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 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 alpha, containing both a canonical and a noncanonical E-box (CAAGTG) 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 alpha, 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.
Walhout
A J
AJ
Laboratory for Physiological Chemistry, Utrecht University, PO Box 80042, 3508 TA Utrecht, Netherlands.
van der Vliet
P C
PC
Timmers
H T
HT
D000818:Animals; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D009955:Ornithine Decarboxylase / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000235:genetics; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D051381:Rats; D013947:Thymosin / Q000031:analogs & derivatives / Q000235:genetics; D014157:Transcription Factors
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C499330:MAX protein, human; C499329:Max protein, rat; C067850:Myc associated factor X; D011498:Protein Precursors; D016271:Proto-Oncogene Proteins c-myc; D014157:Transcription Factors; C040782:prothymosin alpha; D013947:Thymosin; D009955:Ornithine Decarboxylase