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

β-连环蛋白和 APC 调节 Wnt 靶基因转录的机制

• 批准号: 8100176
• 负责人: KATHERINE A JONES
• 金额: $ 44.65万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8100176
• 关键词: Adenomatous Polyposis Coli; Adenomatous Polyposis Coli Protein; Binding; Biological Assay; Cell Nucleus; Cells; Chromatin; Chromatin Remodeling Factor; Colon Carcinoma; Colorectal Cancer; Complex; Data; Dominant-Negative Mutation; Down-Regulation; Enhancers; Epithelial; Event; Felis catus; Fluorescence Resonance Energy Transfer; Gene Expression Regulation; Gene Targeting; Genetic Transcription; Grant; HCT116 Cells; HTATIP gene; Homebound Persons; Human; ISWI; In Vitro; Intestines; Link; Maps; Mediating; Methylation; Modeling; Mutation; N-terminal; Nuclear; Nuclear Proteins; Pathway interactions; Phosphorylation; Physiological; Proteins; Proteolysis; Proteomics; RNA; RNA Interference; Recruitment Activity; Repression; Research Personnel; Role; T cell transcription factor 1; TRRAP gene; Testing; Transcription Elongation; Tumor Suppressor Proteins; adenoma; beta catenin; c-myc Genes; cancer cell; chromatin remodeling; design; in vivo; inhibitor/antagonist; interest; mutant; programs; promoter; research study; trafficking; ubiquitin ligase

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.

描述（由申请人提供）：大多数人结直肠癌细胞（CRC）在腺瘤性结肠息肉病（APC）肿瘤抑制因子中含有突变，APC肿瘤抑制因子控制转录辅激活因子β-连环蛋白（β-cat）的稳定性。Nuclear <$-cat与LEF-1/TCF蛋白结合诱导c-myc和其他Wnt靶基因。我们有兴趣了解<$-cat和其他Wnt通路调节剂如何在细胞核中发挥作用。我们最近确定了一个新的作用APC肿瘤抑制因子作为Wnt基因转录的直接阻遏物，并介导在体内Wnt靶基因的辅激活因子和辅阻遏复合物的循环交换的一个因素。这种转录下调是通过APC与CtBP辅阻遏物和Δ TrCP泛素连接酶的特异性结合介导的。该基金的具体目标是：1）绘制<$-cat和WSTF：ISWI和TRRAP：TIP 60之间的直接相互作用，并评估这些染色质重塑复合物在<$-cat转录和H3 K4甲基化中的功能。FRET研究将建立B-cat和WSTF在体内相互作用的生理相关性。2)研究Wnt途径调节剂Pygopus的功能。我们发现Pygopus通过其PHD结构域直接与H3 K4 Me 3染色质结合。我们将测试Pygo将H3 K4 Me 3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。我们将确定与Pygopus的N-末端同源结构域相互作用的核蛋白，并表征其对<$-cat：LEF-1在体内和体外转录的影响。固定化染色质珠测定将分析通过<$-cat逐步招募因子。对分离的腺瘤进行ChIP实验将探索这种新的Wnt转录模型与结肠癌的相关性。3)表征APC在抑制Wnt靶基因中的作用。我们将确定APC是如何被招募到Wnt靶基因，并表征一种突变型APC，该突变型APC无法结合CtBP，因为它有能力区分APC在阻遏与蛋白水解营业额之间的作用。HCT 116，CtBP-/-和GSK 3B-/-细胞中的ChIP研究将评估<$-cat磷酸化和其他因子在辅调节因子交换中的作用。最后，我们将研究APC抑制ErbB 2转录的机制，并确定ErbB 2是否是结肠癌中重要的Wnt靶基因。大多数人结直肠癌细胞含有腺瘤性结肠息肉病（APC）肿瘤抑制基因突变。我们最近发现APC在细胞核中的基因表达调控中的新作用。重要的是，我们发现人类结肠癌中的突变APC蛋白无法执行此功能。本文概述的研究旨在了解这些事件如何发挥作用，并确定结肠癌细胞中Wnt通路抑制剂的重要新靶点。