Yin Yang 1 Intronic Binding Sequences and Splicing Elicit Intron-Mediated Enhancement of Ubiquitin C Gene Expression
Figure 3
Mutagenesis of YY1, but not Sp1, intron binding sites negatively affects UbC promoter activity.
(A) The schematic diagram shows the UbC intron region (nt +65/+876). ODNs and the relative positions of the putative transcription factor binding motifs are illustrated below: Sp1 binding sites are represented by open ovals and identified with a single-letter code, from (a) to (d); YY1 binding sites are represented by filled rectangles and named (e) and (f). Sequences of the different Sp1 and YY1 binding sites are shown and nucleotide substitutions introduced by mutagenesis are highlighted. (B) HeLa transient transfections with wild-type (P3) and the different Sp1 mutant luciferase constructs were carried out and luciferase expression evaluated by RT and quantitative RealTime-PCR at 48 h post-transfection as detailed under “Materials and Methods”. Promoter activity of the wild-type P3 was set to 100% and promoter activity of the mutants was expressed as a percentage of the wild-type construct. (C), (D) HeLa transient transfections with wild-type (P3) and the different YY1 mutant luciferase constructs were carried out and assayed as in B. Data presented are the means (±SE) of at least four different experiments, with two independent plasmid preparations. Asterisks indicate statistical differences (*, p<0.05; **, p<0.01; ***, p<0.001 versus P3). The statistically significant difference between YY1mut e–f and YY1mut e (C) is also indicated (*, p<0.05).