Sequence Specificity and Transcriptional Output of the C-clamp, an Auxiliary DNA Binding Domain in LEF/TCF Transcription Factors

Colon cancer is the second leading cause of cancer-related death in the United States. It is initiated primarily through aberrant activation of the Wingless/int (Wnt) signaling pathway, a vital developmental pathway in metazoans. The Lymphoid Enhancer Factor/T Cell Factor (LEF/TCF) family of transcription factors are downstream DNA binding proteins in the Wnt signaling pathway. LEF/TCFs have a sequence specific High Mobility Group (HMG) box that binds Wnt Response Elements (WREs). The E" tail isoforms of LEF/TCFs are alternatively spliced to include a second DNA binding domain called the C-clamp. We show by Cyclic Amplification and Selection of Targets (CASTing) and Electrophoretic Mobility Shift Assays (EMSA) that the C-clamp binds GC-rich Helper" elements. Luciferase assays reveal that the C-clamp-Helper site interaction is required for efficient activation of Wnt target gene promoters by TCF1E in a variety of cell types, including colon cancer cells.;To further elucidate C-clamp function in colon cancer cells, we engineered DLD1 cell lines to inducibly express dominant negative TCF1E wild-type (dnTCF1E WT) or a mutated form that lacks C-clamp DNA binding activity (dnTCF1E mut). Microarray analysis after induction of dnTCF1EWT and dnTCF1Emut indicates that the C-clamp is not required for regulation of the majority of Wnt target genes. Rather, the C-clamp is required for induction of p21, a cell cycle inhibitor that forces a stall in cell growth. dnTCF1EWT uniquely regulates the expression of multiple genes that are known to control p21 expression, including the transcriptional repressor SP5. Luciferase assays reveal that SP5 is regulated by the C-clamp/Helper site interaction. Our results indicate that dnTCF1E WT induces p21 expression partly through downregulation of SP5, a known transcriptional repressor of p21 expression.;To investigate the role of the C-clamp/Helper site interaction in a genome-wide binding profile of dnTCF1E, we performed time-course ChIP-seq experiments on dnTCF1EWT and dnTCF1Emut in DLD-1 colon cancer cells. We identified 1046 high confidence dnTCF1EWT binding sites and 691 high confidence dnTCF1Emut binding sites. De novo motif enrichment revealed the presence of the Wnt Response Element (WRE) in genomic regions occupied by both dnTCF1EWT and dnTCF1Emut. However, Helper sites were significantly enriched in dnTCF1EWT-bound regions but not in regions occupied by dnTCF1E mut. The majority of Helper-enriched peaks occurred in CpG islands with no apparent enrichment of the WRE.;In special cases, such as in the SP5, CDX1, and LEF1 promoters, the C-clamp-Helper interaction synergizes with the HMG box-WRE interaction to control Wnt target gene expression. We also observed that the C-clamp contributes to the overall affinity of dnTCF1E for DNA and that the C-clamp may facilitate scanning along the DNA template to facilitate rapid binding to target sites.;Gene expression was assessed in concert with ChIP-seq experiments using 4'Thiouridine-seq, an RNA-seq based approach which allows for assessment of rapid changes in gene expression. We identified 688 genes that showed a decrease in transcription rate as early as 2.5 hours after induction of dnTCF1E WT. Most of these genes, showed a further decrease in transcription rate after 9.5 hours of induction and increased dnTCF1EWT expression. Importantly, the downregulated genes were significantly enriched with ChIP-seq peaks, demonstrating that ChIP-seq and 4'Thiouridine-seq datasets can be successfully correlated.