We have cloned the promoter of the avian β3 integrin gene. Using a probe comprising the 5'-untranslated region of an avian macrophage β3 cDNA, characterized by 5' rapid amplification of cDNA ends, several clones were isolated from an avian genomic library. One major and one minor transcriptional start site were identified at +1 and -47 base pairs, respectively, with the latter coinciding with a consensus sequence of an initiator. DNA sequence analysis of 800 base pairs 5' of the transcriptional start site fails to reveal either a TATA or CAAT box. In addition to an initiator, the first 200 base pairs contain consensus sequences for the binding of AP-1 and SP-1. A 3.5-kilobase fragment located immediately upstream of the transcriptional start site exhibits functional promoter activity, and deletion analysis reveals both suppressor and enhancer elements. In light of our observation that 1,25-dihydroxyvitamin D3 (D3) accelerates β3 transcription, we determined whether the avian β3 promoter contains a vitamin D response element (VDRE). Transfected reporter constructs containing the first 1.5 kilobases upstream of the major β3 transcriptional start site respond to D3 with enhanced luciferase activity. Analysis of this region reveals a classical VDRE consensus sequence, located at -756 to -770. The following observations support the hypothesis that this sequence represents a functional VDRE: 1) a 600-base pair genomic fragment or a 29- base pair oligomer, each containing the putative VDRE, respond to D3 when transfected into HD11 cells; 2) a 67-base pair DNA fragment derived from genomic DNA and containing the candidate β3 VDRE specifically binds the vitamin D receptor-retinoid X receptor β complex; and 3) avian osteoclast precursor-derived nuclear extracts bind to a synthetic oligomer containing the β3 VDRE-like sequence and, in turn, are specifically displaced by unlabeled β3 VDRE and anti-vitamin D receptor antibody.
|Number of pages||10|
|Journal||Journal of Biological Chemistry|
|State||Published - Dec 1 1993|