Assembly of a Functional Beta Interferon Enhanceosome Is Dependent on ATF-2–c-jun Heterodimer Orientation

Abstract

Heterodimeric transcription factors, including the basic region-leucine zipper (bZIP) protein ATF-2–c-jun, are well-characterized components of an enhanceosome that mediates virus induction of the human beta interferon (IFN-β) gene. Here we report that within the IFN-β enhanceosome the ATF-2–c-jun heterodimer binds in a specific orientation, which is required for assembly of a complex between ATF-2–c-jun and interferon regulatory factor 3 (IRF-3). We demonstrate that correct orientation of the ATF-2–c-jun binding site is required for virus induction of the IFN-β gene and for IRF-3-dependent activation of a composite ATF-2– c-jun–IRF site in the IFN-β promoter. We also show that in vitro the DNA-bound ATF-2–c-jun heterodimer adopts a fixed orientation upon the binding of IRF-3 at an adjacent site in the IFN-β enhancer and that the DNA-binding domain of IRF-3 is sufficient to mediate this effect. In addition, we show that the DNA-binding domain of ATF-2 is necessary and sufficient for selective protein-protein interactions with IRF-3. Strikingly, in vivo chromatin immunoprecipitation experiments with IFN-β reporter constructs reveal that recruitment of IRF-3 to the IFN-β promoter upon virus infection is dependent on the orientation of the ATF-2–c-jun heterodimer binding site. These observations demonstrate functional and physical cooperativity between the bZIP and IRF transcription factor families and illustrate the critical role of heterodimeric transcription factors in formation of the IFN-β enhanceosome.

ACKNOWLEDGMENTS

J.V.F. and B.S.P. contributed equally to this work.

We thank Paul Clemons for his work in the initial stages of the ATF-2–c-jun protein-DNA cross-linking experiments and Aseem Ansari for suggesting the AzP bromide photo-cross-linking technique. The IRF-3 E₅ expression vector was prepared by C.H.L. and Marc Wathelet. We thank Michael Green and Paula Pitha for generously providing GST–ATF-2 and GST–IRF-3 expression vectors. We thank Tae Hoon Kim for critical reading of the manuscript and for sharing unpublished observations, and we thank Judith Grisham for editorial assistance. Special thanks go to Stephen C. Harrison, in whose laboratory E.F. is a postdoctoral fellow.

This work was supported by grant AI20642 from the NIH to T.M. J.V.F. acknowledges the support of a National Defense Science and Engineering Graduate Research Fellowship. E.F. is supported by the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation Fellowship, DRG 1547.