A Tandem GC Box Motif Is Necessary for Lipopolysaccharide-Induced Transcription of the Type II TNF Receptor Gene

John R. Bethea, Yoshihiro Ohmori, Thomas A. Hamilton

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

LPS induces the expression of the gene encoding the type II TNF-α receptor in mononuclear phagocytes. To elucidate the nuclear signaling mechanisms involved in this response, a 772-bp fragment of the TNFRII gene promoter was analyzed by deletion and site-specific mutagenesis following transient transfection in the macrophage-like cell line RAW264.7. A region located between -100 and -75 relative to the transcription start site was found to be essential for LPS sensitivity. This contained a GC-rich region composed of two tandemly arrayed Sp-1 sites. While mutation of this region confirmed that it was essential for LPS sensitivity, the sequence was not able to confer LPS sensitivity upon a heterologous promoter. Internal deletion and site-specific mutagenesis of the 100-bp fragment identified regions immediately flanking an initiator region (Inr) site that were also necessary for sensitivity to LPS. Electrophoretic mobility shift assays demonstrated that the GC box bound Sp-1 and Sp-3, although the level of binding activity did not vary with LPS stimulation. An oligonucleotide probe containing nucleotide positions -45 to -18 also bound several protein complexes, but these were not enhanced by LPS. These findings indicate that the tandem GC box is necessary, but not sufficient, for LPS-mediated transcription of the TNFRII gene. A second apparently novel motif, located within 15 nucleotides of the Inr, is also necessary.

Original languageEnglish
Pages (from-to)5815-5823
Number of pages9
JournalJournal of Immunology
Volume158
Issue number12
StatePublished - Jun 15 1997

Fingerprint

Tumor Necrosis Factor Receptors
Site-Directed Mutagenesis
Lipopolysaccharides
Nucleotides
GC Rich Sequence
Oligonucleotide Probes
Transcription Initiation Site
Electrophoretic Mobility Shift Assay
Phagocytes
Genes
Transfection
Macrophages
Gene Expression
Cell Line
Mutation
Proteins

ASJC Scopus subject areas

  • Immunology

Cite this

A Tandem GC Box Motif Is Necessary for Lipopolysaccharide-Induced Transcription of the Type II TNF Receptor Gene. / Bethea, John R.; Ohmori, Yoshihiro; Hamilton, Thomas A.

In: Journal of Immunology, Vol. 158, No. 12, 15.06.1997, p. 5815-5823.

Research output: Contribution to journalArticle

Bethea, John R. ; Ohmori, Yoshihiro ; Hamilton, Thomas A. / A Tandem GC Box Motif Is Necessary for Lipopolysaccharide-Induced Transcription of the Type II TNF Receptor Gene. In: Journal of Immunology. 1997 ; Vol. 158, No. 12. pp. 5815-5823.
@article{42168b4804074c1090f7458290b052fa,
title = "A Tandem GC Box Motif Is Necessary for Lipopolysaccharide-Induced Transcription of the Type II TNF Receptor Gene",
abstract = "LPS induces the expression of the gene encoding the type II TNF-α receptor in mononuclear phagocytes. To elucidate the nuclear signaling mechanisms involved in this response, a 772-bp fragment of the TNFRII gene promoter was analyzed by deletion and site-specific mutagenesis following transient transfection in the macrophage-like cell line RAW264.7. A region located between -100 and -75 relative to the transcription start site was found to be essential for LPS sensitivity. This contained a GC-rich region composed of two tandemly arrayed Sp-1 sites. While mutation of this region confirmed that it was essential for LPS sensitivity, the sequence was not able to confer LPS sensitivity upon a heterologous promoter. Internal deletion and site-specific mutagenesis of the 100-bp fragment identified regions immediately flanking an initiator region (Inr) site that were also necessary for sensitivity to LPS. Electrophoretic mobility shift assays demonstrated that the GC box bound Sp-1 and Sp-3, although the level of binding activity did not vary with LPS stimulation. An oligonucleotide probe containing nucleotide positions -45 to -18 also bound several protein complexes, but these were not enhanced by LPS. These findings indicate that the tandem GC box is necessary, but not sufficient, for LPS-mediated transcription of the TNFRII gene. A second apparently novel motif, located within 15 nucleotides of the Inr, is also necessary.",
author = "Bethea, {John R.} and Yoshihiro Ohmori and Hamilton, {Thomas A.}",
year = "1997",
month = "6",
day = "15",
language = "English",
volume = "158",
pages = "5815--5823",
journal = "Journal of Immunology",
issn = "0022-1767",
publisher = "American Association of Immunologists",
number = "12",

