Promoter characterization of the human and mouse epilysin (MMP-28) genes.
Epilysin (MMP-28) is a recently cloned member of the matrix metalloproteinase family (Lohi et al., J. Biol. Chem. 276 (2001) 10134). It is expressed at highest levels in the skin by basal and suprabasal keratinocytes, and in testis by developing germ cells. To characterize the epilysin promoter, we isolated a 3.0 kb fragment of human genomic DNA containing 5'-flanking sequence of the epilysin gene, and a corresponding 660 bp fragment from the mouse. The 5'-flanking sequences contain no typical TATA-boxes or CCAAT sequences close to the translation initiation sites. RNase protection assay revealed that two transcription start sites are utilized in the human epilysin gene, situated 210 and 230 bp upstream from the translation start site. The promoter contains a GT-box, situated 300 bp upstream from the translation start site, with homology to the consensus binding site for transcription factors of the Sp family. This site is perfectly conserved between the human and mouse promoters. For reporter gene assays a series of constructs with fragments of increasing length of the epilysin promoter were coupled to the firefly luciferase gene. Reporter gene assays indicated that deletion or mutation of the GT-box dramatically reduces the transcriptional activity both in keratinocytes and in spermatogonia. Gel mobility shift assays showed that several nuclear proteins bind specifically to this sequence. Supershift assays with antibodies specific for members of the Sp family identified Sp1 and Sp3 as components of these protein/DNA complexes and hence as possible regulators of the epilysin gene. Our results indicate that the epilysin promoter has distinctive structural and functional features, which may control the unique expression and regulation patterns of the epilysin gene.
Illman
S A
SA
Department of Pathology, Haartman Institute and Biomedicum Helsinki, University of Helsinki and Helsinki University Hospital, FIN-00014 Helsinki, Finland.
Keski-Oja
J
J
Lohi
J
J
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002461:Cell Line, Transformed; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D020782:Matrix Metalloproteinases / Q000235:genetics*; D053505:Matrix Metalloproteinases, Secreted; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D024363:Transcription Initiation Site
D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA; D008156:Luciferases; C424663:MMP28 protein, human; D020782:Matrix Metalloproteinases; D053505:Matrix Metalloproteinases, Secreted