FIG. 4.

Functional role of the dβNRE-S element. (A) Schematic of β1285 wt and β1285dβNRE-Sm1 forms of a 1,285-bp human βMyHC proximal promoter. Site-directed mutations involved nucleotides previously shown to comprise consensus Pur protein binding sites (GGN)n. (B) Promoter activities of wild-type and dβNRE-S mutant reporter genes (1 μg) determined in C2C12 myotubes. Mutation of the dβNRE-S element increased expression of the 1,285-bp βMyHC Luc reporter gene expression in C2C12 muscle cells. Data are reported as luciferase-normalized relative light units (RLU) (firefly/Renilla) and are expressed as the mean ± standard error (n = 8). *, P < 0.0001; all comparisons are to β1285 wt. (C) siRNA targeting either Purα or Purβ led to decreased levels of its target. C2C12 myoblasts were transfected with control siRNA (nontargeting [NT]) or siRNA targeting Purα, Purβ, or Purα and Purβ as described in Methods and Materials. IP90 was used as a loading control. Western blot analysis revealed that Purα and Purβ siRNAs effectively decreased endogenous levels of both Purα and Purβ. (D) Knockdown of endogenous Purα or Purβ protein results in increased expression of the β1285 wt reporter gene in C2C12 myotubes. Data are reported as luciferase-normalized RLU (firefly/Renilla) and are expressed as the mean ± standard error (n = 3). *, P < 0.0001; all comparisons are against β1285 wt activity in the presence of NT siRNA.