A role for Octamer binding protein motifs in the regulation of the proximal preprotachykinin-A promoter.
We have recently developed cell line models that express the endogenous rat preprotachykinin-A (rPPT) gene and support reporter gene expression directed by the rPPT promoter. These are the neuronal derived cell line NF2C and the pancreatic cell lines RINm5F and a derivative RIN-1027-B2. Reporter gene activity in these cell lines is similar to that observed in primary cultures of rat dorsal root ganglion neurons. Analysis of reporter gene expression supported by various fragments of the rPPT promoter demonstrated that although -865 to +92 supported expression, addition of fragments between +92 and +500 led to repression of expression. Two previously defined octamer binding motifs, present both 5' and 3' of the major transcriptional start site, have been postulated to be potential enhancers of rPPT activity and we have now used these cell lines to determine the role of these regions in the rPPT promoter. Site specific mutagenesis of these elements has shown not only that both sites are potential enhancers of gene expression but also that the 3' element binds multiple factors, of which at least one has repressor function. Binding of this repressor protein over or adjacent to the 3' octamer binding protein site leads to the observed repression of promoter activity in the -865 to +500 construct relative to the to -865 to +92 the fragment.
Fiskerstrand
C E
CE
Department of Veterinary Pathology, University of Edinburgh, EH9 1QH.
Newey
P
P
McGregor
G P
GP
Gerrard
L
L
Millan
F
F
Quinn
J P
JP
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006367:HeLa Cells; D051863:Host Cell Factor C1; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000166:cytology; D050811:Octamer Transcription Factor-1; D010179:Pancreas / Q000166:cytology; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011498:Protein Precursors / Q000235:genetics*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000737:chemistry; D015320:Tachykinins / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics; D014162:Transfection
D004268:DNA-Binding Proteins; C497692:HCFC1 protein, human; D051863:Host Cell Factor C1; D050811:Octamer Transcription Factor-1; C494165:POU2F1 protein, human; C494168:Pou2f1 protein, rat; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D015320:Tachykinins; D014157:Transcription Factors; C044206:preprotachykinin; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase