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J Mol Biol. 1999 Jun 11;289(3):459-71.

Interaction of interferon regulatory factor-1 and nuclear factor kappaB during activation of inducible nitric oxide synthase transcription.

Author information

1
Division of Cardiology, Department of Medicine, School of Medicine, The Johns Hopkins University School of Medicine, 720 Rutland Ave, Baltimore, MD, 21205, USA.

Abstract

We investigated the molecular mechanism for the synergistic induction of inducible nitric oxide synthase transcription by TNF-alpha and IFN-gamma. Since TNF-alpha and IFN-gamma stimulate cells in part by activating NF-kappaB and IRF-1, we hypothesized that these two transcription factors interact with each other. IRF-1 and NF-kappaB co-localize in the nucleus of stimulated macrophages. Co-immunoprecipitation experiments show that IRF-1 and NF-kappaB interact in stimulated but not resting cells. Super-shift experiments show that IRF-1 and NF-kappaB interact while binding to their respective DNA binding sites. These results demonstrate the existence of a physical interaction between IRF-1 and NF-kappaB proteins in vivo. We next suggested that this interaction between IRF-1 and NF-kappaB 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 kappaB site. Furthermore, stimulated nuclear extracts depleted of IRF-1 and NF-kappaB cannot induce cyclization. We conclude that IRF-1 and NF-kappaB 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-gamma synergistically augments inducible nitric oxide synthase transcription.

PMID:
10356322
DOI:
10.1006/jmbi.1999.2752
[Indexed for MEDLINE]

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