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. 1998 Sep;18(9):5073–5081. doi: 10.1128/mcb.18.9.5073

FIG. 1.

FIG. 1

c-jun does not activate the A-TRE. (A) Sequence comparison of the A- and C-TREs and A-TRE mutants. The core TRE motifs are boxed, the center of the palindrome where the C- and A-TREs diverge is in bold, and mutations of the A-TRE are underlined. (B) Comparison of activity of the A- and C-TREs in HeLa cells and in F9 cells transfected with c-jun. F9 cells were transfected with 3 μg of TRE-human GH reporter and 5 μg of either pRSV-neo (control) or pRSV-c-jun using calcium phosphate precipitation. In HeLa cells, pRSV-neo was used to keep total DNA at 8 μg in both cell types. Results (mean ± standard deviations of six to eight independent determinations) are expressed as fold induction relative to the −135 bp ANF parent vector. (C) The A-TRE is inducible only by heterodimers in F9 cells. Cotransfections of F9 cells with 3× TRE reporter plasmids (3 μg) and various AP-1 vectors (5 μg in total) were performed, and results are expressed as fold induction relative to activity of the respective 3× A-TRE reporter cotransfected with pRSV-neo. Results (n = 2 from a typical experiment of more than six) are expressed as fold induction relative to 3× A-TRE activity in cells cotransfected with pRSV-neo. c-j, c-jun; cf, c-fos; jD, junD. (D) Heterodimers activate the A-TRE in primary cardiocyte cultures. Cardiocytes were transfected with a single-copy A-TRE (or mutant) reporter (3 μg) in the presence of expression vectors for jun-fos (2.5 μg of each), and media were assayed for GH after 48 h. Results are relative to cotransfection with pRSV-neo. The effect of a half-site mutation of the A-TRE (MUT4) is also shown. (E) junD–c-fos activates the ANF promoter. HeLa cells or cardiocytes were transfected with a reporter construct (3 μg) containing 700 bp of the rat ANF promoter in the absence (neo) or presence of junD and c-fos (1 μg of each). (F) Mutation of two base pairs confers basal activity to the A-TRE. HeLa cells and ventricular cardiocytes were transfected with 3 μg of 1× A-TRE (or mutant) expression vectors as shown, and the media were assayed for GH. The sequences of the mutants are shown in Fig. 1A. Results are shown as fold induction relative to the −135 bp ANF parent reporter plasmid. (G) Mutant A-TRE oligonucleotides were incubated with purified c-jun (5 to 15 ng) and subjected to gel shift analysis as described above.