Role of Mastermind in Notch Transcriptional Activation

描述（由申请人提供）：Notch信号转导通路控制细胞命运决定，对神经发生和造血至关重要，并参与包括白血病在内的多种癌症。信号传导导致Notch受体的胞内结构域（ICD）的蛋白水解释放，Notch受体是CSL增强子结合蛋白的专用转录共激活因子。我们的实验室最近开发了一种无细胞系统，可以在一个简单的模型增强子上重现Notch转录。这些研究表明，ICD仅与第二种Notch共激活因子Mastermind （MAM）一起激活转录。我们发现MAM招募CBP/p300组蛋白乙酰转移酶以及包括激酶、CDK8在内的中介复合物的其他成分。最后，我们发现与MAM共表达也促进了体内ICD的磷酸化和降解。这些发现表明，MAM可能招募一种核激酶来偶联ICD的激活和周转。在这里，我建议通过以下具体目标来研究mam相关的共激活蛋白在Notch转录中的作用以及ICD蛋白水解降解激活的协调：1 .定义Notch共激活蛋白Mastermind家族的保守性质；评估CDK8蛋白激酶活性是否有助于Notch转录和/或体内和体外ICD的周转，以及III。研究c-ski相互作用蛋白（SKIP）在体外Notch转录中的作用。通过这些研究，我们希望更好地了解Notch反激活机制及其在ICD修饰和破坏中的作用，这些研究将促进我们对Notch增强子复合物在转化过程中诱导内源性靶基因的机制的理解。

DESCRIPTION (provided by applicant): The Notch signal transduction pathway controls cell fate decisions important for neurogenesis and hematopoiesis and is involved in various cancers including leukemia. Signaling results in the proteolytic release of the intracellular domain (ICD) of the Notch receptor, a dedicated transcriptional co-activator of CSL enhancer-binding proteins. Our lab recently developed a cell-free system that recapitulates Notch transcription on a simple model enhancer. These studies revealed that the ICD activates transcription only in conjunction with a second Notch co-activator, Mastermind (MAM). We showed that MAM recruits the CBP/p300 histone acetyltransferase as well as other components of the mediator complex including the kinase, CDK8. Finally, we showed that co-expression with MAM also promotes the phosphorylation and degradation of the ICD in vivo. These findings suggest that MAM may recruit a nuclear kinase to couple activation with turnover of the ICD. Here I propose to investigate the role of MAM-associated co-activator proteins in Notch transcription and the co-ordination of activation with proteolytic degradation of the ICD through the following specific aims: I. Define the conserved properties of the Mastermind family of Notch co-activators, II. Assess whether the protein kinase activity of CDK8 contributes to Notch transcription and/or the turnover of the ICD in vivo and in vitro, and III. Examine the role of the c-ski-interacting protein (SKIP) in Notch transcription in vitro. Through these studies we hope to better understand the Notch transactivation mechanism and its role in the modification and destruction of the ICD and taken together, these studies will advance our understanding of the mechanism by which the Notch enhancer complex induces endogenous target genes during transformation.