Engineered p14ARF Promoter for Targeted Gene Expression

The human p14^ARF gene promoter region was originally identified as a 5.6-kb CpG-rich region 5′ upstream of the p14 first exon (1β) [10 (link)]. However, investigators have used the first 800 bases immediately upstream of the transcription initiation site, and have shown that this region is sufficient to confer AP1 and E2F regulation in a sensitive reporter system [9 (link)]. This region, which we termed the minimal human p14^ARF promoter (p14^ARFmin), was the starting promoter region in our p14^ARF promoter constructs. The modified p14^ARFmin promoter was amplified by PCR from human genomic DNA using the published sequence [9 (link)]. Forward primer, 5′- CCATTAATGTCGACAGCTCCGGCAGCGC-3′ and reverse primer, 5′-ACTCGAGATCTCCGCCCCG.
CAGGCGCGCA-3′ were used along with the restriction sites AseI and XhoI to replace the CMV promoter of pIRES vector with the p14^ARF promoter.
Since attempts at expressing GFP in the mut Ras/mut p53 pancreatic cell lines were unsuccessful with the p14^ARF promoter (data not shown), a promoter analysis program (Cister: Cis-element Cluster finder, Boston University) was used to detect the presence and efficacy of various transcription factor binding elements and promoter features. We did not use the whole p14^ARF promoter because it was too large (5.6 kb) for use in an adenovirus vector, which holds a maximum of 5 kb. While this program identified an E2F potential binding site [9 (link)], its homology to the consensus E2F site was low. Additionally, an AP-1 site, the “TATA” box, and the transcription initiation site, while present, also showed poor homology with consensus sites. Two reporter plasmids, pAP1-Luc and pE2F-Luc (BD Bioscience Clontech, Palo Alto, CA, USA), were then used to transfer each of the transcription factor binding elements, E2F and Ap1 enhancers, to a 5′ region upstream of the p14^ARF promoter. The E2F enhancer site, containing 4 tandem repeat E2F elements totaling 70 bp, and the AP1 enhancer site, containing 4 tandem repeat Ap1 elements totaling 50 bp, were amplified by PCR and inserted upstream of the p14^ARF promoter. Sequencing was performed to confirm the direction of orientation and the integrity of the promoter’s structure. Additionally, using designed primers and serial PCR amplifications, the sequence of the downstream p14^ARF was modified by inserting the TATA box and transcription initiation sequence, which shares more homology with the consensus TATA box sequence than that found in the p14^ARF promoter. The TATA box was important in transcription because the TATA factor binding to the region recruits additional factors to initiate transcription. The constructed promoter Ap1e-E2Fe-p14^ARF-TATA–transcription initiation site was called the modified p14^ARFmin promoter, or simply called the p14^ARF. The modified promoter was inserted into the replaced CMV promoter site to make the respective plasmids p14^ARF-p14 or tBID or p14-IRES-tBID and p14-IRES-GFP (Figure 1A–D).