AbstractsBiology & Animal Science

Abstract. The c-Myb transcription factor plays a central role in regulation of growth, differentiation and survival of hematopoietic cells. Despite being studied for many years, the understanding of how c-Myb functions at the molecular level remains incomplete. An important resource for mechanistic studies has been the point mutations found in AMV v-Myb generated in the chicken system. Recently, two independent genetic screens in mice identified three point mutations (D152V, M303V and D384V) in c-myb affecting specific stages of hematopoiesis (Carpinelli et al., 2004; Sandberg et al., 2005). These mutations led to a range of hematopoietic defects, including an increase in hematopoietic stem cell number. While the cellular consequences of these mutations were identified, the molecular mechanisms involved were not thoroughly investigated. We have generated the mutations M303V and D384V in the context of the human c-Myb protein and have initiated studies of their mechanisms of action. In this work we focus on the interaction with the coactivator p300. The M303V mutation lies in the transactivation domain (TAD) which interacts with the KIX domain in p300 (Zor et al., 2004). c-Myb also interacts with another domain in p300, the CH2/HAT-domain (Sano and Ishii, 2001; Tomita et al., 2000). This interaction occurs through the negatively regulatory domain of c-Myb, which contains the D384V mutation. To identify mechanisms affected by the mutations M303V and D384V in c-Myb several questions were addressed. First we focused on transcriptional activity of the mutated c-Myb protein and analysed whether the M303V and D384V mutations changed the activity of the protein. We have performed reporter-effector assays to analyse the activity of M303V and D384V-mutated c-Myb in mammalian cells, and results indicated that the M303V and D384V mutations both reduce transcriptional activity of c-Myb. We further were interested in whether the M303V and D384V mutations affected the binding of c-Myb to the coactivator p300. The interaction studies by GST-pulldown assays revealed that the M303V and D384V mutations weaken interaction between c-Myb and p300. We also analysed whether the c-Myb mutants had lost their response to p300. We found that the c-Myb M303V and c-Myb D384V single mutants are still able to respond to the coactivator p300, although slightly less than wild-type c-Myb. Finally, we considered whether the c-Myb with both mutations was p300-responsive. We found that the double mutant c-Myb M303V-D384V can no longer respond to the coactivator p300. In addition, we compared the subcellular localization of wild-type c-Myb with the double mutant c-Myb M303V-D384V, but no remarkable differences were observed. Our conclusion is that the M303V and D384V mutations in c-Myb decrease the transcriptional activity of the protein and weakens its interaction with p300. Lost p300 response of the double mutant c-Myb M303V-D384V suggests that each of the mutated sites in c-Myb disrupt one of the two interaction surfaces for p300.