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*
Department of Hematology, Molecular Medicine Unit, Atomic Bomb Disease Institute, Nagasaki University School of Medicine, and
Division of Cytokine Signaling, Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Medicine, Nagasaki, Japan
The present study investigates the regulatory mechanisms involved
in the cooperation between IFN-
and TNF-
to promote transcription
from IFN regulatory factor-1 (IRF-1). A transient transfection analysis
revealed that the region between -218 and -144, where +1 is the
transcription start site, as well as previously reported downstream
elements, pp
B and IFN- activation site/B, were required for
the optimal response to the two cytokines. A subsequent DNase I
footprint analysis showed that the region between -171 and -144 was
inducibly protected with stimulation by TNF-, and this protection
was significantly enhanced with the combination of IFN- and TNF-.
In an EMSA with the protected region as a probe, a TNF--inducible
complex (C1) and an IFN--inducible complex (C2), but no
synergy-specific DNA-protein complexes, were recognized. The C1 complex
consisted of a pre-existing factor (p65/p50), whereas the C2 complex
consisted of a newly synthesized IRF-1-related factor. A methylation
interference assay revealed the critical G residues (from -167 to
-151) for the DNA-protein complex formation specific to the cytokine
response, and within this region the novel B sequence, the promoter
distal B (pdB) element (5'-GGGGAAGTAC-3'), was identified.
Because the base substitutions over the pdB region (from -171 to
-144) affected not only the TNF--response but also that of IFN-,
this region might contribute to the cooperative action of the NF-B
subunits with the IRF-1-related factor. Finally, we demonstrated that
none of the cis-acting elements, ppB, pdB, or
IFN- activation site/B, is dispensable for the optimal synergism
in response to IFN- and TNF-.
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