Mammalian two-hybrid assays for studies of interaction of p300 with transcription factors

Abstract

The two-hybrid system is a powerful genetic assay that allows the interaction between two proteins to be detected in vivo. It was originally described in 1989 and since then it has been one of the main techniques used to identify interactions between proteins from different cellular organisms. Here we describe the methods to study the interaction of p300 with other transcription factors, specifically between p300 and two transcription factors related to hypoxia and inflammation, HIF-1α and NF-κB-p65, respectively.

Figure 1

Schematic representation of the CheckMate Mammalian Two-Hybrid System. The CheckMate/Flexi Vector System relies upon three plasmids that are co-transfected into mammalian cells. The pFN10A (ACT) Flexi Vector contains a herpes simplex virus VP16 transcriptional activation domain upstream of the cloning site, and the pFN11A (BIND) Flexi Vector contains the yeast GAL4 DNA-binding domain upstream of the cloning site. The pFN11A (BIND) Flexi Vector also expresses the Renilla reniformis luciferase under the control of the SV40 promoter, allowing normalization for differences in transfection efficiency. The third vector, pGL4.31, contains five GAL4 binding sites upstream of a minimal TATA box, which is upstream of a firefly luciferase gene that acts as a reporter for interactions between proteins “X” and “Y”. “X” = CAD-HIF-1α or CAD-NF-κB-p65; “Y” = p300. Adapted from CheckMate Mammalian Two-Hybrid System Technical Manual (Promega).

Figure 2

p65 competes with HIF-1α for p300. hFOB cells were (A) co-transfected with VP16-p300, Gal4-CAD-HIF-1α fusion constructs (125 ng) and p65-CMV (375 ng) for 18 h. Cells were then exposed to Normoxia (N) or DFO-induced Hypoxia (H). Luciferase activity was measured 24 h post-treatment. Statistically significant difference compared to p300 + CAD-HIF-1α + Empty Vector (normoxia) (p ≤ 0.05). (B) cotransfected with VP16-p300, Gal4-CAD-p65 fusion constructs (125 ng) and HIF-1α-WT (375 ng) for 18 h. Cells were then exposed to N or H. Luciferase activity was measured 24 h post-treatment. Statistically significant difference compared to p300 + CAD-p65 + Empty vector (N) (p ≤ 0.05). Data are shown as the mean ± SE of three independent experiments performed in triplicate.