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Fig. 1. Schematic representation of GalDBD (A) and VP16 (B) fusion constructs used to analyze the interaction of Ref-1 with HLF and HIF-la. The amino acid compositions of the basic regions for HLF and HIF-la are included. bHLH, basic helix-loop-helix domain.

1-265), or HIF-la amino acids 1-245 (GalDBD/HIF-1 a 1-245). The VP16 chimeras contain the 78-amino acid activation domain from the herpes simplex virus fused in frame with either full-length Ref-1 (VP16/Ref-l), HLF amino acids 1-265 (VP16/HLF 1-265), or HLF 1-265 cysteine (amino acid residue 25)-to-serine point mutant (VP16/HLF 1-265 C25S). The luciferase reporter construct G5Elb-Luc contains five copies of the Gal4-binding site upstream of the minimal Elb promoter-driven firefly luciferase gene.

Two-Hybrid Assay

Several methods to transfect plasmid DNA into mammalian cells are available, including calcium phosphate, DEAE-dextran, and liposome-mediated transfec-tion. To analyze the in vivo interaction of Ref-1 with HLF we use the monkey kidney COS-1 cell line in conjunction with the liposomal transfection reagent Lipofect AMINE 2000 (Life Technologies, Rockville, MD) and the Dual-Luciferase reporter assay system (Promega, Madison, WI). Although we have obtained similar results with other transfection reagents (l,2-dioleoyl-3-trimethylammonium-propane, DOTAP; Boehringer Mannheim, Germany) and cell lines (human embryonic kidney, HEK293T), in our hands COS-1 cells and the LipofectAMINE 2000 reagent routinely give high transfection efficiencies, resulting in consistent two-hybrid results. A description of general mammalian cell tissue culture techniques is beyond the scope of this chapter; readers who require further information on these techniques are therefore referred to methods and protocols given elsewhere.11

For two-hybrid assays COS-1 cells are cultured in a humidified incubator (95% air/5% C02) at 37° in Dulbecco's modified Eagle's medium (DMEM, GIBCO-BRL, Gaithersburg, MD) supplemented with 10% (v/v) fetal calf serum (FCS; GIBCO-BRL). Before transfection, cells are trypsinized and plated onto 24-well tissue culture plates (Falcon; Becton Dickinson Labware, Lincoln Park, NJ) at a density of 5 x 104 viable cells per well in a final volume of 450 ¡A of DMEM-FCS. After 24 hr (50-60% cell confluency), transfections are carried out with LipofectAMINE 2000 reagent as follows.

1. For each transfection (or well of cells), 425 ng of plasmid DNA consisting of 300 ng of G5Elb-Luc reporter, 50 ng of GalDBD or GalDBD chimera, 50 ng of VP 16 or VP 16 chimera, and 25 ng of Renilla internal control pRL-TK plasmid (Promega) is diluted into DMEM without FCS to a final volume of 25 fi\. The pRL-TK control plasmid provides constitutive expression of Renilla luciferase from the herpes simplex virus thymidine kinase (TK) promoter and its addition serves as an internal control of transfection efficiency.

2. For each well of cells to be transfected, dilute 1 /xl of LipofectAMINE 2000 reagent into 25 /il of DMEM without FCS.

3. Combine the 25 /xl of plasmid-DMEM mix (step 1) with 25 ¡A of diluted

LipofectAMINE 2000 reagent (step 2). Incubate the mixture at room temperature for 20 min to allow plasmid DNA-LipofectAMINE 2000 reagent complexes to form. Note: Diluted LipofectAMINE 2000 from step 2 must be combined with plasmid DNA mix within 10 min of preparation.

4. After incubation add the plasmid DNA-LipofectAMINE 2000 reagent complexes (50 //l) directly to each well. To obtain consistency and high trans-fection efficiencies it is important to disperse the plasmid DNA-LipofectAMINE 2000 reagent complexes thoroughly and evenly throughout each well. This is best done by gently rocking the plate back and forth three or four times.

5. Return the transfected cells to the incubator and incubate for 24 to 48 hr. We have found that it is not necessary to remove the complexes or change the medium during the assay, as prolonged cell exposure to LipofectAMINE 2000 does not affect the transfection activity.

6. After transfection wash all wells once with 500 >Ltl of phosphate-buffered saline solution and harvest with Dual-Luciferase lysis buffer (100 /il/well; Promega). Lysed extracts (3 /zl) are then analyzed for luciferase activity in a Turner Designs (Sunnyvale, CA) T20/20 luminometer, using the Dual-Luciferase reporter assay system, and measured G5Elb-Luc reporter activity is normalized to the activity of the internal control (pRL-TK).

Results and Discussion

Typical results of a two-hybrid assay showing Ref-1 interaction with HLF are presented in Fig. 2. A fusion protein containing the N-terminal domain of HLF encompassing the basic DNA-binding region attached to the activation domain of VP16 (VP16/HLF 1-265) is assayed for interaction with GalDBD/Ref-1 fusion protein in COS-1 cells. Coexpression of GalDBD/Ref-1 with VP16/HLF 1-265 results in an approximately 4-fold increase in reporter gene activity over that seen when GalDBD alone and VP16/HLF 1-265 are coexpressed, suggesting that GalDBD/Ref-1 can interact with VP16/HLF 1-265 via the Ref-1 portion of the chimera. Because it is possible that the Ref-1 fusion may interact with the activation domain of VP 16, it is also important to assay the GalDBD fusion protein with nonchimeric VP 16. Therefore, Fig. 2 also shows reporter gene activity when GalDBD/Ref-1 or GalDBD are coexpressed with nonchimeric VP16. Clearly, when GalDBD/Ref-1 is coexpressed with nonchimeric VP 16 the activity remains at the low basal level seen when GalDBD alone is coexpressed with VP 16, and thus the interaction of Ref-1 with the VP16/HLF 1-265 fusion is specific for the HLF 1-265 portion of the fusion construct.

Oxidoreductase factors such as Ref-1 are thought to interact by forming transient intermediate disulfide linkages with cysteine residues in target proteins.2'12 In analogy with the mechanism proposed for the interaction of Ref-1 with Fos and Jun, we have previously suggested that Ref-1 enhances HLF DNA-binding

12 A. Holmgren, Arinu. Rev. Biochem. 54, 237 (1985).

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