Identification and characterization of the promoter for the gene encoding human tripeptidyl-peptidase II.
Tripeptidyl-peptidase II (TPP II) is a ubiquitously expressed exopeptidase. The expression of this enzyme is increased, e.g. in some tumor cells, but the regulation of the expression of the gene has not been investigated previously. The gene encoding human TPP II (TPP2) is 82 kb and consists of 30 exons. An 8 kb NcoI fragment covering the 5'-flanking region of the TPP2 gene, including the initiation codon, was cloned into a luciferase-containing reporter vector. Human embryonic kidney cells (HEK-293 cells) and murine fibroblasts (NIH3T3 cells) were transiently transfected with the construct. Through sequential deletions and analysis of short PCR-fragments, the promoter could be localized to a 215 bp fragment upstream of the initiation codon. This region is GC-rich, lacks a TATA-box and contains two inverted CCAAT-boxes and a GC-box. Electrophoretic mobility shift assays showed that nuclear proteins bind to the promoter fragment. The 85 bp 5'-end of the promoter fragment is essential for transcriptional activation. Out of this a 44 bp fragment suffices to compete with binding of nuclear proteins to the 215 bp fragment. Supershift assays demonstrated that the CCAAT-binding factor (CBF; NF-Y) is involved in the formation of a complex with the 215 bp fragment. Although Sp1 binds to the promoter fragment in vitro, it was found to bind to the 3'-end of the 215 bp fragment which is not essential for transcription. The potential role of Sp1 in transcription of TPP2 therefore remains to be established.
Lindås
Ann-Christin
AC
Uppsala University, BMC, Department of Biochemistry, Box 576, SE-751 23 Uppsala, Sweden.
Tomkinson
Birgitta
B
D000626:Aminopeptidases; D000818:Animals; D001483:Base Sequence; D023081:CCAAT-Binding Factor / Q000378:metabolism; D002478:Cells, Cultured; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic; D012697:Serine Endopeptidases / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014162:Transfection
D023081:CCAAT-Binding Factor; D016329:Sp1 Transcription Factor; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C048001:tripeptidyl-peptidase 2; D012697:Serine Endopeptidases