A gene encoding a fluorescent protein from the stony coral Lobophyllia hemprichii has been cloned in Escherichia coli and characterized by biochemical and biophysical methods. The protein, which we named EosFP, emits strong green fluorescence (516 nm) that changes to red (581 nm) upon near-UV irradiation at Ϸ390 nm because of a photo-induced modification involving a break in the peptide backbone next to the chromophore. Single-molecule fluorescence spectroscopy shows that the wild type of EosFP is tetrameric, with strong Fö rster resonance coupling among the individual fluorophores. We succeeded in breaking up the tetramer into AB and AC subunit dimers by introducing the single point mutations V123T and T158H, respectively, and the combination of both mutations yielded functional monomers. Fusion constructs with a variety of proteins were prepared and expressed in human cells, showing that normal biological functions were retained. The possibility to locally change the emission wavelength by focused UV light makes EosFP a superb marker for experiments aimed at tracking the movements of biomolecules within the living cell.
. The Notch gene encodes a transmembrane receptor that gave the name to the evolutionary highly conserved Notch signaling cascade. It plays a pivotal role in the regulation of many fundamental cellular processes such as proliferation, stem cell maintenance and differentiation during embryonic and adult development. After specific ligand binding, the intracellular part of the Notch receptor is cleaved off and translocates to the nucleus, where it binds to the transcription factor RBP-J. In the absence of activated Notch, RBP-J represses Notch target genes by recruiting a corepressor complex. Here, we review Notch signaling with a focus on gene regulatory events at Notch target genes. This is of utmost importance to understand Notch signaling since certain RBP-J associated cofactors and particular epigenetic marks determine the specificity of Notch target gene expression in different cell types. We subsequently summarize the current knowledge about Notch target genes and the physiological significance of Notch signaling in development and cancer.
Notch proteins are the receptors for an evolutionarily highly conserved signalling pathway that regulates numerous cell fate decisions during development. Signal transduction involves the presenilin‐dependent intracellular processing of Notch and nuclear translocation of the intracellular domain of Notch, Notch‐IC. Notch‐IC associates with the DNA‐binding protein RBP‐Jκ/CBF‐1 to activate transcription of Notch target genes. In the absence of Notch signalling, RBP‐Jκ/CBF‐1 acts as a transcriptional repressor through the recruitment of histone deacetylase (HDAC) corepressor complexes. We identified SHARP as an RBP‐Jκ/CBF‐1‐interacting corepressor in a yeast two‐hybrid screen. In cotransfection experiments, SHARP‐mediated repression was sensitive to the HDAC inhibitor TSA and facilitated by SKIP, a highly conserved SMRT and RBP‐Jκ‐interacting protein. SHARP repressed Hairy/Enhancer of split (HES)‐1 promoter activity, inhibited Notch‐1‐mediated transactivation and rescued Notch‐1‐induced inhibition of primary neurogenesis in Xenopus laevis embryos. Based on our data, we propose a model in which SHARP is a novel component of the HDAC corepressor complex, recruited by RBP‐Jκ to repress transcription of target genes in the absence of activated Notch.
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