Figure 5.

Characterization of the IκBβ promoter. (A) Schematic representation of putative binding sites identified in the IκBβ promoter. The initiating methionine (ATG) is located at +59. (B) Boundaries of the IκBβ promoter were established using luciferase reporter constructs in which the 879 nucleotides upstream of the ATG were progressively deleted at the 5′ end. Data shown are from transient transfection of HeLa cells. Maximal reporter activity was observed upon deletion of 503 of the most 5′ nucleotides in the DEL318 construct, suggesting important positive regulatory elements were located upstream and that a negative regulatory element existed upstream. (C) Contribution of the two downstream SP1 sites to the reporter activity observed for the DEL318 construct was established by mutating the SP1 sites individually and together. Both SP1 sites contribute significantly to reporter activity in Jurkat cells. (D) Demonstration that a negative regulatory element is located between nucleotides −449 and −376. Replacement of this sequence in the BP construct with another segment of DNA of equivalent size results in increased reporter activity in Jurkat cells. (E) NF-κB activation by p65 cotransfection in Jurkat cells leads to increased reporter activity of the BP construct through NF-κB binding to the κB site, as mutation of this site inhibits increased reporter activity.