Identification of positive regulatory element(s) located between −365 and −240 of the human PC P2 promoter.

<p>(A) Schematic diagram of 15 bp internal deletions of −114/−39 the human PC P2 promoter. (B) Transient transfections of a series of 25 bp internal deletion constructs into the INS-1 832/13 cell line and non-beta cell HEK293T cell line were performed to identify the positive regulatory sequences in the hP2 promoter. The luciferase activity of each construct was normalized with β-galactosidase activity. The normalized reporter activity obtained from each construct is shown as a percent relative to those transfected with the wild type −365 hP2 promoter, which was arbitrarily set at 100%. *P value <0.05, **P value <0.01. (C) Gel shift and supershift assays of the biotin-labeled probe of the −78 to −54 region of the hP2 promoter (−340/−315 hP2 probe) using an INS-1 832/13 nuclear extract. The nucleotide sequences of the wild type and mutant of the hP2 promoter −78 to −54 regions are also shown. Lane 1 probes incubated with nuclear extracts from INS-1 832/13; lanes 2–3, 10-fold or 50-fold excess wild-type unlabeled oligonucleotides were incubated with nuclear extracts and probes; lane 4, 50-fold excess amount of mutant unlabeled oligonucleotides were incubated with nuclear extracts and probes; lanes 5–7, nuclear extracts were pre-incubated with anti-USF1 or anti-USF2 or both, respectively, before the probes were added to the reactions. Lanes 8–10, nuclear extracts were pre-incubated with anti-Sp1 or anti-Sp3 or both, respectively, before the probes were added to the reactions. Arrow represents DNA-protein complex, SS = supershift band.