Abstract
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.
Highlights
The oncoprotein MYC is an essential DNA-binding transcription factor of the basic helixloop-helix leucine zipper family that regulates transcription of a large number of genes in metazoans and controls cell cycle, cell growth, metabolism, cell proliferation, differentiation, apoptosis, and cell transformation and is overexpressed in many types of cancer [1, 2]
We show that MYC/MAX complexes bind in a sequence-specific manner to high-affinity E-boxes and close variants and to more degenerate and lower affinity non-Enhancer box (E-box) DNA sequences, such as the palindromic hexamer AACGTT, which we confirmed by electrophoretic mobility-shift assays (EMSAs)
Using an unbiased protein binding microarray (PBM) approach we have identified a set of 27 high-confidence hexamer motifs that are bound by both MYC:MAX and MAX:MAX complexes in vitro
Summary
The oncoprotein MYC is an essential DNA-binding transcription factor of the basic helixloop-helix leucine zipper (bHLHZ) family that regulates transcription of a large number of genes in metazoans and controls cell cycle, cell growth, metabolism, cell proliferation, differentiation, apoptosis, and cell transformation and is overexpressed in many types of cancer [1, 2]. MYC binds DNA as a heterodimer with an obligatory bHLHZ partner called MAX. In contrast to MYC, the MAX protein can homodimerize.
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