Most human colorectal cancer cells (CRC) contain mutations in the adenomatous polyposis coli (APC) tumor suppressor, which controls the stability of the transcriptional co-activator, beta-catenin (?-cat). Nuclear ?-cat, partners with LEF-1/TCF proteins to induce c-myc and other Wnt target genes. We are interested in understanding how ?-cat and other Wnt pathway regulators function in the nucleus. We recently identified a new role for the APC tumor suppressor as a direct represser of Wnt gene transcription, and a factor that mediates the cyclic exchange of coactivator and corepressor complexes at Wnt target genes in vivo. This down-regulation of transcription is mediated through specific binding of APC to the CtBP corepressor and the ?TrCP ubiquitin ligase.
The specific aims of this grant are: 1) Map the direct interactions between the ?-cat and WSTF:ISWI and TRRAP:TIP60, and evaluate the function of these chromatin remodeling complexes in ?-cat transcription and H3K4 methylation in vivo and in vitro. FRET studies will establish the physiological relevance of the interaction between B-cat and WSTF in vivo. 2) Investigate the function of the Wnt pathway regulator, Pygopus. We find that Pygopus binds directly to H3K4Me3 chromatin through its PHD domain. We will test the model that Pygo links H3K4Me3 to the Wnt enhancer complex, and enhances chromatin remodeling activities needed for transcription elongation. We will identify nuclear proteins that interact with the N-terminal homology domain of Pygopus, and characterize their effects on ?-cat:LEF-1 transcription in vivo and in vitro. An immobilized chromatin bead assay will analyze the step-wise recruitment of factors by ?-cat. ChIP experiments on isolated adenomas will probe the relevance of this new model of Wnt transcription to colon cancer. 3) Characterize the role of APC in the repression of Wnt target genes. We will determine how APC is recruited to Wnt target genes, and characterize a mutant APC that is unable to bind CtBP for its ability to distinguish between the role of APC in repression versus proteolytic turnover of ?- cat. ChIP studies in HCT116, CtBP-/-, and GSK3B-/- cells will assess the role of ?-cat phosphorylation and other factors in coregulator exchange. Lastly, we will examine the mechanism of APC repression of ErbB2 transcription, and determine whether ErbB2 is an important Wnt target gene in colon cancers. Most human colorectal cancer cells contain mutations in the adenomatous polyposis coli (APC) tumor suppressor. We have recently found new role for APC in the nucleus in the regulation of gene expression. Importantly, we find that mutant APC proteins in human colon cancers are unable to carry out this function. The studies outlined here are designed to understand how these events function, and identify important new targets for inhibitors of the Wnt pathway in colon cancer cells.
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