A unique DNA binding domain converts T-cell factors into strong Wnt effectors

Area of Science:

• Molecular Biology
• Genetics
• Cancer Research

Background:

• Wnt signaling regulates gene expression via LEF/TCF transcription factors binding to Wnt response elements (WREs) and beta-catenin recruitment.
• Alternative splicing generates potent TCF-1 and TCF-4 isoforms with a C-terminal "E" tail, enhancing LEF1 and CDX1 activation.

Purpose of the Study:

• To elucidate the mechanism behind the unique potency of alternatively spliced TCF isoforms.
• To identify and characterize a novel DNA-binding motif responsible for enhanced Wnt target gene regulation.

Main Methods:

• Surface Plasmon Resonance (SPR) to analyze C-clamp DNA-binding properties.
• Cyclic Amplification and Selection of Targets (CASTing) to assess WRE recognition and binding site selection.
• Site-directed mutagenesis to investigate the role of the C-clamp in gene regulation.

Main Results:

• A 30-amino-acid motif, the "cysteine clamp" (C-clamp), was identified as an auxiliary DNA-binding motif.
• The C-clamp binds double-stranded DNA with high affinity (Kd = 16 nM) but does not alter WRE specificity.
• The C-clamp mediates binding to a downstream GC element, enhancing TCF-1E's regulatory activity on Wnt targets crucial for colon cancer cell growth.

Conclusions:

• The C-clamp is a sequence-specific DNA-binding motif that confers unique DNA-binding and regulatory activities to E-tail isoforms of TCF transcription factors.
• Mutations in the C-clamp disrupt beta-catenin's regulation of the LEF1 promoter and impair TCF-1's role in colon cancer cell proliferation.
• E-tail TCF isoforms employ dual DNA-binding activities for precise regulation of Wnt target genes vital for cellular growth.