Biochemical and Cell Biological Study for Regulators of Notch Signaling

Notch信号已被证明在许多不同的细胞命运决定中起着重要作用。一些功能丧失的研究表明，Notch信号在造血的多个发育阶段发挥着重要作用，即最终造血细胞的产生，胸腺T细胞命运的承诺，胸腺CD4 CD8双阴性T细胞向双阳性T细胞的发育，以及脾边缘带B细胞的发育。在此之前，我们已经鉴定了一个人类主脑(Mam)蛋白家族，由三个成员(Mam-1、Mam-2和Mam-3)组成，并由此阐明了它们的生化作用机制。所有的MAM蛋白都与细胞核中Notch和CSL蛋白的胞内域的DNA结合复合体结合并稳定下来，以激活靶启动子的转录。在这个项目中，我们已经产生了一种MAM-1缺陷的小鼠，并发现MAM-1缺陷会破坏脾MZB细胞的发育，这是一个严格依赖于Notcl2、CSL蛋白和Delta 1配体的亚群。MAM-1缺乏也会导致早期胸腺细胞发育的部分受损，而不会影响依赖Notch1的最终造血过程的产生。我们还证明了在原代培养的MAM-1缺陷细胞中，通过组成活性形式的Notch转录激活目标启动子，使其减少几倍。这些结果表明，在一定程度上，Mam-1在淋巴生成的Notch依赖阶段是必需的，从而支持Mam是哺乳动物典型Notch途径的重要组成部分的观点。我们还发现，非典型Notch靶向Hes3启动子可以被一个与Mam结构相似的基因(MAMLD1)的产物激活，该基因的缺失会导致人类尿道下裂。

Notch signaling has been shown to play an important role in a number of different cell fate decisions. Several loss-of-function studies have revealed that Notch signaling plays an essential role in the multiple developmental stages for hematopoiesis, namely, generation of the definitive hematopoiesis, commitment to T cell fate in the thymus, development from thymic CD4 CD8 double negative (DN) T cells to double positive T cells, and development of splenic marginal zone (MZ) B cells. Previously, we have identified a human Mastermind (Mam) family of proteins, consisting of three members (Mam-1, Mam-2, and Mam-3) and thus elucidated their biochemical mechanisms of action. All of the Mam proteins bind to and stabilize the DNA-binding complex of the intracellular domains of Notch and CSL proteins in the nucleus to activate transcription from target promoters. In this term of project, we have generated a line of Mam-1-deficient mice and found that Mam-1 deficiency abolishes the development of splenic MZB cells, a subset strictly dependent on Notcli2, a CSL protein and Delta 1 ligand. Mam-1 deficiency also causes a partially impaired development of early thymocyres, while not affecting the generation of definitive hematopoiesis, processes that are dependent on Notch1. We also demonstrate the transcriptional activation of a target promoter by constitutively active forms of Notch to decrease severalfold in primarily cultured Mam- 1-deficient cells. These results indicate that Mam-1 is thus required to some extent for Notch-dependent stages in lymphopoiesis, thus supporting the notion that Mam is an essential component of the canonical Notch pathway in mammals.We also found that the non-canonical Notch target Hes3 promoter can be activated by a product of a gene (MAMLD1) that holds structural similarity with Mam and of which deletion causes hypospadia in humans.