Structure-Function Analysis of the C-clamp of TCF/Pangolin in Wnt/ß-catenin Signaling

Abstract

The evolutionarily conserved Wnt/ß-catenin (Wnt/ß-cat) pathway plays an important role in animal development in metazoans. Many Wnt targets are regulated by members of the TCF/LEF1 (TCF) family of transcription factors. All TCFs contain a High Mobility Group (HMG) domain that bind specific DNA sequences. Invertebrate TCFs and some vertebrate TCF isoforms also contain another domain, called the C-clamp, which allows TCFs to recognize an additional DNA motif known as the Helper site. While the C-clamp has been shown to be important for regulating several Wnt reporter genes in cell culture, its physiological role in regulating Wnt targets is less clear. In addition, little is known about this domain, except that two of the four conserved cysteines are functionally important. Here, we carried out a systematic mutagenesis and functional analysis of the C-clamp from the Drosophila TCF/Pangolin (TCF/Pan) protein. We found that the C-clamp is a zinc-binding domain that is sufficient for binding to the Helper site. In addition to this DNA-binding activity, the C-clamp also inhibits the HMG domain from binding its cognate DNA site. Point mutations were identified that specifically affected DNA-binding or reduced the inhibitory effect. These mutants were characterized in TCF/Pan rescue assays. The specific DNA-binding activity of the C-clamp was essential for TCF/Pan function in cell culture and in patterning the embryonic epidermis of Drosophila, demonstrating the importance of this C-clamp activity in regulating Wnt target gene expression. In contrast, the inhibitory mutation had a subtle effect in cell culture and no effect on TCF/Pan activity in embryos. These results provide important information about the functional domains of the C-clamp, and highlight its importance for Wnt/ß-cat signaling in Drosophila.

Bipartite Recognition of DNA by TCF/Pangolin Is Remarkably Flexible and Contributes to Transcriptional Responsiveness and Tissue Specificity of Wingless Signaling

“…Previous work has shown that TCFs containing C-clamp domains recognize two distinct DNA sequence motifs, HMG sites (via the HMG domain) and Helper sites (via the C-clamp) [44]–[46], [48], [78]. The close proximity of these motifs suggested that they act as HMG-Helper site pairs, which we confirmed through site-directed mutagenesis (Figure 1C).…”

An embryonic system to assess Wnt transcriptional targets

“…The opposite condition was the expression of a dominant-negative allele (tcfΔN-short). This form of TCF lacks the Arm binding region, the Groucho binding sequence (GBS) and the C-clamp that are required for β-catenin binding, Gro repressor binding, and additional target specificity respectively (Supplemental Figure 1) 15,26 . This makes tcfΔN-short a strong dominant negative form of TCF as it is unable to release DNA, interact with Gro or recruit the transactivating domain of β-catenin effectively blocking both activation and de-repression and leading to small, un-patterned embryos (Fig.…”

Wnt Signaling Polarizes C. elegans Asymmetric Cell Divisions During Development

“…Conversely, in cells where basal target gene expression level is low, POP-1 derepression must be coupled with POP-1 activation via SYS-1 (Kidd et al, 2005, Shetty et al, 2005). In addition to basal transcription factors, POP-1 target genes are influenced by a second “Helper” DNA sequence that binds to a distinct POP-1 DNA binding domain termed the C-clamp to specifically facilitate transcriptional activation (Bhambhani et al, 2014, Ravindranath and Cadigan, 2014). The C-clamp and Helper DNA sequences are dispensable for target gene repression, however.…”