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

基本信息

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
项目类别：
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

项目摘要

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，该通路的不当激活与 Wnt 信号通路导致 ¿ -连环蛋白，然后激活参与细胞增殖和生长的靶基因，其中一个靶基因是 c-Myc，它也是正常肠细胞分化所必需的，因此，了解 ¿ c-Myc 转录的连环蛋白调节对于阐明正常肠道发育和结直肠癌的发病机制至关重要。 c-Myc 的连环蛋白激活被认为是通过位于 c-Myc 启动子上游的序列发生的。我的研究结果表明，使用我们实验室开发的无偏倚的全基因组筛选，该结果表明一个相当强大的 ¿ -连环蛋白结合位点存在于 c-Myc 转录终止位点下游 2 KB 处，我的初步结果表明，与通常认为的相反，该 3' 结合区域提供了主要的 ¿ -连环蛋白对 c-Myc 表达的调节。对该元件及其独特特性的表征将为 c-Myc 调节提供新的见解。我们将通过测量包含两种突变组合的报告基因的活性来测试这种结合是否具有功能。 5' TCF 结合位点和 3' 增强子周围的序列分析揭示了预测的 FOXO 和 AP-1 结合位点，我认为 AP-1 和/或特定 FOXO 因子与 3' 位点的结合会促进。一个染色质变化促进-连环蛋白结合。这将通过染色质免疫沉淀 (ChIP) 测定来测试。与 3' 元件相关的连环蛋白与与 5' 启动子区域结合的因子相互作用，我们将进行染色质构象捕获 (CCC) 分析，我们将使用体细胞遗传方法，利用腺相关病毒的同源重组来进行分析。确定天然 3' 增强子如何促进结直肠癌细胞系中的 c-Myc 表达为了消除肠道内的 3' 增强子，我们将使用 Cre: LoxP 技术，使用 ¿ -受肠道特异性 CYP1A1 启动子控制的萘黄酮敏感 Cre 重组酶使我们能够评估删除 3' 增强子对 c-Myc 水平、肠道细胞迁移、增殖和代谢的影响。 : c-Myc 表达的调节 ¿ β-连环蛋白是肠道基本生长过程的基础。该途径的异常会导致结直肠癌。我们的研究探讨了 β-连环蛋白调节正常胃肠道发育和稳态以及肠道恶性肿瘤发病机制的基本机制。