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

描述（申请人提供）：Wnt信号通路对正常肠道生长和发育至关重要，该通路的不适当激活与结直肠癌相关。Wnt信号传导导致β-连环蛋白的核积累，然后激活参与细胞增殖和生长的靶基因。一个这样的靶点是c-Myc，其也是正常肠细胞分化所需的。因此，理解β-catenin对c-Myc转录的调控对于阐明正常肠道发育和结直肠癌的发病机制是必不可少的。人们认为c-Myc的连环蛋白激活通过位于c-Myc启动子上游的序列发生。我的研究结果，使用我们实验室开发的无偏见的全基因组筛选，表明一个更强大的连环蛋白结合位点存在于c-Myc转录终止位点下游的两个酶。我的初步结果表明，与通常认为的相反，这个3'结合区提供了c-Myc表达的主要<$-catenin调节。该元件及其独特性质的表征将为c-Myc调控提供新的见解。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有功能。3'元件周围序列的分析揭示了预测的FOXO和AP-1结合位点。我推测AP-1和/或特定FOXO因子与3'位点的结合促进了染色质的变化，从而促进了β-连环蛋白的结合。这将通过染色质免疫沉淀（ChIP）试验进行检测。以确定是否为了确定与3'元件相关的β-连环蛋白与结合到5'启动子区的因子相互作用，我们将进行染色质构象捕获（CCC）测定。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，以确定天然3'增强子如何有助于结肠直肠癌细胞系中的c-Myc表达。为了在肠道内特异性消除3'增强子，我们将使用Cre：LoxP技术，利用在精氨酸特异性CYP 1A 1启动子控制下的对萘啶酮敏感的Cre重组酶。该小鼠品系将允许我们评估删除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：c-Myc表达的调节由连环蛋白构成肠道基本生长过程的基础。这一途径的缺失导致结直肠癌。我们的研究致力于阐明β-连环蛋白调节正常胃肠道发育和体内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.