Identification of a Family of Mastermind-Like Transcriptional Coactivators for Mammalian Notch Receptors

The molecular mechanisms by which Notch receptors induce diverse biological responses are not fully understood. We recently cloned a mammalian homologue of the Mastermind gene of Drosophila melanogaster, MAML1 (Mastermind-like-1 molecule) and determined that it functions as a transcriptional coactivator for Notch receptors. In this report, we characterize two additional genes in this Mastermind-like gene family: MAML2 and MAML3. The three MAML genes are widely expressed in adult tissues but exhibit distinct expression patterns in mouse early spinal cord development. All MAML proteins localize to nuclear bodies, share a conserved basic domain in their N termini that binds to the ankyrin repeat domain of Notch, and contain a transcriptional activation domain in their C termini. Moreover, as determined by using coimmunoprecipitation assays, each MAML protein was found to be capable of forming a multiprotein complex with the intracellular domain of each Notch receptor (ICN1 to -4) and CSL in vivo. However, MAML3 bound less efficiently to the ankyrin repeat domain of Notch1. Also, in U20S cells, whereas MAML1 and MAML2 functioned efficiently as coactivators with each of the Notch receptors to transactivate a Notch target HES1 promoter construct, MAML3 functioned more efficiently with ICN4 than with other forms of ICN. Similarly, MAML1 and MAML2 amplified Notch ligand (both Jagged2 and Delta1)-induced transcription of the HES-1 gene, whereas MAML3 displayed little effect. Thus, MAML proteins may modify Notch signaling in different cell types based on their own expression levels and differential activities and thereby contribute to the diversity of the biological effects resulting from Notch activation.

Notch受体（Notch receptors）介导多样生物学应答的分子机制尚未完全阐明。本课题组此前成功克隆了黑腹果蝇（Drosophila melanogaster）Mastermind基因的哺乳动物同源基因——Mastermind样蛋白1（Mastermind-like-1 molecule, MAML1），并证实其可作为Notch受体的转录辅激活因子发挥功能。本研究在此基础上，对Mastermind样基因家族中另外两个成员MAML2与MAML3进行了系统表征。三个MAML基因在成体组织中广泛表达，但在小鼠早期脊髓发育过程中呈现出截然不同的表达模式。所有MAML蛋白均定位于核体（nuclear bodies），其N端均携带一段保守的碱性结构域，可结合Notch受体的锚蛋白重复序列结构域，且C端均含有转录激活结构域。此外，通过免疫共沉淀实验证实，所有MAML蛋白均可在体内与各Notch受体的胞内结构域（ICN1至ICN4）以及CSL蛋白形成多蛋白复合物。但MAML3与Notch1锚蛋白重复序列结构域的结合效率相对较低。在U20S细胞中，MAML1与MAML2可高效协同各Notch受体，激活Notch靶基因HES1的启动子报告质粒；而MAML3对ICN4的辅助激活效率显著高于其对其他ICN亚型的作用。类似地，MAML1与MAML2可增强Notch配体（包括Jagged2与Delta1）诱导的HES-1基因转录，而MAML3几乎无此效应。综上，MAML蛋白可通过自身表达水平与活性差异，在不同细胞类型中调控Notch信号通路，进而参与介导Notch激活所引发的多样生物学效应。