Regulation of rat ornithine decarboxylase promoter activity by binding of transcription factor Sp1.
Ornithine decarboxylase (ODC) is the rate-limiting enzyme of polyamine biosynthesis. We investigated the transcriptional regulation of the rat ODC gene using transient expression assays. The 5'-flanking region (-1156 to +13) of the ODC gene was sufficient to mediate strong basal expression of a luciferase reporter. Sequences between -345 and -93 contributed to basal promoter activity. This region, containing five potential Sp1 binding sites, was analyzed by electrophoretic mobility shift assays. Three specific DNA-protein complexes were identified using H35 nuclear extracts and the -345/-93 ODC probe. Binding to all three was eliminated by competition with an oligonucleotide containing an Sp1 binding site, but not by a mutant Sp1 oligonucleotide. Preincubation with an antibody against Sp1 supershifted complexes associated with one or more of Sp1 binding sites 1-4 as well as with site 5. DNase I footprinting revealed two protected regions: PR-I (-92 to -130) and PR-II (-304 to -332). PR-I contains a putative binding site for Sp1 that was protected by recombinant Sp1 protein. Transfection studies in Schneider SL2 cells demonstrated that the ODC promoter is transactivated up to 350-fold by Sp1 and that this transactivation is dependent on the presence of Sp1 binding sites 1-4. Thus, although the ODC promoter binds multiple nuclear proteins, Sp1 or a related protein appears to be a critical determinant of ODC transcription, possibly through cooperative interactions between Sp1 and additional transcription factors.
Kumar
A P
AP
University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville 78957.
Mar
P K
PK
Zhao
B
B
Montgomery
R L
RL
Kang
D C
DC
Butler
A P
AP
D000818:Animals; D000941:Antigens / Q000276:immunology; D001667:Binding, Competitive; D015971:Gene Expression Regulation, Enzymologic; D009687:Nuclear Proteins / Q000378:metabolism; D009955:Ornithine Decarboxylase / Q000235:genetics* / Q000276:immunology; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D000941:Antigens; D009687:Nuclear Proteins; D016329:Sp1 Transcription Factor; D009955:Ornithine Decarboxylase