SUMMARY Specific recognition of DNA by transcription factors is essential for precise gene regulation. In Wingless (Wg) signaling in Drosophila, target gene regulation is controlled by TCF, which binds to specific DNA sequences through a HMG domain [1]. However, there is considerable degeneracy in what constitutes a TCF binding site [2–5], raising the possibility that it is not sufficient for target location. Some isoforms of human TCF contain a domain termed the C-clamp that mediates binding to an extended sequence in vitro [6]. However, the significance of this extended sequence for the function of Wnt response elements (WREs) is unclear. In this report, we identified a new cis-regulatory element named the TCF Helper site (Helper site) that is essential for activation of several WREs. This motif greatly augments the ability of TCF binding sites to respond to Wg signaling. Drosophila TCF contains a C-clamp that enhances in vitro binding to TCF-Helper site pairs and is required for transcriptional activation of WREs containing Helper sites. A genome-wide search for clusters of TCF and Helper sites identified two new WREs. Our data suggest that DNA recognition by fly TCF occurs through a bipartite mechanism involving both the HMG domain and C-clamp, which enables TCF to locate and activate WREs in the nucleus.
To package a cell's genetic material into the relatively small space provided by nuclei, linear DNA molecules are tightly wrapped around histone proteins, forming nucleosomes that are then packed together to form a higher-order structure called chromatin. This highly compacted DNA-protein assembly efficiently packages DNA but also acts as a steric barrier to transcription factors and other proteins that need to access generegulatory regions (45).
Signaling pathways usually activate transcriptional targets in a cell type-specific manner. Notable exceptions are pathway-specific feedback antagonists, which serve to restrict the range or duration of the signal. These factors are often activated by their respective pathways in a broad array of cell types. For example, the Wnt ligand Wingless (Wg) activates the naked cuticle (nkd) gene in all tissues examined throughout Drosophila development. How does the nkd gene respond in such an unrestricted manner to Wg signaling? Analysis in cell culture revealed regions of the nkd locus that contain Wg response elements (WREs) that are directly activated by the pathway via the transcription factor TCF. In flies, Wg signaling activates these WREs in multiple tissues, in distinct but overlapping patterns. These WREs are necessary and largely sufficient for nkd expression in late stage larval tissues, but only contribute to part of the embryonic expression pattern of nkd. These results demonstrate that nkd responsiveness to Wg signaling is achieved by several WREs which are broadly (but not universally) activated by the pathway. The existence of several WREs in the nkd locus may have been necessary to allow the Wg signaling-Nkd feedback circuit to remain intact as Wg expression diversified during animal evolution.
C‐terminal‐binding protein (CtBP) is a well‐characterized transcriptional co‐repressor that requires homo‐dimerization for its activity. CtBP can both repress and activate Wingless nuclear targets in Drosophila. Here, we examine the role of CtBP dimerization in these opposing processes. CtBP mutants that cannot dimerize are able to promote Wingless signalling, but are defective in repressing Wingless targets. To further test the role of dimerization in repression, the positions of basic and acidic residues that form inter‐molecular salt bridges in the CtBP dimerization interface were swapped. These mutants cannot homo‐dimerize and are compromised for repression. However, their co‐expression leads to hetero‐dimerization and consequent repression of Wingless targets. Our results support a model where CtBP is a gene‐specific regulator of Wingless signalling, with some targets requiring CtBP dimers for inhibition while other targets utilize CtBP monomers for activation of their expression. Functional interactions between CtBP and Pygopus, a nuclear protein required for Wingless signalling, support a model where monomeric CtBP acts downstream of Pygopus in activating some Wingless targets.
The highly conserved Wingless/Wnt signaling pathway controls many developmental processes by regulating the expression of target genes, most often through members of the TCF family of DNA-binding proteins. In the absence of signaling, many of these targets are silenced, by mechanisms involving TCFs that are not fully understood. Here we report that the chromatin remodeling proteins ISWI and ACF1 are required for basal repression of Wg target genes in Drosophila. This regulation is not due to global repression by ISWI and ACF1 and is distinct from their previously reported role in chromatin assembly. While ISWI is localized to the same regions of Wingless target gene chromatin as TCF, we find that ACF1 binds much more broadly to target loci. This broad distribution of ACF1 is dependent on ISWI. ISWI and ACF1 are required for TCF binding to chromatin, while a TCF-independent role of ISWI-ACF1 in repression of Wingless targets is also observed. Finally, we show that Wingless signaling reduces ACF1 binding to Wg targets, and ISWI and ACF1 regulate repression by antagonizing histone H4 acetylation. Our results argue that Wg signaling activates target gene expression partly by overcoming the chromatin barrier maintained by ISWI and ACF1.
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