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 provides insight into how B-Myb functions as a site-specific transcription factor.

B-Myb 是脊椎动物 Myb 转录因子家族中高度保守的成员，在细胞周期进程和增殖中起着关键作用。Myb 蛋白通过使用其 DNA 结合域 (DBD) 与 Myb 结合位点 (MBS) 序列特异性相互作用来激活 Myb 依赖性启动子。B-Myb 对 MBS 启动子的反式激活受到其负调节域 (NRD) 的抑制，并且 Cdk2-CyclinA 对 NRD 的磷酸化减轻了激活 B-Myb 依赖性启动子的抑制。然而，B-Myb 介导的转录的自身抑制和激活的结构机制尚未得到很好的表征。在这里，我们确定 B-Myb NRD 中的一个区域（残基 510-600）直接与 DBD 相关联并抑制 DBD 与 MBS DNA 序列的结合。我们使用生物物理分析证明 NRD 在 T515、T518 和 T520 处的磷酸化足以破坏 NRD 和 DBD 之间的相互作用，从而导致对 MBS DNA 的亲和力增加，并在细胞分析中增加 B-Myb 依赖性启动子激活. 我们对 B-Myb 自动调节和磷酸化激活作用的生化表征提供了对 B-Myb 如何作为位点特异性转录因子发挥作用的深入了解。