Figure 6.

The two E-boxes of the proximal PTF1-binding site are redundant. (A) Mutational disruption of the first (E1m) or second (E2m) E-box had no effect on the binding of the PTF1a/E12 heterodimer (cf. lanes 2,6,10), the RBPJ form of the trimer (lanes 3,7,11), or the RBPJL form of the trimer (lanes 4,8,12). (Lanes 14–16) Mutation of both E-boxes (E12m) eliminated binding of the dimer and both forms of the trimer. The positions of the monomer RBPJ (1), the PTF1a/E12 heterodimer (2), and the trimers (3) are indicated. The PTF1 subunits were synthesized by cell-free translation. (B) The E-box mutations shown in A were incorporated into the −389 RbpL-Luc plasmid to test whether both E-boxes were also required for activity of the Rbpjl promoter in transfected acinar or kidney cells. Mutational inactivation of either E-box had no effect on acinar activity, whereas inactivation of both nearly eliminated it. None of the mutations affected activity of the promoter in 293 kidney cells. (C) A proper stereochemical relationship between the TC-box and an E-box is required for binding the complete PTF1 trimer in EMSAs. To simplify the complexity of two E-boxes, we mutated the proximal one (which had no discernible effect on complex binding or activity) and tested the effects of altered spacing between the TC-box and the remaining E-box by inserting 6 bp (E2m + 6 bp) or deleting 6 bp (E2m − 6 bp) (see the diagram in D). Either alteration decreased binding of the PTF1 trimer, but not the PTF1a/E12 heterodimer. (D) Proper spacing between the TC-box and an E-box is required for the acinar activity of the promoter. The spacing changes described for C were incorporated into −389 RbpL-Luc for transfection into 266-6 acinar cells. Either alteration decreased acinar activity by at least 90%. Error bars are standard deviations.