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至关重要的细胞命运决定，在人类癌症中受到错误调控。信号导致Notch受体的胞内域(ICD)的蛋白水解性释放，Notch受体是CSL增强子结合蛋白的专用转录辅助激活因子。我们实验室最近开发了一种基于染色质的系统，它可以在体外重现Notch转录。我们发现ICD缺乏一个内在的激活结构域，而是招募了MasterMind(MAM)，它提供了两个不同的调节域。MAM染色质特异的激活结构域招募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，它也将Notch转录与Notch增强子内ICD的修饰和蛋白水解性转换连接起来。MAM的这个区域还动员CBP/p300形成核包涵体，类似于神经退行性疾病中突变的多谷氨酰胺重复蛋白形成的包涵体。这些发现表明，MAM是一种新型的转录调节因子，其功能是将激活和周转结合起来，确保Notch增强子复合体是短暂的。 我们计划通过以下目标进一步定义Notch的调控机制：1)分析调控染色质模板上Notch转录的起始和延伸的机制，并确定与MAM激活结构域相互作用的蛋白质(S)；2)评估MAM诱导核包涵体形成的过程，确定这些结构的组成并评估与泛素化-蛋白分解机制的联系；3)分析MAM在Notch ICD蛋白水解性周转中的作用。我们将确定使ICD磷酸化的核激酶，并确定Sel-10泛素连接酶是否被招募到Notch增强子；以及4)在无细胞体外转录系统中表征天然(周期性)Notch增强子的结构和调节模式。这些研究将有助于阐明Notch反式激活机制及其在ICD修饰和破坏中的作用，并可能提出阻断人类癌症中Notch信号的方法。

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.