Promoter characterization of the human and mouse epilysin (MMP-28) genes

Epilysin (MMP-28) is a recently cloned member of the matrix metalloproteina se family (Lohi et al., J. Biol. Chem. 276 (2001) 10134). It is expressed a t 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 sequ ence of the epilysin gene, and a corresponding 660 bp fragment from the mou se. The 5'-flanking sequences contain no typical TATA-boxes or CCAAT sequen ces close to the translation initiation sites. RNase protection assay revea led that two transcription start sites are utilized in the human epilysin g ene, situated 210 and 230 bp upstream from the translation start site. The promoter contains a GT-box, situated 300 bp upstream from the translation s tart site, with homology to the consensus binding site for transcription fa ctors of the Sp family. This site is perfectly conserved between the human and mouse promoters. For reporter gene assays a series of constructs with f ragments of increasing length of the epilysin promoter were coupled to the firefly luciferase gene. Reporter gene assays indicated that deletion or mu tation of the GT-box dramatically reduces the transcriptional activity both in keratinocytes and in spermatogonia. Gel mobility shift assays showed th at 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 pr omoter has distinctive structural and functional features, which may contro l the unique expression and regulation patterns of the epilysin gene. (C) 2 001 Elsevier Science B.V. All rights reserved.