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

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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.