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 luci...

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Bibliographic Details
Published in:The Journal of biological chemistry 1995-03, Vol.270 (9), p.4341-4348
Main Authors: Kumar, Addanki P., Mar, Penny K., Zhao, Biwei, Montgomery, Raechelle L., Kang, Dong-Chul, Butler, Andrew P.
Format: Article
Language:English
Subjects:
Animals
Antigens - immunology
Binding, Competitive
Gene Expression Regulation, Enzymologic
Nuclear Proteins - metabolism
Ornithine Decarboxylase - genetics
Ornithine Decarboxylase - immunology
Promoter Regions, Genetic
Protein Binding
Rats
Sp1 Transcription Factor - metabolism
Transcriptional Activation
Tumor Cells, Cultured
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Summary: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 trans-activated up to 350-fold by Sp1 and that this trans-activation 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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.270.9.4341