Abstract
Some members of the Wnt family of extracellular glycoproteins regulate target gene expression by inducing stabilization and nuclear accumulation of beta-catenin, which functions as a transcriptional activator after binding to transcription factors of the T-cell factor/lymphoid enhancer factor (TCF/LEF) family. Three different members of this family have been identified in Xenopus laevis thus far that differ in their ability to influence mesodermal differentiation and to activate expression of the Wnt target gene fibronectin. Here we report on the isolation and characterization of additional variants of XTCF-4. We show that the differential ability of these proteins and other members of the TCF family to activate target genes is neither due to preferential interaction with transcriptional cofactors of the groucho family or SMAD4 nor to different DNA binding affinities. Expression of these proteins in an epithelial cell line reveals differences in their ability to form a ternary complex with DNA and beta-catenin. Interestingly, formation of this ternary complex was not sufficient to activate target gene expression as previously thought. Our experiments identify two amino acid sequence motifs, LVPQ and SFLSS, in the central domain of XTCF-4 that regulate the formation of the DNA-TCF-beta-catenin complex or activation of target genes, respectively. Biochemical studies reveal that the phosphorylation state of these XTCF-4 variants correlates with their ability to form a ternary complex with beta-catenin and DNA but not to activate target gene expression. The described variants of XTFC-4 with their different properties in complex formation provide strong evidence that in addition to the regulation of beta-catenin stability the isoforms of TCF/LEF transcription factors and their posttranslational modifications define the cellular response of a Wnt/wingless signal.
Highlights
Wnt-1 was originally identified as a proto-oncogene in mice [1] and was found to be a vertebrate homolog of the Drosophila segment polarity gene wingless
Given the similarity with respect to the LVPQ and SFLSS motifs between XTFC-4A and XTCF-3 on the one hand and XTCF-4C and mLEF-1 on the other, we investigated the ability of XTCF-4A and-C to activate target gene expression
By using different biochemical approaches, we provide evidence that these differences are neither due to preferential interaction with corepressors of the groucho family or CtBP nor to differential binding of the transcriptional activator SMAD4
Summary
Wnt-1 was originally identified as a proto-oncogene in mice [1] and was found to be a vertebrate homolog of the Drosophila segment polarity gene wingless. The identification of c-myc as a target gene of TCF-4/-catenin links Wnt signaling with cell proliferation and with carcinogenesis [5]. Another important target gene of TCF/LEF in this context leading to cell cycle deregulation was identified as cyclin D1 [6]. Sequencespecific DNA binding is mediated by the HMG box, and the target motif of these factors is given by the sequence (C/G)TTTG(A/T)(A/T) [7] Members of this family are unable to activate transcription of reporter gene constructs that carry multiple copies of their minimal binding motifs [8] and lack activation properties on their own. The contradictory results may be explained by different splice variants of LEF-1 that were used by Roose et al [22] and Levanon et al [24], respectively
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