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: 7882420
• 负责人: KATHERINE A JONES
• 金额: $ 46.04万
• 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/7882420
• 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; ERBB2 gene; Enhancers; Epithelial; Event; Felis catus; Fluorescence Resonance Energy Transfer; Gene Expression Regulation; Gene Targeting; Genetic Transcription; Grant; HCT116 Cells; Homebound Persons; Human; In Vitro; Intestines; Link; Maps; Mediating; Methylation; Modeling; Mutation; N-terminal; Nuclear; Nuclear Protein; 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(申请人提供)：大多数人类结直肠癌细胞(CRC)含有腺瘤性息肉病(APC)肿瘤抑制基因突变，该基因控制转录共激活因子β-连环蛋白(β-CAT)的稳定性。核-CAT，与Lef-1/Tcf蛋白配对，诱导c-myc和其他Wnt靶基因。我们有兴趣了解？-CAT和其他Wnt途径调节因子在细胞核中如何发挥作用。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靶基因的辅活化子和辅抑制子复合体的循环交换的因子。这种转录下调是通过APC与CtBP辅阻遏子和TrCP泛素连接酶的特异性结合来实现的。这项资助的具体目的是：1)定位？-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体在体内和体外？-CAT转录和H3K4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学相关性。2)研究Wnt途径调节因子Pygopus的功能。我们发现，Pygopus通过其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增强子复合体，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的N-末端同源结构域相互作用的核蛋白，并研究它们在体内和体外对？-CAT：Lef-1转录的影响。固定化染色质珠法将分析因子在CAT中的逐步募集。对分离的腺瘤进行的芯片实验将探索这种新的Wnt转录模式与结肠癌的相关性。3)研究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，并表征一个无法结合CtBP的突变体APC，因为它能够区分APC在抑制和蛋白水解性转换中的作用。在HCT116、CtBP-/-和GSK3B-/-细胞中进行的芯片研究将评估β-CAT磷酸化和其他因素在辅助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腺瘤性息肉病(APC)肿瘤抑制基因突变。我们最近发现了APC在核中调控基因表达的新作用。重要的是，我们发现人类结肠癌中突变的APC蛋白不能执行这一功能。这里概述的研究旨在了解这些事件是如何发挥作用的，并确定结肠癌细胞中Wnt途径抑制剂的重要新靶点。