A44
Direct c-Myc DNA binding mediates p15ink4b transcriptional repression
Marie-Eve Beaulieu
Université de Sherbrooke, Quebec, Canada
c-Myc has a central role in tumor growth. As a heterodimer with Max, c-Myc activates target genes that promote cell proliferation and represses the transcription of cell cycle inhibitors (e.g. p15ink4b). While the mechanism of c-Myc transactivation is well understood, no clear mechanism has been evidenced to explain transrepression by c-Myc. However, it has been shown that the interaction between c-Myc and Miz-1 is important for the transrepression of p15ink4b. On the other hand, results presented in the literature suggest that this interaction cannot account for the totality of c-Myc transrepression activity on p15ink4b. This raises the possibility of the implication of a direct interaction between c-Myc/Max and the target promoter DNA. Interestingly, examination of the promoter sequence failed to identify canonical E-box (CACGTG) sequences but revealed the presence of a noncanonical E-box (CAGCTG), which could constitute a c-Myc-binding site. We provide the first evidences that c-Myc/Max bind directly to this noncanonical E-box and that DNA binding is important in the repression of p15ink4b by c-Myc. Indeed, EMSA and Circular Dichroism analysis revealed that c-Myc/Max binding to the noncanonical E-box is specific, albeit with a reduced affinity compared to a canonical E-box. Moreover, luciferase assays in transient transfected HeLa cells reveal that the mutation of the noncanonical E-box abolishes p15ink4b transrepression by c-Myc. These results support a mechanism for c-Myc repression of p15ink4b in which both Miz-1 binding and DNA binding are important.
