The members of the TCF/LEF family of DNA-binding proteins are components of diverse gene regulatory networks. As nuclear effectors of Wnt/?-catenin signaling they act as assembly platforms for multimeric transcription complexes that either repress or activate gene expression. Previously, it was shown that several aspects of TCF/LEF protein function are regulated by post-translational modification. The association of TCF/LEF family members with acetyltransferases and deacetylases prompted us to investigate whether vertebrate TCF/LEF proteins are subject to acetylation. Through co-expression with p300 and CBP and subsequent analyses using mass spectrometry and immunodetection with anti-acetyl-lysine antibodies we show that TCF4 can be acetylated at lysine K??? by CBP. K??? acetylation is restricted to TCF4E splice variants and requires the simultaneous presence of ?-catenin and the unique TCF4E C-terminus. To examine the functional consequences of K??? acetylation we substituted K??? with amino acids representing the non-acetylated and acetylated states. Reporter gene assays based on Wnt/?-catenin-responsive promoter regions did not indicate a general role of K??? acetylation in transactivation by TCF4E. However, in the presence of CBP, non-acetylatable TCF4E with a K???R substitution was more susceptible to inhibition by the HBP-1 repressor protein compared to wild-type TCF4E. Acetylation of K??? using a bacterial expression system or amino acid substitutions at K??? alter the electrophoretic properties of TCF4E::DNA complexes. This result suggests that K??? acetylation leads to a conformational change that may also represent the mechanism whereby acetylated TCF4E acquires resistance against HBP1. In summary, TCF4 not only recruits acetyltransferases but is also a substrate for these enzymes. The fact that acetylation affects only a subset of TCF4 splice variants and is mediated preferentially by CBP suggests that the conditional acetylation of TCF4E is a novel regulatory mechanism that diversifies the transcriptional output of Wnt/?-catenin signaling in response to changing intracellular signaling milieus.
Alternative splicing can produce multiple protein products with variable domain composition from a single gene. The mouse Tcf7l2 gene is subject to alternative splicing. It encodes TCF4, a member of the T-cell factor (TCF) family of DNA-binding proteins and a nuclear interaction partner of beta-catenin which performs essential functions in Wnt growth factor signalling. Multiple TCF4 isoforms, potentially exhibiting cell-type-specific distribution and differing in gene regulatory properties, could strongly influence tissue-specific Wnt responses. Therefore, we have examined mouse Tcf7l2 splice variants in neonatal tissues, embryonic stem cells and neural progenitors. By polymerase chain reaction amplification, cloning and sequencing, we identify a large number of alternatively spliced transcripts and report a highly flexible combinatorial repertoire of alternative exons. Many, but not all of the variants exhibit a broad tissue distribution. Moreover, two functionally equivalent versions of the C-clamp, thought to represent an auxiliary DNA-binding domain, were identified. Depending upon promoter context and precise domain composition, TCF4 isoforms exhibit strikingly different transactivation potentials at natural Wnt/beta-catenin target promoters. However, differences in C-clamp-mediated DNA binding can only partially explain functional differences among TCF4 variants. Still, the cell-type-specific complement of TCF4 isoforms is likely to be a major determinant for the context-dependent transcriptional output of Wnt/beta-catenin signalling.
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