SEQUENCES FLANKING THE E-BOX CONTRIBUTE TO COOPERATIVE BINDING BY C-MYC MAX HETERODIMERS TO ADJACENT BINDING-SITES/

Previously, we have shown that c-Myc/Max heterodimers. bind cooperativ ely to the two adjacent, canonical E-boxes (CACGTG) located in the rat ornithine decarboxylase (ODC) gene. In order to study this in more de tail, we changed the length of the linker that separates the two E-box es, as well as their flanking sequences. We found that high affinity, cooperative binding requires a minimal linker length of 1-4 tip and th at the binding affinity is influenced by E-box flanking sequences. Bin ding to the c-Myc responsive element of prothymosin alpha, containing both a canonical and a noncanonical E-box (CA AGTG) was also studied. As shown by DNAseI footprinting analysis, only the canonical E-box is bound by c-Myc/Max and c-Max/Max dimers. Replacing the noncanonical si te with a canonical E-box only partially restored high affinity, coope rative binding. By making hybrid fragments between ODC and prothymosin alpha, we found that nucleotides in the linker between the E-boxes in fluence the affinity of c-Myc/Max heterodimers. Taken together, our re sults show that E-box sequences and sequences in the linker separating both E-boxes influence cooperative, high affinity binding by c-Myc/Ma x dimers. (C) 1998 Elsevier Science B.V.