Analyzing In Vivo Conformational Changes of a Transcription Factor, c-Myb, Using Fluorescence Resonance Energy Transfer (FRET) technology

The c-myb proto-oncogene was first identified as the normal cellular counterpart to the v-myb oncogene identified in Avian Myeloblastosis virus (AMV), a retrovirus that transforms immature hematopoietic cells in tissue culture and induces leukemias in animals.  However, the normal c-Myb protein is not oncogenic, in part because of conformational and auto-inhibitory interactions that keep its transforming activity in check. In addition, recent studies have shown that the transcriptional activity of c-Myb protein changes during the cell cycle, perhaps because of changes in post-translational modifications and protein interactions that help direct it to different sets of target gene promoters in different stages of the cell cycle. We hypothesize that c-Myb protein undergoes conformational changes that are controlled by cell cycle regulators and by upstream signaling pathways, for example the signals that direct cells either to proliferate or to undergo differentiation. To address this, we are utilizing FRET technology to elucidate whether the intramolecular interactions of c-Myb or its interactions with various co-factors and co-activators change in vivo during the cell cycle or in response to extracellular signals that trigger a switch from proliferation to differentiation. The goal is to determine how the conformations and protein-protein interactions of c-Myb change in cells that are progressing through the cell cycle, or in response to signals that control cell fate.