Identification of novel, functional genetic variants in the human matrix metalloproteinase-2 gene: role of Sp1 in allele-specific transcriptional regulation.
Matrix metalloproteinase-2 (MMP-2) is an enzyme with proteolytic activity against matrix and nonmatrix proteins, particularly basement membrane constituents. Thus, any naturally occurring genetic variants that directly affect gene expression and/or protein function would be expected to impact on progression of pathological processes involving tissue remodeling. We scanned a 2-kilobase pair promoter region and all 13 exons of the human MMP-2 gene, from a panel of 32 individuals, and we identified the position, nature, and relative allele frequencies of 15 variant loci as follows: 6 in the promoter, 1 in the 5'-untranslated region, 6 in the coding region, 1 in intronic sequence, and 1 in the 3'-untranslated region. The majority of coding region polymorphisms resulted in synonymous substitutions, whereas three promoter variants (at -1306, -790, and +220) mapped onto cis-acting elements. We functionally characterized all promoter variants by transient transfection experiments with 293, RAW264.7, and A10 cells. The common C --> T transition at -1306 (allele frequency 0.26), which disrupts an Sp1-type promoter site (CCACC box), displayed a strikingly lower promoter activity with the T allele. Electrophoretic mobility shift assays confirmed that these differences in allelic expression were attributable to abolition of Sp1 binding. These data suggest that this common functional genetic variant influences MMP-2 gene transcription in an allele-specific manner and is therefore an important candidate to test for association in a wide spectrum of pathologies for which a role for MMP-2 is implicated, including atherogenesis and tumor invasion and metastasis.
Price
S J
SJ
Department of Cardiovascular Medicine, Henry Wellcome Building for Genomic Medicine and Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
Greaves
D R
DR
Watkins
H
H
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000483:Alleles; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D016208:Databases, Factual; D005091:Exons; D017930:Genes, Reporter; D014644:Genetic Variation; D006801:Humans; D007438:Introns; D020778:Matrix Metalloproteinase 2 / Q000235:genetics*; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D016329:Sp1 Transcription Factor; D020778:Matrix Metalloproteinase 2