Design and properties of a Myc derivative that efficiently homodimerizes

Abstract

bHLH and bHLHZip are highly conserved structural domains mediating DNA binding and speci®c proteinprotein interactions. They are present in a family of transcription factors, acting as dimers, and their selective dimerization is utilized to switch on and o cell proliferation, dierentiation or apoptosis. Myc is a bHLHZip protein involved in growth control and cancer, which operates in a network with the structurally related proteins Max, Mad and Mnt. It does not form homodimers, working as a heterodimer with Max; Max, instead, forms homodimers and heterodimers with Mad and Mnt. Myc/Max dimers activate gene transcription, while Mad/Max and Mnt/Max complexes are Myc/Max antagonists and act as repressors. Modifying the molecular recognition of dimers may provide a tool for interfering with Myc function and, in general, for directing the molecular switches operated via bHLH(Zip) proteins. By molecular modelling and mutagenesis, we analysed the contribution of single amino acids to the molecular recognition of Myc, creating bHLHZip domains with altered dimerization speci®city. We report that Myc recognition speci®city is encoded in a short region within the leucine zipper; mutation of four amino acids generates a protein, Omomyc, that homodimerizes eciently and can still heterodimerize with wild type Myc and Max. Omomyc sequestered Myc in complexes with low DNA binding eciency, preventing binding to Max and inhibiting Myc transcriptional activator function. Consistently with these results, Omomyc produced a proliferation arrest in NIH3T3 cells. These data demonstrate the feasibility of interfering with fundamental biological processes, such as proliferation, by modifying the dimerization selectivity of a bHLHZip protein; this may facilitate the design of peptides of potential pharmacological interest.

Omomyc expression in skin prevents Myc-induced papillomatosis

“…Omomyc DNA, carrying an ATG start codon, 7,8 was amplified from the pBPOmomyc construct by PCR primed using oligonucleotides containing the NotI restriction site. The resulting Omomyc sequence was fused to the Involucrin promoter, into the NotI site of the Involucrin expression cassette.…”

“…Overexpressed Omomyc competes with Max for Myc and thereby prevents Myc from binding to the E-box and activating transcription. 7 In contrast, Myc-dependent trans-repression, which does not involve binding to the E-box, is unaffected, or even enhanced, by Omomyc. 8 The strong proapoptotic activity of Omomyc mutant protein may spring from its ability to differentially influence activated and repressed Myc transcriptional targets.…”

Myc-mediated transformation: the repression connection

“…The link between Myc gene regulation and transformation has been explored using various altered forms of Myc, and studies have demonstrated that Myc/Max heterodimerization is critical for transactivation, transrepression, and transformation (Amati et al, 1993a;Desbarats et al, 1996;Facchini et al, 1997;Soucek et al, 1998). For example, MycEG and MaxEG, c-Myc and Max proteins with modi®ed leucine zipper domains that can heterodimerize with each other but not with endogenous proteins, must be expressed together to transform cells and to transactivate gene expression from a Myc/Max DNA binding site (Amati et al, 1993a).…”

OmoMYC blunts promoter invasion by oncogenic MYC to inhibit gene expression characteristic of MYC-dependent tumors

“…OmoMYC comprises the carboxy-terminal basic region, helix-loop-helix and leucine zipper of MYC and carries four point mutations in the leucine zipper that are intended to alter its dimerization properties 20 . Here, we present an analysis of the OmoMYC structure, its genome-wide chromatin binding and gene regulation to understand its astounding ability to discriminate physiological from oncogenic functions of MYC.…”