FIG. 2.

Effect of the elimination of the two enhancer AP-1 sites on MIEP activity. (A) The nucleotide sequences and locations of the two AP-1 sites lying within the MIEP enhancer are shown in shaded boxes. Below the wild-type sequence, the specific point mutations introduced in each AP-1 element are indicated in lowercase letters. Coordinates refer to the HCMV ie1/ie2 transcriptional start site. (B) Structures of the luciferase (Luc) reporter plasmids containing MIEP sequences from −1144 to +112 without (pMIEP.Luc) or with the AP-1⁻²³⁹ site (pMIEPAp1⁻²³⁹.Luc), the AP-1⁻²³⁹ and AP-1⁻¹⁷⁴ sites (pMIEPAp1^{−239/−174}.Luc), or the NF-κB sites (pMIEPNFkB.Luc) altered. AP-1 and NF-κB recognition sites are marked by circles and rectangles, respectively, and their positions within the MIEP are indicated. Open symbols, wild-type sequence; black symbols, mutated sequences. (C) U937 cells were electroporated with the indicated luciferase reporter constructs along with the internal control plasmid pRL-TK. Fifteen hours later, cells were serum starved for 24 h and then treated with TPA (20 ng/ml; +) or vehicle (DMSO; −) for 6 h. Cells were lysed, and luciferase activity was determined. Each value represents the average ± standard deviation of four determinations. The results are presented as the fold induction, taking as 1.00 the activity exhibited by the cells transfected with plasmid pMIEP.Luc in the absence of TPA. (D) Similar to the experiment shown in panel C, except that the results are presented as percentages of TPA stimulation of cells transfected with the corresponding reporter construct relative to the activity of TPA-induced cells transfected with pMIEP.Luc (100%). (E) MEFs were transfected with pMIEP.Luc, pMIEPAp1^{−239/−174}.Luc, or pMIEPNFkB.Luc and processed as indicated for panel D. Fold induction of the pMIEP.Luc after TPA stimulation ranged from 4 to 9 in different experiments carried out in MEFs.