Abstract
B-Myb is a highly conserved member of the vertebrate Myb family of transcription factors that plays a critical role in cell-cycle progression and proliferation. Myb proteins activate Myb-dependent promoters by interacting specifically with Myb-binding site (MBS) sequences using their DNA-binding domain (DBD). Transactivation of MBS promoters by B-Myb is repressed by its negative regulatory domain (NRD), and phosphorylation of the NRD by Cdk2-CyclinA relieves the repression to activate B-Myb-dependent promoters. However, the structural mechanisms underlying autoinhibition and activation of B-Myb-mediated transcription have been poorly characterized. Here, we determined that a region in the B-Myb NRD (residues 510-600) directly associates with the DBD and inhibits binding of the DBD to the MBS DNA sequence. We demonstrate using biophysical assays that phosphorylation of the NRD at T515, T518, and T520 is sufficient to disrupt the interaction between the NRD and the DBD, which results in increased affinity for MBS DNA and increased B-Myb-dependent promoter activation in cell assays. Our biochemical characterization of B-Myb autoregulation and the activating effects of phosphorylation provide insight into how B-Myb functions as a site-specific transcription factor.