Repression of the LEF1 alternative promoter in colon cancer

Lymphoid Enhancer Factor 1 (LEF-1) mediates Wnt signaling by recruiting the transactivator beta-catenin to Wnt target genes for regulation. Many of these target genes control cell proliferation and survival and are overexpressed in colon cancer. LEF-1 is also aberrantly expressed in colon cancers because promoter 1 (P1) which produces a full-length LEF-1 (FL-LEF-1) is activated. A second promoter in intron 2 (P2) which produces a dominant negative LEF-1 (dnLEF-1) is inactive. FL-LEF-1 is a growth promoting form because it mediates Wnt activation of proliferation genes, whereas truncated dnLEF-1, an isoform that is missing the beta-catenin binding domain, is a growth inhibiting form that can oppose Wnt signaling and reduce expression of proliferation genes. This thesis aims to understand the mechanism(s) behind the repression of alternative promoter P2.;We show that both P1 and P2 are bona fide targets of the Wnt pathway because these promoters can be activated by TCF-beta-catenin complexes. Because both P1 and P2 have Wnt response elements in their promoters, we speculate that Wnts coordinately regulate transcription of both promoters in normal cells and in so doing, establish a system of positive and negative regulatory feedback in normal development. However, in colon cancer P1 is overactive and is regulated by TCF-beta-catenin. Despite its equal potential for regulation by TCF-beta-catenin, P2 is inactive and TCF-beta-catenin complexes do not occupy the promoter. Detailed mapping by deletion analysis and a study of acetylation patterns of chromatin modification between P1 and P2 have revealed a distal regulatory element upstream of P2. This element is a repressor and it specifically inhibits P2 transcription and not P1. Further study revealed that the repressor directly targets a P2-specific element. We identified Yin Yang 1 (YY1) transcription factor as the P2-specific factor necessary for repression and showed that YY1 occupies silent LEF1 P2 in the colon cancer genome. To understand the mechanism of repression, we characterized the distal repressor element. A thorough deletion and mutational analysis of the repressor region identified sequences important for repression. Biochemical and DNA affinity purification followed by mass spectrometry analysis identified repressor binding candidates such as CHERP, ZEB2, components of the chromatin remodeling complexes, co-repressors, and other repressor proteins. We present a preliminary validation and characterization of a subset of these putative repressor proteins. Lastly, we also present preliminary data that suggest a non-coding antisense RNA might be involved in P2 repression. We propose that careful, balanced expression of growth-promoting FL-LEF-1 and growth-inhibitory dnLEF-1 is important for controlled gene expression during normal development. We further propose that disruption of this balanced expression through the mechanisms discovered here contribute to misregulated Wnt signaling in colon cancer.