Sequences flanking the E-box contribute to cooperative binding by c-Myc/Max heterodimers to adjacent binding sites

• Published in: Biochimica et biophysica acta Vol. 1397; no. 2; pp. 189 - 201
• Main Authors: Walhout, A.J.M, van der Vliet, P.C, Timmers, H.Th.M
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 29-04-1998
• Subjects: Animals; Base Sequence; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Basic-Leucine Zipper Transcription Factors; Binding Sites; c-Myc; Cooperativity; DNA binding; DNA-Binding Proteins - metabolism; E-box; Helix-Loop-Helix Motifs; Humans; Ornithine decarboxylase; Ornithine Decarboxylase - genetics; Promoter Regions, Genetic; Protein Precursors - genetics; Prothymosin α; Proto-Oncogene Proteins c-myc - metabolism; Rats; Thymosin - analogs & derivatives; Thymosin - genetics; Transcription Factors; E-box; Ornithine decarboxylase; Cooperativity; c-Myc; DNA binding; Prothymosin α
• ISSN: 0167-4781; 0006-3002; 1879-2634
• DOI: 10.1016/S0167-4781(97)00227-3

Previously, we have shown that c-Myc/Max heterodimers, bind cooperatively to the two adjacent, canonical E-boxes (CACGTG) located in the rat ornithine decarboxylase (ODC) gene. In order to study this in more detail, we changed the length of the linker that separates the two E-boxes, as well as their flanking sequences. We found that high affinity, cooperative binding requires a minimal linker length of 1–4 bp and that the binding affinity is influenced by E-box flanking sequences. Binding to the c-Myc responsive element of prothymosin
α, 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 site with a canonical E-box only partially restored high affinity, cooperative binding. By making hybrid fragments between ODC and prothymosin
α, we found that nucleotides in the linker between the E-boxes influence the affinity of c-Myc/Max heterodimers. Taken together, our results show that E-box sequences and sequences in the linker separating both E-boxes influence cooperative, high affinity binding by c-Myc/Max dimers.