Regulation of rat ornithine decarboxylase promoter activity by binding of transcription factor Sp1

Addanki P Kumar, P. K. Mar, B. Zhao, R. L. Montgomery, D. C. Kang, A. P. Butler

Research output: Contribution to journalArticle

44 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)4341-4348
Number of pages8
JournalJournal of Biological Chemistry
Volume270
Issue number9
DOIs
StatePublished - 1995
Externally publishedYes

Fingerprint

Sp1 Transcription Factor
Ornithine Decarboxylase
Rats
Binding Sites
Oligonucleotides
Assays
Genes
Recombinant proteins
Electrophoretic mobility
5' Flanking Region
Deoxyribonuclease I
Biosynthesis
Polyamines
Electrophoretic Mobility Shift Assay
Transcription
Nuclear Proteins
Luciferases
Recombinant Proteins
Transfection
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Regulation of rat ornithine decarboxylase promoter activity by binding of transcription factor Sp1. / Kumar, Addanki P; Mar, P. K.; Zhao, B.; Montgomery, R. L.; Kang, D. C.; Butler, A. P.

In: Journal of Biological Chemistry, Vol. 270, No. 9, 1995, p. 4341-4348.

Research output: Contribution to journalArticle

Kumar, Addanki P ; Mar, P. K. ; Zhao, B. ; Montgomery, R. L. ; Kang, D. C. ; Butler, A. P. / Regulation of rat ornithine decarboxylase promoter activity by binding of transcription factor Sp1. In: Journal of Biological Chemistry. 1995 ; Vol. 270, No. 9. pp. 4341-4348.
@article{b5e6cc19f6e44f899f9745236549dcd0,
title = "Regulation of rat ornithine decarboxylase promoter activity by binding of transcription factor Sp1",
abstract = "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.",
author = "Kumar, {Addanki P} and Mar, {P. K.} and B. Zhao and Montgomery, {R. L.} and Kang, {D. C.} and Butler, {A. P.}",
year = "1995",
doi = "10.1074/jbc.270.9.4341",
language = "English (US)",
volume = "270",
pages = "4341--4348",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "9",

}

TY - JOUR

T1 - Regulation of rat ornithine decarboxylase promoter activity by binding of transcription factor Sp1

AU - Kumar, Addanki P

AU - Mar, P. K.

AU - Zhao, B.

AU - Montgomery, R. L.

AU - Kang, D. C.

AU - Butler, A. P.

PY - 1995

Y1 - 1995

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0028924259&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028924259&partnerID=8YFLogxK

U2 - 10.1074/jbc.270.9.4341

DO - 10.1074/jbc.270.9.4341

M3 - Article

C2 - 7876196

AN - SCOPUS:0028924259

VL - 270

SP - 4341

EP - 4348

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 9

ER -