Myb binding sites within the N-ras promoter repress transcription

In vitro and in vivo methods were combined to determine the function of the three Myb binding sites (NrasI, NrasII and NrasIII) within the promoter region of the mouse N-ras gene. We found that the c-Myb DNA-binding domain can bind with high affinity to NrasI and NrasII, but with a reduced affinity to NrasIII. In contrast, the full length v-Myb protein from BM2 cells only bound to the middle one of the three sites, NrasII. Both c-Myb and v-Myb functioned as repressors and reduce the basal activity of the N-ras promoter by 60%, as determined by transient transfection experiments using different regions of the N-ras promoter. This repression required a functional Myb DNA-binding domain and could not be overcome by fusion to the potent VP16 activation domain. In electrophoretic mobility shift assays, the v-Myb protein is shown to be present in different conformations depending on its binding to the NrasII or the mim-1A site. The v-Myb conformation is thus suggested to play a critical role in the regulation of v-Myb activity.