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

Abstract

The T-cell factor (TCF) family of transcription factors are major mediators of Wnt/β-catenin signaling in metazoans. All TCFs contain a High Mobility Group (HMG) domain that possesses specific DNA binding activity. In addition, many TCFs contain a second DNA binding domain, the C-clamp, which binds to DNA motifs referred to as Helper sites. While HMG and Helper sites are both important for the activation of several Wnt dependent cis-regulatory modules (W-CRMs), the rules of what constitutes a functional HMG-Helper site pair are unknown. In this report, we employed a combination of in vitro binding, reporter gene analysis and bioinformatics to address this question, using the Drosophila family member TCF/Pangolin (TCF/Pan) as a model. We found that while there were constraints for the orientation and spacing of HMG-Helper pairs, the presence of a Helper site near a HMG site in any orientation increased binding and transcriptional response, with some orientations displaying tissue-specific patterns. We found that altering an HMG-Helper site pair from a sub-optimal to optimal orientation/spacing dramatically increased the responsiveness of a W-CRM in several fly tissues. In addition, we used the knowledge gained to bioinformatically identify two novel W-CRMs, one that was activated by Wnt/β-catenin signaling in the prothoracic gland, a tissue not previously connected to this pathway. In sum, this work extends the importance of Helper sites in fly W-CRMs and suggests that the type of HMG-Helper pair is a major factor in setting the threshold for Wnt activation and tissue-responsiveness.

TCF7L1 and TCF7 differentially regulate specific mouse ES cell genes in response to GSK-3 inhibition

“…Presumably, 3xFLAG-TCF7 outcompetes 3xFLAG-TCF7L1 for binding, even though 3xFLAG-TCF7L1 protein is more abundant. Indeed, so called 'E-tail' isoforms of TCF7 and TCF7L2, contain a cysteine-clamp that binds to a secondary DNA motif referred to as a 'helper site', which is essential for TCF7/TCF7L2-mediated repression and Wnt responsiveness (23,28,29). We observed multiple helper sites associated with these particular Wnt target genes.…”

“…In support of this notion, in mouse hair follicle stem cells, TCF7L1 and the E-tail containing TCF7L2, demonstrated a significant amount of overlap in co-bound genes (30). Mechanistically, this could be attributed to suboptimal helper site configurations at specific Wnt target genes (28). The genomic binding profile of TCF7L1 was the first of the TCF/LEFs to be characterized in mESCs, demonstrating a large overlap in binding with master transcriptional regulators of pluripotency, namely POU5F1, SOX2, and NANOG (11,12).…”

The Role of the C-Clamp in Wnt-Related Colorectal Cancers

“…Helper-like sites have also been found in the promoter regions of several other human Wnt targets [42,53], including genes (e.g., SP5 , CDX2, and MYC ) that are upregulated in colorectal cancer (CRC) [49,53]. Taken together, the data support a model where the DNA binding specificity of TCFs is enhanced by the presence of a C-clamp, which allows bipartite recognition via HMG domain–HMG site and C-clamp–Helper site interactions [2,55]. …”

Wingless Signaling: A Genetic Journey from Morphogenesis to Metastasis

“…Approximate positions of wg mutations are indicated by stars (those implicated in Wg protein transport are within the blue box), and the position of the nonconserved insert region of Wg is indicated in gray (van den Heuvel et al 1993;Bejsovec and Wieschaus 1995;Dierick and Bejsovec 1998). R = A/G) that is bound by a separate DNA-binding domain in Tcf, called the C-clamp Parker et al 2008;Archbold et al 2014).…”