Characterization of human cathepsin L promoter and identification of binding sites for NF-Y, Sp1 and Sp3 that are essential for its activity.
Cathepsin L is a cysteine protease whose overexpression in human melanoma cells increases their tumorigenicity and switches their phenotype from non-metastatic to highly metastatic. Regulation of the transcription of the gene encoding human cathepsin L has not been yet studied and only preliminary data exist on the promoter regulation of the gene encoding rodent cathepsin L. In the present study we identified molecular elements involved in the transcriptional regulation of human cathepsin L in melanoma cells. The sequence of the 5'-flanking region of the gene encoding human cathepsin L was determined up to 3263 bp upstream of the translation start site. The major transcription intiation site was located. Three mRNA splice variants, differing in their 5' untranslated ends, were identified. Regulatory regions crucial for cathepsin L promoter activity were characterized between -1489 and -1646 bp. In this region, two GC boxes (-1590/-1595 and -1545/-1550) and a CCAAT motif (-1571/-1575) were involved in specific DNA-protein interactions. An electrophoretic mobility-shift assay demonstrated that Sp1 and Sp3 transcription factors bound to these GC boxes, and only the transcription factor nuclear factor Y (NF-Y) bound to the CCAAT motif. Mutagenesis studies demonstrated that these binding sites contributed at least 85% of cathepsin L promoter activity. Thus structural and functional analysis demonstrated that binding sites for NF-Y, Sp1 and Sp3 are essential for transcription of the gene encoding human cathepsin L in melanoma cells.
Jean
Didier
D
Immunochimie des Régulations Cellulaires et des Interactions Virales, INSERM U.354, Centre INSERM, Hôpital Saint-Antoine, 184 rue du Faubourg Saint-Antoine, 75012 Paris, France.
Guillaume
Nathalie
N
Frade
Raymond
R
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D023081:CCAAT-Binding Factor / Q000378:metabolism*; D056668:Cathepsin L; D002403:Cathepsins / Q000235:genetics*; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008545:Melanoma / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D023081:CCAAT-Binding Factor; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; C402244:nuclear factor Y; D051705:Sp3 Transcription Factor; D008156:Luciferases; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L