Abstract
We report here the characterization of the human tissue inhibitor of metalloproteinases-2 (TIMP-2) gene. The gene is 83 kilobase pairs (kb) long with exon-intron splicing sites located in preserved positions among the three members of the TIMP family. A 2.6-kb genomic DNA fragment flanking the 5'-end of the gene contains several regulatory elements including five Sp1, two AP-2, one AP-1, and three PEA-3 binding sites. Despite the presence of a complete AP-1 consensus at position -281, the promoter did not respond to 12-O-tetradecanoylphorbol-13-acetate treatment. However, 12-O-tetradecanoylphorbol-13-acetate response was generated by insertion of a similar AP-1 consensus at position -71, indicating the importance of the positioning of this motif. The promoter contains a typical CpG island; however, methylation of this island did not seem to influence gene expression. Analysis of the 3'-end of the gene revealed that the two mRNAs for TIMP-2 (1.2 and 3.8 kb) differ by the selection of their polyadenylation signal sites, but selection of these sites does not affect RNA stability. In summary, the TIMP-2 gene has several features observed in housekeeping genes, and differs significantly from TIMP-1 and TIMP-3 genes. These differences are likely to explain the specific roles that these inhibitors play in the regulation of matrix metalloproteinases.
Highlights
Matrix metalloproteinases (MMP)1 are a large family of secreted neutral endopeptidases with a broad spectrum of proteolytic activity for several components of the extracellular matrix (ECM)
As is the case in many genes that belong to a same family, we found that the exon-intron boundaries were preserved in the three members of tissue inhibitors of metalloproteinases (TIMP) family
In contrast to the TIMP-1 gene, which is contained within a 4.5-kb HindIII genomic fragment, the human tissue inhibitor of metalloproteinases-2 (TIMP-2) (hTIMP-2) gene is much larger and contains a first intron of 57 kb
Summary
Isolation of TIMP-2 Genomic Clones—A human TIMP-2 cDNA probe containing the full (1,035-nt) sequence [24] was used to screen a human placenta library prepared in the cosmid pWE 15 vector (Stratagene, La Jolla, CA) and a human chromosome 17 library in the cosmid SuperCos I vector (originally provided by Dr Larry Deaven at Los Alamos National Laboratory). The positive clones from the tertiary screening were examined by Southern blot analysis, after digestion with EcoRI, using a 5Ј-end probe (a 2.6-kb PstI fragment containing exon 1), a 3Ј-end probe (a 1.4-kb EcoRI fragment containing the last exon) derived from a EMBL 3 library [23] and oligoprobes corresponding to TIMP-2 cDNA sequences extending from amino acid 21 to 27 (oligo YDC1: 5Ј-GCCAAAGCGGTCAGTGAGAAG3Ј) and from amino acid 76 to 81 (oligo 1332: 5Ј-CTTTCCTCCAACGTCCAG-3Ј). DNase Footprinting—DNase footprinting assays were carried out using a BamHI-NarI fragment (nt positions Ϫ519 to Ϫ188) of the hTIMP-2 promoter encompassing the AP-1 binding consensus sequence (positions Ϫ288 to Ϫ281) This fragment was [␣-32P]dCTP-end-labeled at the NarI site using Klenow enzyme and gel-isolated. Quantitative analysis of the mRNA was performed by measuring the intensity of the radioactive signals on a GS-250 Molecular Imager (Bio-Rad)
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