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

描述(申请人提供)：Wnt信号通路对于正常的肠道生长和发育是必不可少的，该通路的不适当激活与结直肠癌有关。WNT信号导致连环蛋白的核积聚，然后激活与细胞增殖和生长有关的靶基因。其中一个靶点是c-Myc，它也是正常肠道细胞分化所必需的。因此，了解-catenin对c-Myc转录的调控对于阐明肠道的正常发育和结直肠癌的发病机制都是至关重要的。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。我的发现，使用我们实验室开发的无偏见的全基因组筛查，表明在c-Myc转录终止位点下游存在一个相当强大的连接素结合位点。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表达的主要连环蛋白调节。对该元件及其独特性质的表征将为c-Myc的调控提供新的见解。我们将通过测量包含两个5‘TCF结合位点和3’增强子的各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。对3‘元件周围序列的分析揭示了预测的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，从而促进了连环蛋白的结合。这将通过染色质免疫沉淀(CHIP)分析进行测试。为了确定与3‘元件相关的β-连环蛋白是否与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学方法，使用腺相关病毒的同源重组，来确定自然的3‘增强子如何在结直肠癌细胞系中促进c-Myc的表达。为了消除肠道内特异性的3‘增强子，我们将使用Cre：loxP技术，利用肠道特异性CYP1A1启动子控制下的萘黄酮敏感的Cre重组酶。这个小鼠品系将使我们能够评估删除3‘增强子对c-Myc水平、肠道细胞迁移、增殖和新陈代谢的影响。与公共健康相关：连环蛋白对c-Myc表达的调节是肠道基本生长过程的基础。这一途径的异常会导致结直肠癌。我们的研究涉及b-连环蛋白调节正常胃肠道发育和内环境平衡的基本机制，以及肠道恶性肿瘤的发病机制。

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.