IRF-1 and NF-κB p50/cRel bind to distinct regions of the proximal murine IL-12 p35 promoter during costimulation with IFN-γ and LPS

Jutta I. Kollet, Thomas M Petro

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

LPS and IFN-γ, which activate NF-κB cRel/p50 and IFN regulatory factor-1 (IRF-1), respectively, costimulate expression of the IL-12 p35 subunit in macrophages. The murine p35 promoter proximal to exon 2 is active during costimulation with IFN-γ and LPS because it contains κB and IRF elements (E) with significant homology to the human p35 promoter. IFN-γ or LPS stimulate nuclear localization of IRF-1 or cRel/p50, respectively, in the RAW 264.7 macrophage cell line. EMSAs reveal that IFN-γ/LPS stimulates within 2 h, in RAW 264.7 cells or peritoneal macrophages, nuclear localization of proteins that target nt -137/-93 of the p35 promoter. DNA affinity assays utilizing nuclear extracts from RAW 264.7 cells show that NF-κB cRel and p50 bind to the κB-E within nt -122 to -93 of the p35 exon 2 promoter while IRF-1 binds to the IRF-E within nt -157 to -113 but not the one within nt -122 to -93. In addition, p50/cRel attachment to the κB-E was not dependent upon IRF-1 association with the IRF-E, and vice versa. Chromosome immunoprecipitation assays confirm inducible recruitment of IRF-1 and cRel to the endogenous p35 exon 2 promoter in both RAW 264.7 and primary macrophages costimulated with IFN-γ and LPS. IFN-γ, IFNγ/LPS, or overexpression of IRF-1 plus cRel activated the wild-type p35 promoter reporter but not the p35 promoter reporter mutated at nt -110/-101 or in the presence of IRF-1 siRNA. Thus, cRel with IRF-1 induce p35 expression through a small region of the p35 exon 2 promoter during IFN-γ and LPS costimulation of macrophages.

Original languageEnglish (US)
Pages (from-to)623-633
Number of pages11
JournalMolecular Immunology
Volume43
Issue number6
DOIs
StatePublished - Feb 1 2006

Fingerprint

Interleukin-12
Exons
Macrophages
Peritoneal Macrophages
Nuclear Proteins
Immunoprecipitation
Small Interfering RNA
Chromosomes
DNA
RAW 264.7 Cells

Keywords

  • IL-12
  • IRF-1
  • Macrophages
  • NF-κB
  • Transcription factors

ASJC Scopus subject areas

  • Molecular Biology
  • Immunology

Cite this

IRF-1 and NF-κB p50/cRel bind to distinct regions of the proximal murine IL-12 p35 promoter during costimulation with IFN-γ and LPS. / Kollet, Jutta I.; Petro, Thomas M.

In: Molecular Immunology, Vol. 43, No. 6, 01.02.2006, p. 623-633.

Research output: Contribution to journalArticle

@article{ec103ff185f04fff836a03f117d919a7,
title = "IRF-1 and NF-κB p50/cRel bind to distinct regions of the proximal murine IL-12 p35 promoter during costimulation with IFN-γ and LPS",
abstract = "LPS and IFN-γ, which activate NF-κB cRel/p50 and IFN regulatory factor-1 (IRF-1), respectively, costimulate expression of the IL-12 p35 subunit in macrophages. The murine p35 promoter proximal to exon 2 is active during costimulation with IFN-γ and LPS because it contains κB and IRF elements (E) with significant homology to the human p35 promoter. IFN-γ or LPS stimulate nuclear localization of IRF-1 or cRel/p50, respectively, in the RAW 264.7 macrophage cell line. EMSAs reveal that IFN-γ/LPS stimulates within 2 h, in RAW 264.7 cells or peritoneal macrophages, nuclear localization of proteins that target nt -137/-93 of the p35 promoter. DNA affinity assays utilizing nuclear extracts from RAW 264.7 cells show that NF-κB cRel and p50 bind to the κB-E within nt -122 to -93 of the p35 exon 2 promoter while IRF-1 binds to the IRF-E within nt -157 to -113 but not the one within nt -122 to -93. In addition, p50/cRel attachment to the κB-E was not dependent upon IRF-1 association with the IRF-E, and vice versa. Chromosome immunoprecipitation assays confirm inducible recruitment of IRF-1 and cRel to the endogenous p35 exon 2 promoter in both RAW 264.7 and primary macrophages costimulated with IFN-γ and LPS. IFN-γ, IFNγ/LPS, or overexpression of IRF-1 plus cRel activated the wild-type p35 promoter reporter but not the p35 promoter reporter mutated at nt -110/-101 or in the presence of IRF-1 siRNA. Thus, cRel with IRF-1 induce p35 expression through a small region of the p35 exon 2 promoter during IFN-γ and LPS costimulation of macrophages.",
keywords = "IL-12, IRF-1, Macrophages, NF-κB, Transcription factors",
author = "Kollet, {Jutta I.} and Petro, {Thomas M}",
year = "2006",
month = "2",
day = "1",
doi = "10.1016/j.molimm.2005.04.004",
language = "English (US)",
volume = "43",
pages = "623--633",
journal = "Molecular Immunology",
issn = "0161-5890",
publisher = "Elsevier Limited",
number = "6",

