sequence of the BMP-2 gene is well conserved in the promoter region between two species. Transient transfection experiments with the reporter fused with various lengths of the BMP-2 promoter sequence demonstrated that there exist enhancer elements in an 1.1 kb GC-rich fragment covering both promoter regions. It is noteworthy that the enhancer elements are 5'-flanked by a 790 bp strong repressor element that is characterized by numerous AT stretches. This intriguing organization may be amenable to the tight control of the expression of BMP-2 that is essential for development or bone morphogenesis.