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

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. © 2014 archbold et al.