Paola Pellanda

Myc oncoproteins are transcription factors of the basic-Helix-Loop-Helix Leucine-zipper (bHLH-LZ) family. As pre-requisite for DNA binding, Myc has to dimerize with the bHLH-LZ partner Max and together they bind preferentially to the E-box motif CACGTG. 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 residues engaged in sequence-specific contacts (H359 and E363) were mutated to Alanine (MycHEA), and compared this with a mutant in which three residues involved in DNA backbone interactions were mutated to Alanine (MycRA). While both mutants showed impaired E-box recognition in vitro, their over-expression in murine fibroblasts revealed very different genome-interaction profiles, MycRA showing no detectable DNA binding, and MycHEA retaining about half of the binding sites seen with Mycwt.
The analysis of the binding intensity of Mycwt and MycHEA at their binding sites revealed that, while Mycwt bound more strongly the sites containing the E-box, MycHEA bound to DNA was sequence-independent, confirming that the mutant lost the sequence-specific recognition ability. In spite of this residual DNA-binding activity, MycHEA was profoundly impaired in its biological functions, undistinguishable from MycRA, strongly suggesting that the E-box recognition is essential for Myc’s biological function.