}

TY - JOUR

T1 - IRF-1 and NF-κB p50/cRel bind to distinct regions of the proximal murine IL-12 p35 promoter during costimulation with IFN-γ and LPS

AU - Kollet, Jutta I.

AU - Petro, Thomas M

PY - 2006/2/1

Y1 - 2006/2/1

N2 - LPS and IFN-γ, which activate NF-κB cRel/p50 and IFN regulatory factor-1 (IRF-1), respectively, costimulate expression of the IL-12 p35 subunit in macrophages. The murine p35 promoter proximal to exon 2 is active during costimulation with IFN-γ and LPS because it contains κB and IRF elements (E) with significant homology to the human p35 promoter. IFN-γ or LPS stimulate nuclear localization of IRF-1 or cRel/p50, respectively, in the RAW 264.7 macrophage cell line. EMSAs reveal that IFN-γ/LPS stimulates within 2 h, in RAW 264.7 cells or peritoneal macrophages, nuclear localization of proteins that target nt -137/-93 of the p35 promoter. DNA affinity assays utilizing nuclear extracts from RAW 264.7 cells show that NF-κB cRel and p50 bind to the κB-E within nt -122 to -93 of the p35 exon 2 promoter while IRF-1 binds to the IRF-E within nt -157 to -113 but not the one within nt -122 to -93. In addition, p50/cRel attachment to the κB-E was not dependent upon IRF-1 association with the IRF-E, and vice versa. Chromosome immunoprecipitation assays confirm inducible recruitment of IRF-1 and cRel to the endogenous p35 exon 2 promoter in both RAW 264.7 and primary macrophages costimulated with IFN-γ and LPS. IFN-γ, IFNγ/LPS, or overexpression of IRF-1 plus cRel activated the wild-type p35 promoter reporter but not the p35 promoter reporter mutated at nt -110/-101 or in the presence of IRF-1 siRNA. Thus, cRel with IRF-1 induce p35 expression through a small region of the p35 exon 2 promoter during IFN-γ and LPS costimulation of macrophages.

AB - LPS and IFN-γ, which activate NF-κB cRel/p50 and IFN regulatory factor-1 (IRF-1), respectively, costimulate expression of the IL-12 p35 subunit in macrophages. The murine p35 promoter proximal to exon 2 is active during costimulation with IFN-γ and LPS because it contains κB and IRF elements (E) with significant homology to the human p35 promoter. IFN-γ or LPS stimulate nuclear localization of IRF-1 or cRel/p50, respectively, in the RAW 264.7 macrophage cell line. EMSAs reveal that IFN-γ/LPS stimulates within 2 h, in RAW 264.7 cells or peritoneal macrophages, nuclear localization of proteins that target nt -137/-93 of the p35 promoter. DNA affinity assays utilizing nuclear extracts from RAW 264.7 cells show that NF-κB cRel and p50 bind to the κB-E within nt -122 to -93 of the p35 exon 2 promoter while IRF-1 binds to the IRF-E within nt -157 to -113 but not the one within nt -122 to -93. In addition, p50/cRel attachment to the κB-E was not dependent upon IRF-1 association with the IRF-E, and vice versa. Chromosome immunoprecipitation assays confirm inducible recruitment of IRF-1 and cRel to the endogenous p35 exon 2 promoter in both RAW 264.7 and primary macrophages costimulated with IFN-γ and LPS. IFN-γ, IFNγ/LPS, or overexpression of IRF-1 plus cRel activated the wild-type p35 promoter reporter but not the p35 promoter reporter mutated at nt -110/-101 or in the presence of IRF-1 siRNA. Thus, cRel with IRF-1 induce p35 expression through a small region of the p35 exon 2 promoter during IFN-γ and LPS costimulation of macrophages.

KW - IL-12

KW - IRF-1

KW - Macrophages

KW - NF-κB

KW - Transcription factors

UR - http://www.scopus.com/inward/record.url?scp=28944440894&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=28944440894&partnerID=8YFLogxK

U2 - 10.1016/j.molimm.2005.04.004

DO - 10.1016/j.molimm.2005.04.004

M3 - Article

VL - 43

SP - 623

EP - 633

JO - Molecular Immunology

JF - Molecular Immunology

SN - 0161-5890

IS - 6

ER -