Characterization of the human p11 promoter sequence.

ABSTRACT

p11 is expressed in many different cell types, and serves a variety of regulatory functions. In order to better understand the transcriptional control of this protein, the 5' promoter region of the human p11 gene was cloned and sequenced. After confirming the transcription start point (TSP) using 5' rapid amplification of cDNA ends analysis, the 5' promoter was analysed. The sequence lacks a TATA box, but contains a variety of putative regulatory elements. There are two GAS sites, two AP-1 sites, two overlapping Sp-1 sites, and a gamma-IRE site clustered between -1080 and -1450. There is another cluster of putative regulatory sites between the TSP and -550 which contains two Sp-1 sites, two AP-2 sites, one GAS site, one NF-kappaB site, an incomplete CAAT box (8/9) and an overlapping Sp-1/AP-2 site at -17 to -26. Reporter gene constructs containing 4225 and 1498 bases 5' of the TSP demonstrated excellent unidirectional transcriptional activity in both constructs. Reporter genes containing serial 5' deletions were compared to the -1498 construct. The reporter gene which contained base pairs (bp) -36 to +89 had almost no activity. The reporter gene containing -188 to +89 had 50% of the -1498 construct, indicating that this sequence contains at least the minimal promoter. The Sp-1/AP-2 site near the transcription start site was studied by electrophoretic mobility shift and reporter gene assays. Addition of HeLa cell nuclear extract to labeled double-stranded (ds) oligonucleotide containing this sequence resulted in a gel shift which was inhibited by excess unlabeled ds oligonucleotide and by a consensus cold Sp-1 ds oligonucleotide, indicating specific Sp-1 binding. Excess AP-2 or NF-kappaB ds oligonucleotide had no effect on nuclear protein binding to the sequence. Mutation of the p11 wild-type Sp-1/AP-2 sequence eliminated both nuclear protein binding and the sequences ability to compete with native sequence for nuclear binding protein. A -1048 to +89 reporter construct containing a mutated Sp-1/AP-2 site resulted in a 40% decrease in transcriptional activity. Therefore, the 5' flanking sequence of the p11 gene exhibits promoter activity which may be localized to a variety of controlling regions, of which the proximal Sp-1/AP-2 site appears to be important for basal activity via its Sp-1 binding ability.