A Novel Mechanism for LEF1 Promoter Regulation in Colon Cancer

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DESCRIPTION (provided by applicant): In the United States, colon cancer is the third leading cause of cancer-related deaths with African-Americans having a higher incidence and mortality rate than any other ethnicity. This year, the American Cancer Society estimates that 102,480 Americans will be diagnosed with colon cancer and of those cases, 50,830 individuals will succumb to the disease. To date, colorectal cancer is the most notorious cancer linked to overactive, aberrant Wnt signaling. Most cases derive from inactivating mutations in the adenomatous polyposis coli gene, which encodes a key component of the b-catenin Destruction Complex. The increase in stable, cytoplasmic, b- catenin results in an influx of this regulator int the nucleus where it works with Lymphoid Enhancer Factor/T Cell Factors (LEF/TCFs) to activate transcription of Wnt target genes, such as CMYC and LEF1. The LEF1 locus contains two promoters. Promoter 1 (P1), which is a Wnt target, produces full-length Lymphoid Enhancer Factor-1 (FL-LEF-1), an isoform that works with b-catenin: promoter 2 (P2) produces dominate negative LEF-1 (dnLEF-1), an isoform that lacks the b-catenin binding domain, and can therefore down-regulate Wnt target genes. Importantly, only the Wnt-targeted FL-LEF-1 isoform is expressed in colon cancer. The dnLEF-1 producing P2 promoter is actively silenced by an unknown mechanism involving an upstream repressor element and the multifunctional protein, Yin Yang 1 (YY1). Since re-introduction of dnLEF-1 can oppose Wnt signaling by binding to Wnt target genes for downregulation, it is important to elucidate the mechanism of its transcriptional repression and determine how this regulator can be re-expressed in cancer. The following aims and methods are proposed: 1) Determine whether YY1 uses an RNA-dependent mechanism to silence P2. RNA immunoprecipitation assays and in vitro biochemical approaches are proposed. 2) Define the nucleotide context and associated proteins of the upstream repressor element. A transient transfection assay for repression will be used to define the core sequences within the element. DNA affinity chromatography and mass spectrometry will identify proteins interacting with these key sequences 3) Test the hypothesis that transcription from upstream P1 affects transcriptional activity of downstream P2. P1 transcription will be modulated using a custom designed TALE-repressor. Defining the mechanism of P2 repression will lead to a better understanding of expression from multi-promoter gene loci, and thus highlight the potential for broad impact and new therapeutic approaches in cancer and other diseases.