Figure 3.

Msx2 proximal promoter bearing 550 bp of the upstream sequences exhibited maximum activity and was transactivated by YY1. Five constructs of the Msx2 promoter were generated and cloned into luciferase reporter vector pGL3 (A). These are represented schematically on the left. A schematic representation of the genomic promoter region of the Msx2 gene with restriction enzyme sites and their respective positions is shown for referencing the various constructs. The promoter activities of these constructs were assayed by the luciferase reporter and compared with the negative control using the basal vector pGL3, and the positive control using p800-Luc, which contains the PAI-1 gene promoter (74). In this assay, the Msx2SS-Luc construct displayed greater activity than the other Msx2 promoter constructs. Subsequently, different amounts of YY1 (x-axis) were co-transfected with Msx2 promoter reporter constructs into P19 cells cultured on 10-cm plates, and luciferase reporter activities were assayed from Msx2SS-Luc (B) or Msx2KS-Luc (C) constructs, 24 h post-transfection. Increasing amounts of YY1 resulted in significantly elevated levels of Msx2 promoter activities when compared with control in which an expression vector with no YY1 insert (empty vector) was used. *P < 0.01.