http://repub.eur.nl/res/aut/12090/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/35185/ 2007-10-01T00:00:00Z http://repub.eur.nl/res/pub/10281/ 2004-01-01T00:00:00Z The thymus is seeded by very small numbers of progenitor cells that undergo massive proliferation before differentiation and rearrangement of TCR genes occurs. Various signals mediate proliferation and differentiation of these cells, including Wnt signals. Wnt signals induce the interaction of the cytoplasmic cofactor beta-catenin with nuclear T cell factor (TCF) transcription factors. We identified target genes of the Wnt/beta-catenin/TCF pathway in the most immature (CD4-CD8-CD34+) thymocytes using Affymetrix DNA microarrays in combination with three different functional assays for in vitro induction of Wnt signaling. A relatively small number (approximately 30) of genes changed expression, including several proliferation-inducing transcription factors such as c-fos and c-jun, protein phosphatases, and adhesion molecules, but no genes involved in differentiation to mature T cell stages. The adhesion molecules likely confine the proliferating immature thymocytes to the appropriate anatomical sites in the thymus. For several of these target genes, we validated that they are true Wnt/beta-catenin/TCF target genes using real-time quantitative PCR and reporter gene assays. The same core set of genes was repressed in Tcf-1-null mice, explaining the block in early thymocyte development in these mice. In conclusion, Wnt signals mediate proliferation and cell adhesion, but not differentiation of the immature thymic progenitor pool. http://repub.eur.nl/res/pub/13000/ 2002-02-26T00:00:00Z beta-catenin mediates Wnt signaling by acting as the essential co-activator for TCF transcription factors. Wnt signaling increases the half-life and therefore the absolute level of beta-catenin in responding cells. The current model states that these changes in beta-catenin stability set the threshold for Wnt signaling. However, we find that pharmacological inhibition of proteasome activity by ALLN leads to accumulation of cytosolic beta-catenin but not to increased TCF-mediated transcription. In addition, in temperature-sensitive ubiquitylation mutant CHO cells inhibition of ubiquitylation increases beta-catenin levels, but does not induce transcriptional activation of TCF reporter genes. Using an antibody specific for beta-catenin dephosphorylated at residues Ser37 and Thr41, we show that Wnt signals specifically increase the levels of dephosphorylated beta-catenin, whereas ALLN does not. We conclude that changes in the phosphorylation status of the N-terminus of beta-catenin that occur upon Wnt signaling independently affect the signaling properties and half-life of beta-catenin. Hence, Wnt signals are transduced via N-terminally dephosphorylated beta-catenin.