Direct Visualization of the Binding of c-Myc/Max Heterodimeric b-HLH-LZ to E-Box Sequences on the hTERT Promoter

Abstract

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.

NFX1-123 Increases hTERT Expression and Telomerase Activity Posttranscriptionally in Human Papillomavirus Type 16 E6 Keratinocytes

“…HPV16 E6 is known to regulate hTERT at the promoter through two proximal E boxes that flank the transcript start site and the proximal X1 box, affecting transcription factors and chromatin structure (15,16,23,35,58,64). HPV16 E6, however, also requires the expression of NFX1-123 for full activation of hTERT expression (26).…”

“…Activation of hTERT by E6-E6AP requires two E boxes found at the hTERT promoter flanking the transcriptional start site (55,58,60). The heterodimer c-Myc-Max is known to bind to E boxes as transcriptional activators (35), and indeed c-Myc can be found at the hTERT promoter in epithelial cells in the context of E6-E6AP (59), although its protein levels at the hTERT promoter do not fluctuate significantly with hTERT expression (64). GC-rich sites in the hTERT promoter are also important for Sp1 activation (46), although there is evidence of Sp1-repressive effects as well (62).…”

Role of c-MYC in alternative activation of human macrophages and tumor-associated macrophage biology

“…on May 10, 2018. by guest www.bloodjournal.org From c-MYC/MAX heterodimer binds with high affinity to the CACGTG E-box sequence in the promoter region of target genes and induces their expression. 22,23 In addition, the c-MYC/MAX complex can suppress the expression of certain genes 24 in a process that is mostly related to the protumoral activity of c-MYC. 25 More recently, a key role of this transcription factor in hematopoietic stem-cell function and survival and in lymphoid compartment homeostasis has also been reported.…”

An Optimized Telomerase-Specific Lentivirus for Optical Imaging of Tumors

“…The hTERT promoter contains two E-boxes (CACGTG) that are binding sites for the Myc/Max/Mad network of transcription factors. The oncoprotein c-Myc heterodimerizes with the Max protein to activate hTERT transcription (25). In contrast, Mad1 and Max protein heterodimers result in the repression of hTERT expression (19).…”