Polymorphisms in the osteopontin promoter affect its transcriptional activity.
Understanding the molecular mechanisms that underlie regulation of transcription of the human osteopontin encoding gene (OPN) may help to clarify several processes, such as fibrotic evolution of organ damage, tumorigenesis and metastasis, and immune response, in which OPN overexpression is observed. With the aim to evaluate variants with functional effect on transcription, we have analyzed the promoter region and focused our investigation on three common variants present in the first 500 bp upstream of the transcription start site. Transfection of constructs carrying the four most frequent haplotypes relative to variants at -66, -156, and -443 fused to the luciferase reporter gene in a panel of different cell lines showed that one haplotype conferred a significantly reduced level of reporter gene expression in all tested cell lines. We describe that the -66 polymorphism modifies the binding affinity for the SP1/SP3 transcription factors, the -156 polymorphism is included in a yet uncharacterized RUNX2 binding site, and the -443 polymorphism causes differential binding of an unknown factor. The finding of differential effects of various combination of variants in haplotypes may contribute to explain data of association studies reported in several already published articles. Future association studies using haplotypes instead of single OPN variants will allow to achieve more accurate results referable to differential expression of OPN in several common diseases, in which OPN is considered a candidate susceptibility gene.
Laboratory of Molecular Genetics G. Gaslini Institute, Genova, Italy.
Francesca
F
Giacopelli
Renato
R
Marciano
Angela
A
Pistorio
Paolo
P
Catarsi
Silvana
S
Canini
Gerard
G
Karsenty
Roberto
R
Ravazzolo
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors; D006239:Haplotypes; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D015810:Linkage Disequilibrium; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009362:Neoplasm Metastasis; D053495:Osteopontin; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012689:Sequence Homology, Nucleic Acid; D012795:Sialoglycoproteins / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D050659:Core Binding Factor Alpha 1 Subunit; D017931:DNA Primers; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; D016329:Sp1 Transcription Factor; C506896:Spp1 protein, mouse; D053495:Osteopontin; D008156:Luciferases