Sequence-specific DNA binding by MYC/MAX to low-affinity non-E-box motifs

• Published in: PloS one Vol. 12; no. 7; p. e0180147
• Main Authors: Allevato, Michael, Bolotin, Eugene, Grossman, Mark, Mane-Padros, Daniel, Sladek, Frances M, Martinez, Ernest
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 18.07.2017; Public Library of Science (PLoS)
• Subjects: Assaying; Base Sequence; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors - chemistry; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors - metabolism; Binding sites; Biochemistry; Biology; Biology and Life Sciences; Cancer; Cell cycle; Cell growth; Deoxyribonucleic acid; DNA; DNA - genetics; DNA - metabolism; DNA binding; DNA methylation; DNA microarrays; Electrophoresis; Electrophoretic mobility; Gene expression; Gene Expression Regulation; Genomes; Genomics; Humans; In vitro methods and tests; Loci; Lymphocytes B; Myc protein; Neurosciences; Nucleotide Motifs; Nucleotide sequence; Protein arrays; Protein Binding; Protein Multimerization; Proteins; Proto-Oncogene Proteins c-myc - chemistry; Proto-Oncogene Proteins c-myc - metabolism; Research and Analysis Methods; Stem cells; Studies; Substrate Specificity; Transcription factors; California
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0180147

The MYC oncoprotein regulates transcription of a large fraction of the genome as an obligatory heterodimer with the transcription factor MAX. The MYC:MAX heterodimer and MAX:MAX homodimer (hereafter MYC/MAX) bind Enhancer box (E-box) DNA elements (CANNTG) and have the greatest affinity for the canonical MYC E-box (CME) CACGTG. However, MYC:MAX also recognizes E-box variants and was reported to bind DNA in a "non-specific" fashion in vitro and in vivo. Here, in order to identify potential additional non-canonical binding sites for MYC/MAX, we employed high throughput in vitro protein-binding microarrays, along with electrophoretic mobility-shift assays and bioinformatic analyses of MYC-bound genomic loci in vivo. We identified all hexameric motifs preferentially bound by MYC/MAX in vitro, which include the low-affinity non-E-box sequence AACGTT, and found that the vast majority (87%) of MYC-bound genomic sites in a human B cell line contain at least one of the top 21 motifs bound by MYC:MAX in vitro. We further show that high MYC/MAX concentrations are needed for specific binding to the low-affinity sequence AACGTT in vitro and that elevated MYC levels in vivo more markedly increase the occupancy of AACGTT sites relative to CME sites, especially at distal intergenic and intragenic loci. Hence, MYC binds diverse DNA motifs with a broad range of affinities in a sequence-specific and dose-dependent manner, suggesting that MYC overexpression has more selective effects on the tumor transcriptome than previously thought.