STRUCTURE-FUNCTION ANALYSIS OF MYC/MAX-DNA BINDING

The c-Myc oncoprotein (or Myc) is a transcription factor of the basic-Helix-Loop-Helix Leucine-zipper (bHLH-LZ) family, whose transcriptional activity depends on dimerization with the bHLH-LZ partner Max and DNA binding, mediated by the basic regions of both proteins. Myc/Max dimers bind preferentially to the hexanucleotide motif CACGTG (known as E-box) and variants thereof. The ability of Myc to bind DNA in vivo, however, is not stringently regulated by the presence of the E-box, since many genomic sites targeted by Myc do not contain this motif. Hence, we still need to fully comprehend how Myc recognizes its genomic targets and to what extent sequence-specific DNA binding contributes to this process. Based on the crystal structure of the DNA-bound Myc/Max dimer, we generated a Myc mutant in which two basic region residues engaged in sequence-specific contacts (H359 and E363) were mutated to Alanine (Myc HEA), and compared this with a mutant in which three Arginine residues involved in DNA backbone interactions were mutated to Alanine (Myc RA). While both mutants showed impaired E-box recognition in vitro, their over-expression in murine fibroblasts revealed very different genome-interaction profiles, Myc RA showing no detectable DNA binding, and Myc HEA retaining about half of the binding sites seen with Myc wt. The analysis of the binding intensity of Myc wt and Myc HEA at their binding sites revealed that, as expected, Myc wt bound more strongly the sites containing the E-box, while Myc HEA bound the sites with an E-box as well as the sites without it, confirming that the mutant lost the sequence-specific recognition ability. The interactions retained by the Myc HEA were dramatically reduced with the protein expressed from the endogenous c-myc locus, though genome engineering. Thus, unlike Myc RA, the Myc HEA mutant retained non-specific interactions with genomic DNA (detectable at elevated protein levels) but failed to engage more stably through sequence-specific DNA contacts. In spite of this residual DNA-binding activity, Myc HEA was profoundly impaired in its biological function, undistinguishable from Myc RA: in particular, neither mutant could substitute for wild-type Myc in supporting cell proliferation in murine fibroblasts, whether at normal or supra-physiological levels. While the assessment of transcriptional activities is still ongoing, we conclude that E-box recognition is essential for Myc’s biological function.