ABSTRACT
The members of the TCF/LEF family of DNA-binding proteins are components of diverse gene regulatory networks. As nuclear effectors of Wnt/ß-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 K150 by CBP. K150 acetylation is restricted to TCF4E splice variants and requires the simultaneous presence of ß-catenin and the unique TCF4E C-terminus. To examine the functional consequences of K150 acetylation we substituted K150 with amino acids representing the non-acetylated and acetylated states. Reporter gene assays based on Wnt/ß-catenin-responsive promoter regions did not indicate a general role of K150 acetylation in transactivation by TCF4E. However, in the presence of CBP, non-acetylatable TCF4E with a K150R substitution was more susceptible to inhibition by the HBP-1 repressor protein compared to wild-type TCF4E. Acetylation of K150 using a bacterial expression system or amino acid substitutions at K150 alter the electrophoretic properties of TCF4E::DNA complexes. This result suggests that K150 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/ß-catenin signaling in response to changing intracellular signaling milieus.
Subject(s)
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , DNA/chemistry , DNA/metabolism , High Mobility Group Proteins/metabolism , Nucleic Acid Conformation , Repressor Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Acetylation , Amino Acid Sequence , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/chemistry , CREB-Binding Protein/metabolism , HEK293 Cells , Humans , Lysine/metabolism , Mice , Molecular Sequence Data , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Structure, Tertiary , Substrate Specificity , Transcription Factor 4 , Transcription Factors/chemistry , Transcriptional Activation , beta Catenin/metabolismABSTRACT
Long distance migration of differentiating granule cells from the cerebellar upper rhombic lip has been reported in many vertebrates. However, the knowledge about the subcellular dynamics and molecular mechanisms regulating directional neuronal migration in vivo is just beginning to emerge. Here we show by time-lapse imaging in live zebrafish (Danio rerio) embryos that cerebellar granule cells migrate in chain-like structures in a homotypic glia-independent manner. Temporal rescue of zebrafish Cadherin-2 mutants reveals a direct role for this adhesion molecule in mediating chain formation and coherent migratory behavior of granule cells. In addition, Cadherin-2 maintains the orientation of cell polarization in direction of migration, whereas in Cadherin-2 mutant granule cells the site of leading edge formation and centrosome positioning is randomized. Thus, the lack of adhesion leads to impaired directional migration with a mispositioning of Cadherin-2 deficient granule cells as a consequence. Furthermore, these cells fail to differentiate properly into mature granule neurons. In vivo imaging of Cadherin-2 localization revealed the dynamics of this adhesion molecule during cell locomotion. Cadherin-2 concentrates transiently at the front of granule cells during the initiation of individual migratory steps by intramembraneous transport. The presence of Cadherin-2 in the leading edge corresponds to the observed centrosome orientation in direction of migration. Our results indicate that Cadherin-2 plays a key role during zebrafish granule cell migration by continuously coordinating cell-cell contacts and cell polarity through the remodeling of adherens junctions. As Cadherin-containing adherens junctions have been shown to be connected via microtubule fibers with the centrosome, our results offer an explanation for the mechanism of leading edge and centrosome positioning during nucleokinetic migration of many vertebrate neuronal populations.
Subject(s)
Cadherins/metabolism , Cell Movement , Cerebellum/embryology , Neurons/physiology , Zebrafish Proteins/metabolism , Animals , Cadherins/genetics , Cell Adhesion , Cell Differentiation , Cell Polarity , Centrosome/metabolism , Cerebellum/cytology , Genes, Reporter , Green Fluorescent Proteins , Immunohistochemistry , Microscopy, Electron, Transmission , Neurons/cytology , Zebrafish/embryology , Zebrafish Proteins/geneticsABSTRACT
Taking advantage of a mutant estrogen receptor ligand binding domain (ER(T2)), we developed novel Caspase fusion proteins for inducible apoptosis. We show that Caspase-ER(T2) fusion proteins become specifically activated by the synthetic ligand 4-OH- tamoxifen and rapidly induce apoptotic cell death in human, murine, and zebrafish cells. This novel tool for targeted cell ablation greatly facilitates the generation of disease models as well as developmental and regeneration studies in model organisms.