Introduction

A growing number of transcription factors including activating protein-1 (AP-1), Myb, activating transcription factor/cAMP response element-binding protein (ATF/CREB), nuclear factor-*:B (NF-kB), and p53 have been shown to have their DNA-binding activities modulated by changes in redox status (reviewed by Morel and Barouki1). For the Fos-Jun heterodimer that constitutes the AP-1 complex, the nuclear redox protein Ref-1 imparts DNA-binding activity by reducing specific cysteine sulfhydryl groups in the basic DNA-binding regions of Fos and Jun. Although the precise mechanism by which Ref-1 reduces Fos and Jun is not known, chemical cross-linking studies with bacterially expressed proteins have suggested that a direct cy steine-mediated interaction may occur between Ref-1 and Jun.2 However, in vivo cellular interactions between Ref-1 and Fos or Jun have not been demonstrated, nor have interactions been shown in cell extracts by standard biochemical techniques such as coimmunoprecipitation. This is most likely due to the transient nature of such interactions.

The mammalian hypoxia-inducible factors HIF-la and HIF-like factor (HLF) are two highly related basic helix-loop-helix/Per-Arnt-Sim homology (bHLH/PAS) transcription factors that are rapidly activated by oxygen deprivation to induce a network of genes responsible for maintaining oxygen homeostasis (reviewed by Semenza3). The molecular mechanisms of oxygen sensing and signaling that lead to the activation of HIF-la and HLF are poorly understood. However, evidence suggests the involvement of an oxygen-regulated redox signal that may activate a kinase cascade or modify the HIFs directly.4 In support of this, overexpression of redox factors Ref-1 and thioredoxin enhances the hypoxic response of HIF-la and HLF.5-7 We have shown that Ref-1 can specifically enhance the

1 Y. Morel and R. Barouki, Biochem. J. 342,481 (1999).

2 S. Xanthoudakis, G. G. Miao, and T. Curran, Proc. Natl. Acad. Sci. U.S.A. 91,23 (1994).

3G. L. Semenza, Annu. Rev. Cell Dev. Biol. 15,551 (1999).

4 N. S. Chandel, D. S. McClintock, C. E. Feliciano, T. M. Wood, J. A. Melendez, A. M. Rodriguez, and P. T. Schumacker, J. Biol. Chem. 275,25130 (2000).

5 L. E. Huang, Z. Arany, D. M. Livingston, and H. F. Bunn, J. Biol. Chem. 271,32253 (1996).

6 M. Ema, K. Hirota, J. Mimura, H. Abe, J. Yodoi, K. Sogawa, L. Poellinger, and Y. Fujii-Kuriyama, EMBOJ. 18,1905 (1999).

7 P. Carrero, K. Okamoto, P. Coumailleau, S. O'Brien, H. Tanaka, and L. Poellinger, Mol. Cell. Biol. 20,402 (2000).

in vitro DNA-binding activity of HLF by reducing a cysteine residue in the basic DNA-binding domain of HLF.8

Here we describe a cell-based mammalian two-hybrid assay that is capable of detecting the intracellular interaction of Ref-1 with the basic DNA-binding domain of HLF. By using this assay we are able to demonstrate that the interaction between Ref-1 and HLF is dependent on a redox-sensitive cysteine in the basic region of HLF.

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