Mechanism of beta-catenin and APC-regulated transcription of Wnt target genes

描述（由申请人提供）：大多数人类结直肠癌细胞（CRC）含有大肠腺瘤性息肉病（APC）肿瘤抑制因子的突变，该突变控制转录共激活因子β -连环蛋白（¿-cat）的稳定性。核¿-cat，与LEF-1/TCF蛋白合作诱导c-myc和其他Wnt靶基因。我们有兴趣了解¿-cat和其他Wnt通路调节因子如何在细胞核中起作用。我们最近发现了APC肿瘤抑制因子作为Wnt基因转录的直接抑制因子的新作用，以及在体内介导Wnt靶基因的共激活因子和共抑制因子复合物的循环交换的因子。这种转录下调是通过APC与CtBP共抑制因子和TrCP泛素连接酶的特异性结合介导的。本资助的具体目的是：1)绘制-cat与WSTF:ISWI和TRRAP:TIP60之间的直接相互作用，并评估这些染色质重塑复合物在体内和体外的-cat转录和H3K4甲基化中的功能。FRET研究将在体内建立b猫和WSTF相互作用的生理相关性。2)研究Wnt通路调节因子Pygopus的功能。我们发现Pygopus通过PHD结构域直接与H3K4Me3染色质结合。我们将测试Pygo将H3K4Me3连接到Wnt增强子复合物，并增强转录延伸所需的染色质重塑活性的模型。我们将鉴定与Pygopus n端同源结构域相互作用的核蛋白，并表征它们在体内和体外对¿-cat:LEF-1转录的影响。固定化染色质球试验将分析通过¿-cat逐步招募因子。在分离腺瘤上的ChIP实验将探索这种新的Wnt转录模式与结肠癌的相关性。3)表征APC在Wnt靶基因抑制中的作用。我们将确定APC是如何被招募到Wnt靶基因上的，并描述一个无法结合CtBP的突变APC，因为它能够区分APC在抑制和蛋白水解转化中的作用。在HCT116、CtBP-/-和GSK3B-/-细胞中的ChIP研究将评估¿-cat磷酸化和其他因子在共调节因子交换中的作用。最后，我们将研究APC抑制ErbB2转录的机制，并确定ErbB2是否是结肠癌中重要的Wnt靶基因。大多数人类结直肠癌细胞含有大肠腺瘤性息肉病（APC）肿瘤抑制因子的突变。近年来，我们在细胞核中发现了APC调控基因表达的新作用。重要的是，我们发现突变的APC蛋白在人类结肠癌中不能执行这一功能。这里概述的研究旨在了解这些事件如何起作用，并确定结肠癌细胞中Wnt途径抑制剂的重要新靶点。

DESCRIPTION (provided by applicant): Most human colorectal cancer cells (CRC) contain mutations in the adenomatous polyposis coli (APC) tumor suppressor, which controls the stability of the transcriptional co-activator, beta-catenin (¿-cat). Nuclear ¿-cat, partners with LEF-1/TCF proteins to induce c-myc and other Wnt target genes. We are interested in understanding how ¿-cat and other Wnt pathway regulators function in the nucleus. We recently identified a new role for the APC tumor suppressor as a direct represser of Wnt gene transcription, and a factor that mediates the cyclic exchange of coactivator and corepressor complexes at Wnt target genes in vivo. This down-regulation of transcription is mediated through specific binding of APC to the CtBP corepressor and the ¿TrCP ubiquitin ligase. The specific aims of this grant are: 1) Map the direct interactions between the ¿-cat and WSTF:ISWI and TRRAP:TIP60, and evaluate the function of these chromatin remodeling complexes in ¿-cat transcription and H3K4 methylation in vivo and in vitro. FRET studies will establish the physiological relevance of the interaction between B-cat and WSTF in vivo. 2) Investigate the function of the Wnt pathway regulator, Pygopus. We find that Pygopus binds directly to H3K4Me3 chromatin through its PHD domain. We will test the model that Pygo links H3K4Me3 to the Wnt enhancer complex, and enhances chromatin remodeling activities needed for transcription elongation. We will identify nuclear proteins that interact with the N-terminal homology domain of Pygopus, and characterize their effects on ¿-cat:LEF-1 transcription in vivo and in vitro. An immobilized chromatin bead assay will analyze the step-wise recruitment of factors by ¿-cat. ChIP experiments on isolated adenomas will probe the relevance of this new model of Wnt transcription to colon cancer. 3) Characterize the role of APC in the repression of Wnt target genes. We will determine how APC is recruited to Wnt target genes, and characterize a mutant APC that is unable to bind CtBP for its ability to distinguish between the role of APC in repression versus proteolytic turnover of ¿- cat. ChIP studies in HCT116, CtBP-/-, and GSK3B-/- cells will assess the role of ¿-cat phosphorylation and other factors in coregulator exchange. Lastly, we will examine the mechanism of APC repression of ErbB2 transcription, and determine whether ErbB2 is an important Wnt target gene in colon cancers. Most human colorectal cancer cells contain mutations in the adenomatous polyposis coli (APC) tumor suppressor. We have recently found new role for APC in the nucleus in the regulation of gene expression. Importantly, we find that mutant APC proteins in human colon cancers are unable to carry out this function. The studies outlined here are designed to understand how these events function, and identify important new targets for inhibitors of the Wnt pathway in colon cancer cells.