Mutations in the HNF1beta gene, encoding the dimeric POU-homeodomain transcription factor HNF1beta (TCF2 or vHNF1), cause various phenotypes including maturity onset diabetes of the young 5 (MODY5), and abnormalities in kidney, pancreas and genital tract development. To gain insight into the molecular mechanisms underlying these phenotypes and into the structure of HNF1beta, we functionally characterized eight disease-causing mutations predicted to produce protein truncations, amino acids substitutions or frameshift deletions in different domains of the protein. Truncated mutations, retaining the dimerization domain, displayed defective nuclear localization and weak dominant-negative activity when co-expressed with the wild-type protein. A frameshift mutation located within the C-terminal QSP-rich domain partially reduced transcriptional activity, whereas selective deletion of this domain abolished transactivation. All five missense mutations, which concern POU-specific and homeodomain residues, were correctly expressed and localized to the nucleus. Although having different effects on DNA-binding capacity, which ranged from complete loss to a mild reduction, these mutations exhibited a severe reduction in their transactivation capacity. The transcriptional impairment of those mutants, whose DNA-binding activity was weakly or not affected, correlated with the loss of association with one of the histone-acetyltransferases CBP or PCAF. In contrast to wild-type HNF1beta, whose transactivation potential depends on the synergistic action of CBP and PCAF, the activity of these mutants was not increased by the synergistic action of these two coactivators or by treatment with the specific histone-deacetylase inhibitor TSA. Our findings suggest that the complex syndrome associated with HNF1beta-MODY5 mutations arise from either defective DNA-binding or transactivation function through impaired coactivator recruitment.

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The seven-transmembrane receptor Smoothened is essential for hedgehog signal transduction. In adulthood, the highest density of Smoothened mRNA is found in the granule cell layer of the dentate gyrus. There, Smoothened expression is regulated by the synaptic activity involving the glutamatergic transmission. The precise localization of Smoothened proteins, however, has not yet been reported. Here, we describe Smoothened protein distribution in the hippocampal mossy fibers using specific Smoothened antibodies. We provide evidences for their presynaptic localization, and using electron microscopy, show that Smoothened is located in close association with synaptic vesicles and rarely with the plasma membrane. These findings demonstrate that Smoothened is localized presynaptically and suggest that Smoothened signal transduction may be implicated in the complex aspects of mossy fiber function.