c-Myc transcription in intestinal growth, differentiation, and carcinogenesis

c-Myc 转录在肠道生长、分化和癌变中的作用

• 批准号: 7759165
• 负责人: Gregory S. Yochum
• 金额: $ 30.94万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-10 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7759165
• 关键词: APC gene; Address; Adenomatous Polyposis Coli; Agar; Alleles; Antibodies; Binding; Binding Sites; Biological Assay; Bromodeoxyuridine; CYP1A1 gene; Cell Cycle; Cell Differentiation process; Cell Line; Cell Proliferation; Cells; Chromatin; Colorectal Cancer; Consensus; Defect; Dependovirus; Development; Differentiation and Growth; Elements; Engineering; Enhancers; Estrogen Receptors; Family; Family member; Gene Targeting; Genetic Transcription; Growth; Growth and Development function; HCT116 Cells; HT29 Cells; Histology; Homeostasis; Immigration; Immunohistochemistry; In Situ Hybridization; Intestines; Kinetics; Label; Large Intestine Carcinoma; Lead; Ligand Binding Domain; Malignant Epithelial Cell; Malignant Neoplasms; Measures; Mediator of activation protein; Metabolic; Metabolism; Modeling; Molecular Conformation; Monitor; Mouse Strains; Mutate; Mutation; Nuclear; Nude Mice; Pathogenesis; Pathway interactions; Phenotype; Process; Promoter Regions; Property; Proteins; Regulation; Reporter Genes; Sequence Analysis; Signal Pathway; Signal Transduction; Site; Small Interfering RNA; Somatic Cell Genetics; Subcutaneous Injections; Technology; Testing; Time; Transcription Factor AP-1; Tumorigenicity; activating transcription factor; c-myc Genes; carcinogenesis; cell motility; cell type; chromatin immunoprecipitation; chromatin remodeling; gastrointestinal; genome wide association study; genome-wide; histone acetyltransferase; homologous recombination; in vivo; insight; member; metaplastic cell transformation; promoter; public health relevance; recombinase; tissue fixing; transcription factor

DESCRIPTION (PROVIDED BY APPLICANT): The Wnt signaling pathway is essential for normal intestinal growth and development, and inappropriate activation of this pathway is associated with colorectal cancer. Wnt signaling causes nuclear accumulation of ¿-catenin, which then activates target genes involved in cell proliferation and growth. One such target is c- Myc, which also is required for normal intestinal cell differentiation. Therefore, understanding ¿-catenin regulation of c-Myc transcription is essential for elucidating both normal intestinal development and the pathogenesis of colorectal carcinoma. ¿-catenin activation of c-Myc is thought to occur through sequences located upstream from the c-Myc promoter. My findings, using an unbiased, genome-wide screen developed in our lab, indicate that a considerably more robust ¿-catenin binding site exists two kilobases downstream from the c-Myc transcriptional stop site. My preliminary results suggest that, in contrast to what is commonly believed, this 3' binding region provides the principal ¿-catenin regulation of c-Myc expression. Characterization of this element and its unique properties will provide new insights into c-Myc regulation. We will test whether this binding is functional by measuring the activities of reporter genes containing various combinations of mutations in the two 5' TCF binding sites and the 3' enhancer. Analysis of sequences surrounding the 3' element revealed predicted FOXO and AP-1 binding sites. I hypothesize that binding of AP-1 and/or a specific FOXO factor to the 3' site promotes a chromatin change that facilitates ¿-catenin binding. This will be tested by chromatin immunoprecipitation (ChIP) assays. To determine whether ¿-catenin associated with the 3' element interacts with factors bound to the 5' promoter region, we will perform chromatin conformation capture (CCC) assays. We will use a somatic cell genetic approach, using homologous recombination of an adeno-associated virus, to determine how the native 3' enhancer contributes to c-Myc expression in a colorectal carcinoma cell line. To eliminate the 3' enhancer specifically within the intestine, we will use Cre: LoxP technology, making use of a ¿-naphthoflavone-sensitive Cre recombinase under control of the intestine-specific CYP1A1 promoter. This mouse strain will allow us to assess the effects of deleting the 3' enhancer on c-Myc levels, intestinal cell migration, proliferation, and metabolism. PUBLIC HEALTH RELEVANCE: The regulation of c-Myc expression by ¿-catenin underlies fundamental growth processes in the intestine. Abnormalities in this pathway lead to colorectal cancer. Our studies address the fundamental mechanisms underlying b-catenin regulation of normal gastrointestinal development and homeostasis as well as the pathogenesis of intestinal malignancy.

描述（由申请人提供）：Wnt信号通路对于正常的肠道生长和发育至关重要，并且该途径的不当激活与大肠癌有关。 Wnt信号传导会导致 - 蛋白酶的核积累，然后激活与细胞增殖和生长有关的靶基因。这样的目标是C- MYC，这也是正常肠细胞分化所必需的。因此，理解C -MYC转录的钙蛋白调节对于阐明正常肠发育和结直肠癌的发病机理至关重要。 C-MYC的 - 蛋白质激活被认为是通过C-MYC启动子上游的序列发生的。我的发现是使用实验室中开发出的无偏基因组筛查的，表明一个更坚固的`` - catenin结合位点''位于C-MYC转录停止位点下游的两个千座碱基。我的初步结果表明，与通常认为的相反，这个3'结合区域提供了c -Myc表达的主� -catenin调节。该元素及其独特属性的表征将为C-MYC调节提供新的见解。我们将通过测量在两个5'TCF结合位点和3'增强子中含有各种突变组合的报告基因的活性来测试这种结合是否起作用。分析3'元素周围的序列显示了预测的FOXO和AP-1结合位点。我假设AP -1和/或特定的FOXO因子与3'位点的结合促进了染色质的变化，从而促进 - catenin结合。这将通过染色质免疫沉淀（CHIP）测定进行测试。为了确定与3'元素相关的 - 帕宁蛋白是否与与5'启动子区域结合的因子相互作用，我们将执行染色质会议捕获（CCC）测定。我们将使用与腺相关病毒的同源重组的体细胞遗传方法来确定天然3'增强子在有色癌细胞系中如何促进C-MYC表达。为了消除肠内的3'增强剂，我们将使用CRE：LOXP技术，利用渗透性的CRE重组酶在肠道特异性CYP1A1启动子的控制下使用ood-naphthththhoflavone敏感的CRE重组酶。这种小鼠菌株将使我们能够评估删除3'增强子对C-MYC水平，肠细胞迁移，增殖和代谢的影响。公共卫生相关性：通过渗透对肠道基本增长过程的基础C -MYC表达的调节。这种途径中的异常导致结直肠癌。我们的研究涉及正常胃肠道发育和稳态的B-catenin调节的基本机制以及肠道恶性肿瘤的发病机理。