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The reporter plasmids pBLCAT3 [promoterless; ATCC reference no. 37528 (ATCC, Manassas, VA, USA)] and pBLCAT2 (containing the thymidine kinase promoter; ATCC reference no. 37527) contain the bacterial gene CAT as a reporter (10). Promoter or enhancer sequences inserted into these vectors drive the expression of the CAT gene. The accumulated CAT protein level is then directly proportional to the activity of the promoter/enhancer and can be easily measured (e.g., by ELISA). The CAT assay system provides an easily quantifiable method for evaluating the effect of stimuli on an exogenous gene because of the absence of an endogenous counterpart in eukaryotic cells (13). However, both pBLCAT2 and pBLCAT3 vectors have been reported to have high background activity when they are transiently transfected into F9 embryonal carcinoma cells (14) and rat pheochromocytoma (PC12) cells (1). This background activity may mask the activity of the promoter/enhancer under study. AP1 transcription factors regulate gene expression through AP1 sites in the promoters of many genes [e.g., the matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs)]. AP1 factors include members of the Jun family (c-Jun, JunB, and JunD) that can homodimerize or heterodimerize with Fos family members (c-Fos, FosB, Fra-1, and Fra-2) to transactivate gene expression. These heterodimers and homodimers recognize and bind to AP1 motifs in genes, the consensus of which is 5′TGAGTCA-3′ (6). Non-consensus AP1 sites have previously been reported to be functional in a variety of vector sequences (4,8). An AP1 site at -59/-53 in the promoter of the Timp-1 gene is important for both the basal and inducible expression of the gene (2,9,12). To probe the transactivation potential of varying combinations of Fos and Jun factors on this non-consensus site (5′-TGAGTAA-3′) compared to the exact consensus found, for example, in the MMP-1 gene (5′-TGAGTCA-3′), co-transfection experiments were performed in murine C3H10T1/2 cells. Figure 1 shows the results of transfecting a -95/+47 Timp-1 reporter construct in pBLCAT3 with c-Fos and/or Jun expression vectors [all AP1 expression plasmids were kind gifts of Dr. P.R. Dobner, University of Massachusetts (5)]. The Timp-1 promoter is induced by c-Fos alone, with further induction in the additional presence of c-Jun or JunD. JunB represses the induction seen with c-Fos alone. Substitution of the Timp-1 AP1 motif for the consensus sequence (as above) in the context of the -95/+47 construct shows the same pattern of expression, which demonstrates that the A→C transversion does not alter the response to these AP1 factors. Intriguingly, when the AP1 site is mutated to a nonfunctional sequence (5′-GGAGGCA-3′), the -95/+47 construct loses induction by c-Fos alone or c-Fos and c-Jun but retains transactivation by c-Fos and JunD. No transactivation of any of these constructs was seen when c-Fos was replaced with other Fos family members (e.g., Fra-1, Fra-2, and FosB) (data not shown). To determine whether transactivation of the -95/+47 Timp-1 construct that contained the mutant AP1 site was due to an artifact of the vector backbone, the empty vectors pBLCAT3 and pBLCAT2 were co-transfected with c-Fos and JunD expression plasmids. The results showed that the empty pBLCAT3 vector was potently transactivated by the overexpression of c-Fos and JunD (over 50-fold, although control levels were very low); pBLCAT2 had a higher level of basal activity and was induced by approximately 8-fold. A perfect AP1 consensus site (5′TGAGTCA-3′) was found at position 1569/1575 in the simian virus 40 (SV40) region of the pBLCAT3 vector backbone using Transfac (15). Using the MMP-1 AP1 site oligonucleotide as Benchmarks

Cryptic Promoter Activity of pBLCAT3 Induced by Overexpression of AP1 Factors