Transcriptional activation by AP-2alpha is modulated by the oncogene DEK

Abstract

Cell differentiation and development are highly regulated processes at the transcriptional level. One of the main transcription factors that regulate these processes is AP-2alpha, a cell-type specific protein required for vertebrate development and embryogenesis. AP-2alpha also regulates apoptosis and cell-cycle specific events by interacting with the oncogene c-Myc. In searching for novel AP-2alpha- interacting factors, using an affinity chromatography approach, we have observed that oncoprotein DEK interacts with AP-2alpha in vitro. The existence of an interaction between AP-2alpha and DEK in cellular cultures was demonstrated by expression of a tagged AP-2alpha form followed by immunodetection. By transient co-expression experiments using a reporter for APOE promoter activity we have found that DEK stimulates the transactivation activity of AP-2alpha over APOE promoter. Finally, electrophoretic mobility shift assays suggested that DEK enhances the DNA-binding activity of AP-2alpha. Our data suggest a novel cellular function of DEK as a transcriptional co-activator.

Figure 1

Purification of DEK by AP-2α affinity chromatography. A rat nuclear extract (E) was fractionated and enriched using an AP-2α affinity column (see Materials and Methods). The eluted fractions were pooled and subjected to SDS–PAGE, followed by silver staining (1). The band indicated by an arrow was identified as the rat homologue of human DEK by mass spectrometry.

Figure 2

Interaction of hisAP-2 with human DEK in U-87, D7 and D10 astrocytoma cell lines. (A) Analysis of the expression of hisAP-2 in the stably transfected clones. Nuclear extracts (20 µg) from U-87, D7 and D10 were analysed by western blotting using C-18 antibody. As a positive control we used 1 µg of AP-2r. The arrow indicates the position of hisAP-2 band. (B) Purification of hisAP-2 from the stably transfected cell clones. Purified fractions (30 µl) from U-87, D7 and D10 were analysed by western blotting using the C-18 antibody. The arrow indicates position of hisAP-2. (C) DNA-binding activity of the purified fractions. EMSAs were carried out using 5 and 15 µl of the purified fractions from U-87, D7 and D10 and CXX probe. As a positive control 0.5 and 1 µg of AP-2r were used. The arrow indicates the position of the hisAP-2–DNA complex. (D) Co-purification of DEK with AP-2α. Ten micrograms of the original nuclear extracts and 40 µg of purified fractions from U-87, D7 and D10 were analysed by western blotting using a polyclonal antibody that recognizes the human DEK protein (33). The arrow indicates the position of the DEK band. In (A), (B) and (D), the molecular weight markers are indicated on the left.

Figure 3

Effect of DEK on the transcriptional activity of AP-2α in HepG2 cells. 300 ng of the APOE promoter luciferase vector pXP2-227 were co-transfected with 300 ng of pcDNA3 (grey bars) or with an AP-2α expression vector (black bars) and increasing quantities of 0, 200, 400, 600 or 800 ng of the DEK expression vector combined with decreasing quantities of pcDNA3 of 800, 600, 400, 200 and 0 ng. Luciferase activity was corrected for the transfection efficiency taking into account the activity of 300 ng of β-galactosidase expression vector. Transfection assays were performed in triplicate and results are representative of at least three independent experiments; data are expressed as mean ± SEM.

Figure 4

Effect of DEK on binding of AP-2r to DNA in vitro. CXX probe was incubated with 10 (lanes 2–5), 30 (lanes 6–9) or 100 ng (lanes 10–13) of AP-2r in the presence of either 20 (lanes 3, 7 and 11), 100 (lanes 4, 8 and 12) or 500 ng (lanes 5, 9 and 13) of GST-DEK. As negative controls, CXX probe was incubated with 100 ng of AP-2r in the presence of either 20 (lane 16), 100 (lane 17) or 500 ng (lane 18) of GST, and 500 ng of GST-DEK (lane 14) and GST (lane 19) were incubated with the probe in the absence of AP-2r.