Interaction of Interferon Regulatory Factor-1 and Nuclear Factor κB during activation of inducible nitric oxide synthase transcription

Marta Saura, Carlos Zaragoza, Clare Bao, Audrey McMillan, Charles J. Lowenstein

Research output: Contribution to journalArticle

Abstract

We investigated the molecular mechanism for the synergistic induction of inducible nitric oxide synthase transcription by TNF-α and IFN-γ. Since TNF-α and IFN-γ stimulate cells in part by activating NF-κB and IRF-1, we hypothesized that these two transcription factors interact with each other. IRF-1 and NF-κB co-localize in the nucleus of stimulated macrophages. Co-immunoprecipitation experiments show that IRF-1 and NF-κB interact in stimulated but not resting cells. Super-shift experiments show that IRF-1 and NF-κB interact while binding to their respective DNA binding sites. These results demonstrate the existence of a physical interaction between IRF-1 and NF-κB proteins in vivo. We next suggested that this interaction between IRF-1 and NF-κB bends the DNA of the iNOS promoter region. Using a cyclization assay, we demonstrate that nuclear extracts from stimulated cells accelerate the rate of conversion of a linear to circular DNA, compared to extracts from resting cells. However, stimulated nuclear extracts cannot affect the rate of cyclization of a promoter with a mutant IRE or κB site. Furthermore, stimulated nuclear extracts depleted of IRF-1 and NF-κB cannot induce cyclization. We conclude that IRF-1 and NF-κB interact in vivo, and that this interaction physically bends the indicible nitric oxide synthase promoter DNA. This interaction may explain the mechanism by which IFN-γ synergistically augments inducible nitric oxide synthase transcription.

Original languageEnglish (US)
Pages (from-to)459-471
Number of pages13
JournalJournal of Molecular Biology
Volume289
Issue number3
DOIs
StatePublished - Jun 11 1999

Fingerprint

Interferon Regulatory Factor-1
Nitric Oxide Synthase Type II
Cyclization
Cell Extracts
DNA
Neurofibromin 1
Circular DNA
Immunoprecipitation
Genetic Promoter Regions
Nitric Oxide Synthase
Transcription Factors
Macrophages
Binding Sites

Keywords

  • Inflammation
  • Macrophage
  • NOS2
  • Radicals
  • Virus

ASJC Scopus subject areas

  • Virology

Cite this

Interaction of Interferon Regulatory Factor-1 and Nuclear Factor κB during activation of inducible nitric oxide synthase transcription. / Saura, Marta; Zaragoza, Carlos; Bao, Clare; McMillan, Audrey; Lowenstein, Charles J.

In: Journal of Molecular Biology, Vol. 289, No. 3, 11.06.1999, p. 459-471.

Research output: Contribution to journalArticle

Saura, Marta ; Zaragoza, Carlos ; Bao, Clare ; McMillan, Audrey ; Lowenstein, Charles J. / Interaction of Interferon Regulatory Factor-1 and Nuclear Factor κB during activation of inducible nitric oxide synthase transcription. In: Journal of Molecular Biology. 1999 ; Vol. 289, No. 3. pp. 459-471.
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