Characterization of the promoter of the gene encoding human tripeptidyl-peptidase II and identification of upstream silencer elements.
Tripeptidyl-peptidase II (TPP II) is one of the many proteases involved in the important process of intracellular proteolysis. The widespread distribution and broad substrate specificity suggest that TPP II is encoded by a "house-keeping gene". However, both TPP II protein and mRNA levels vary in different cells. To investigate whether these variations are due to regulation on a genetic level, the promoter of the TPP2 gene has previously been identified. The promoter contains two inverted CCAAT-boxes and an E-box. By means of reporter assays and electrophoretic mobility shift assays the promoter has now been further characterized. It could be concluded that USF-1 (upstream stimulatory factor-1) binds to the E-box in the promoter. The transcription factors NF-Y and USF-1 are present in protein-DNA complexes of different sizes. Mutation of the E-box had no effect, indicating that only binding of NF-Y to the two CCAAT-boxes was important for activation of transcription. However, this does not exclude the possibility that USF-1 can play an important role in transcription in other types of cells. Furthermore, the region upstream of the promoter was investigated due to its ability to inhibit transcription. Several silencer elements were identified and we also showed that Oct-1 binds to one of these elements. Thus, this investigation reveals that TPP II expression could be regulated through both positive and negative regulatory elements. Further studies are required to establish the involvement of different genetic elements, and how the interplay between different transcription factors will affect the transcriptional rate in vivo.
Lindås
Ann-Christin
AC
Uppsala University, Biomedical Center, Department of Biochemistry and Organic Chemistry, Uppsala, Sweden.
Tomkinson
Birgitta
B
D024506:5' Flanking Region / Q000235:genetics; D000626:Aminopeptidases; D020029:Base Pairing / Q000235:genetics; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D024721:E-Box Elements / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics*; D037941:Silencer Elements, Transcriptional / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; D008156:Luciferases; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C048001:tripeptidyl-peptidase 2; D012697:Serine Endopeptidases