}

TY - JOUR

T1 - A Tandem GC Box Motif Is Necessary for Lipopolysaccharide-Induced Transcription of the Type II TNF Receptor Gene

AU - Bethea, John R.

AU - Ohmori, Yoshihiro

AU - Hamilton, Thomas A.

PY - 1997/6/15

Y1 - 1997/6/15

N2 - LPS induces the expression of the gene encoding the type II TNF-α receptor in mononuclear phagocytes. To elucidate the nuclear signaling mechanisms involved in this response, a 772-bp fragment of the TNFRII gene promoter was analyzed by deletion and site-specific mutagenesis following transient transfection in the macrophage-like cell line RAW264.7. A region located between -100 and -75 relative to the transcription start site was found to be essential for LPS sensitivity. This contained a GC-rich region composed of two tandemly arrayed Sp-1 sites. While mutation of this region confirmed that it was essential for LPS sensitivity, the sequence was not able to confer LPS sensitivity upon a heterologous promoter. Internal deletion and site-specific mutagenesis of the 100-bp fragment identified regions immediately flanking an initiator region (Inr) site that were also necessary for sensitivity to LPS. Electrophoretic mobility shift assays demonstrated that the GC box bound Sp-1 and Sp-3, although the level of binding activity did not vary with LPS stimulation. An oligonucleotide probe containing nucleotide positions -45 to -18 also bound several protein complexes, but these were not enhanced by LPS. These findings indicate that the tandem GC box is necessary, but not sufficient, for LPS-mediated transcription of the TNFRII gene. A second apparently novel motif, located within 15 nucleotides of the Inr, is also necessary.

AB - LPS induces the expression of the gene encoding the type II TNF-α receptor in mononuclear phagocytes. To elucidate the nuclear signaling mechanisms involved in this response, a 772-bp fragment of the TNFRII gene promoter was analyzed by deletion and site-specific mutagenesis following transient transfection in the macrophage-like cell line RAW264.7. A region located between -100 and -75 relative to the transcription start site was found to be essential for LPS sensitivity. This contained a GC-rich region composed of two tandemly arrayed Sp-1 sites. While mutation of this region confirmed that it was essential for LPS sensitivity, the sequence was not able to confer LPS sensitivity upon a heterologous promoter. Internal deletion and site-specific mutagenesis of the 100-bp fragment identified regions immediately flanking an initiator region (Inr) site that were also necessary for sensitivity to LPS. Electrophoretic mobility shift assays demonstrated that the GC box bound Sp-1 and Sp-3, although the level of binding activity did not vary with LPS stimulation. An oligonucleotide probe containing nucleotide positions -45 to -18 also bound several protein complexes, but these were not enhanced by LPS. These findings indicate that the tandem GC box is necessary, but not sufficient, for LPS-mediated transcription of the TNFRII gene. A second apparently novel motif, located within 15 nucleotides of the Inr, is also necessary.

UR - http://www.scopus.com/inward/record.url?scp=0031570409&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0031570409&partnerID=8YFLogxK

M3 - Article

C2 - 9190933

AN - SCOPUS:0031570409

VL - 158

SP - 5815

EP - 5823

JO - Journal of Immunology

JF - Journal of Immunology

SN - 0022-1767

IS - 12

ER -