Direct visualization of the binding of c-Myc/Max heterodimeric b-HLH-LZ to E-box sequences on the hTERT promoter.
Myc and Max belong to the b-HLH-LZ family of transcription factors. Heterodimerization between Myc and Max or homodimerization of Max allows these proteins to bind their cognate DNA sequence known as the E-box (CACGTG). Recent evidence has suggested that the c-Myc/Max heterodimeric b-HLH-LZ could interact to form a head-to-tail dimer of dimers and induce complex topologies such as loops in promoters containing more than one E-box sequence. In an attempt to shed light on this hypothesis, the interaction between the heterodimeric b-HLH-LZ of c-Myc/Max and a fragment of the hTERT promoter containing two E-box sequences was studied by atomic force microscopy. Specific binding events were observed at both E-box sites with equal probabilities. In accordance with previous results obtained by EMSA, we observed that the specific binding of the c-Myc/Max b-HLH-LZ bends the promoter. However no looping could be observed in a wide range of concentration encompassing the Ka (association constant) of the putative tetramer and the Ka for the specific binding of the heterodimer. In contrast, experiments performed with a mandatory c-Myc/Max b-HLH-LZ tetramer incubated with the hTERT promoter fragment allowed for the visualization of loops and cross-linked DNA strands originating from specific binding. Altogether, our results indicate that the c-Myc/Max b-HLH-LZ dimer binds specifically and equally to both E-box sites of the hTERT promoter and induces a significant bending of the promoter and that the suggested oligomerization of the c-Myc/Max heterodimeric b-HLH-LZ, if existing, is most likely too weak to induce the formation of a loop in a promoter.
Lebel
Réjean
R
Département de pharmacologie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, QC, Canada J1H 5N4.
McDuff
François-Olivier
FO
Lavigne
Pierre
P
Grandbois
Michel
M
D050976:Basic-Leucine Zipper Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000737:chemistry; D019281:Dimerization; D024721:E-Box Elements / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D018625:Microscopy, Atomic Force; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry / Q000378:metabolism*; D019098:Telomerase / Q000235:genetics*
D050976:Basic-Leucine Zipper Transcription Factors; D003432:Cross-Linking Reagents; C067850:Myc associated factor X; D016271:Proto-Oncogene Proteins c-myc; D004247:DNA; C509186:TERT protein, human; D019098:Telomerase