Mechanism of Notch Transcriptional Activation

描述（由申请人提供）：Notch信号转导通路控制着对神经发生和造血重要的细胞命运决定，并且在人类癌症中被错误调节。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基于染色质的系统，可以在体外重现Notch转录。我们发现ICD缺乏一个内在的激活域，而是招募了Mastermind (MAM)，它提供了两个不同的调控域。MAM染色质特异性激活域招募CBP/p300组蛋白乙酰转移酶。第二个激活结构域是体内Notch转录所必需的，也可以将Notch转录与Notch增强子内ICD的修饰和蛋白水解周转结合起来。MAM的这一区域也将CBP/p300动员到类似于神经退行性疾病中突变的多聚谷氨酰胺重复蛋白形成的核包涵体上。这些发现表明，MAM是一种新的转录调节因子，其功能是将激活与周转结合起来，确保Notch增强子复合物是短暂的。

DESCRIPTION (provided by applicant): The Notch signal transduction pathway controls cell fate decisions important for neurogenesis and hematopoesis, and is misregulated in human cancers. 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 chromatin-based system that recapitulates Notch transcription in vitro. We show that the ICD lacks an intrinsic activation domain, but rather recruits Mastermind (MAM), which provides two distinct regulatory domains. A MAM chromatin-specific activation domain recruits the CBP/p300 histone acetyltransferase. The second activation domain, which is essential for Notch transcription in vivo, also serves to couple Notch transcription to modification and proteolytic turnover of the ICD within the Notch enhancer. This region of MAM also mobilizes CBP/p300 to nuclear inclusion bodies similar to those formed by mutant polyglutamine-repeat proteins in neurodegenerative diseases. These findings indicate that MAM is a novel transcriptional regulator which functions to couple activation to turnover, ensuring that the Notch enhancer complex is short-lived. We plan to further define the Notch regulatory mechanism through the following aims: 1) Analyze the mechanisms that regulate initiation and elongation of Notch transcription on chromatin templates and identify the protein(s) that interact with the MAM activation domains; 2) Assess the process by which MAM induces the formation of nuclear inclusion bodies, determine the composition of these structures and assess connections to the ubiquitination-proteolysis machinery; 3) Analyze the role of MAM in the proteolytic turnover of the Notch ICD. We will identify the nuclear kinase that phosphorylates the ICD and determine if the Sel-10 ubiquitin ligase is recruited to the Notch enhancer; and 4) Characterize the structure and mode of regulation of a natural (periodic) Notch enhancer in the cell-free in vitro transcription system. These studies will help elucidate the Notch transactivation mechanism and its role in the modification and destruction of the ICD, and may suggest approaches for blocking Notch signaling in human cancers.