Fig 2. Additive or weak cooperative transactivation between adjacent κB REs.

A) Yeast-based luciferase assays were performed at moderate to high expression levels of p50TAD or p65. Results were normalized and plotted as in Fig 1. The impact of tandem duplication (2d) of the decameric (1d) κB-RE or the indicated combination of two different κB-REs was evaluated. The REs were chosen based on the results from Fig 1 to include sequences exhibiting different levels of transactivation potentials. B) RE1 and RE6 were inactive as decameric κB-REs and there was no transactivation for two decamers in tandem or high expression of NF-κB proteins (up to 1% galactose). C) Functional interactions between two different κB-REs derived from the MCP-1 promoter. The M1 and M2 decameric κB-REs exhibited different responsiveness to p50TAD and p65, when examined separately, but are both derived from the MCP-1 promoter and they are located in close distance (19 nucleotide spacer) in the natural context. The combined responsiveness of the M1 and M2 κB-REs was examined, taking into account the impact of the distance between the two decamers and orientation relative to the transcriptional start site. Tandem repeats of M1 or M2 were included as controls. Yeast reporter strains, transformed with the indicated expression vector were cultured for 16 hrs with the indicated low amount of galactose. Relative activity refers to the average light units normalized for cell number (measured by optical density at 600nm). Average and standard error of four biological repeats are presented. The NF-κB-independent reporter activity (empty vector) is also presented as reference. Interestingly, the M2+M1 strains exhibited higher basal level of reporter expression. The M1+M2 sp strain contains the M1 and M2 κB decamers separated by 18 nt as in the human gene (see Table A in S1 File).