Figure 5.

Functional characterization of Rfx2 MBS sites. (A). Binding competition for Rfx2 MBS by gel mobility shift assay. The MBS1 oligo was used as labeled probe for the bacterially expressed A-MYB DNA binding domain-maltose binding fusion protein. Lanes show increasing amounts of unlabeled competitor oligonucleotides in the range of 50 to 500 ng. (B). Stimulation of Rfx2 promoter fragments by co-transfection of A-myb expression vector. Transient transfections were carried out with or without the C-terminal truncated A-myb expression vector. The locations of the promoter fragments are indicated in Fig. 4C. Note that the scale for PFB and PFC is amplified 2-fold. (C). Ability of individual Rfx2 MBS multimers to promote expression of a luciferase reporter. Individual MBSs were ligated in a head to tail fashion to generate 5 copies in a minimal promoter luciferase vector. Results are of transient transfection plus or minus co-transfection with the A-myb d304 expression plasmid. Fold activation is indicated above the (+) columns. (D). ChIP assay shows in vivo Rfx2 promoter occupancy by A-MYB. Left: Amplification of Rfx2 promoter following chromatin capture by anti-A-MYB or IgG control. Right: Amplification of unrelated Tcrd locus following chromatin capture.