Franz Oswald

The Notch signaling pathway: Transcriptional regulation at Notch target genes

Abstract.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.

NF-κB2 Is a Putative Target Gene of Activated Notch-1 via RBP-Jκ

ABSTRACT NF-κB2 (p100/p52), a member of the NF-κB/Rel family of transcription factors, is involved in the regulation of a variety of genes important for immune function. Previously, we have shown that the NF-κB2 gene is regulated in a positive and a negative manner. Two κB elements within the NF-κB2 promoter mediate tumor necrosis factor alpha-inducible transactivation. In addition, we have shown that there exists a transcriptional repression in the absence of NF-κB. To identify a DNA binding activity responsible for this transcriptional repression, we have partially purified a nuclear complex, named Rep-κB. Here we further analyze this putative repressive binding activity. Detailed examination of Rep-κB–DNA interaction revealed the sequence requirements for binding to be almost identical to those of recombination signal binding protein Jκ (RBP-Jκ), the mammalian homolog of the protein encoded by Drosophila suppressor of hairless [Su(H)]. In addition, in electromobility shift assays, Rep-κB binding activity is recognized by an antibody directed against RBP-Jκ. By performing transient-transfection assays, we show that human RBP-Jκ represses basal as well as RelA (p65)-stimulated NF-κB2 promoter activity. Studies in Drosophila melanogaster have shown that Su(H) is implicated in the Notch signaling pathway regulating cell fate decisions. In transient-transfection assays we show that truncated Notch-1 strongly induces NF-κB2 promoter activity. In summary, our data clearly demonstrate that Rep-κB is closely related or identical to RBP-Jκ. RBP-Jκ is a strong transcriptional repressor of NF-κB2. Moreover, this repression can be overcome by activated Notch-1, suggesting that NF-κB2 is a novel putative Notch target gene.

p300 Acts as a Transcriptional Coactivator for Mammalian Notch-1

ABSTRACT Notch-1 belongs to a family of transmembrane receptor proteins that direct the decisions as to various cell fates. After ligand binding, a proteolytic cleavage step occurs and the intracellular part of Notch-1, Notch-1-IC, translocates into the nucleus, where it targets the DNA binding protein RBP-Jκ/CBF1. RBP-Jκ mediates repression through recruitment of a histone deacetylase-containing complex. The Notch-1-IC/RBP-Jκ complex overcomes repression and activates the transcription of Notch target genes. We have identified a novel domain in Notch-1-IC, the EP domain, which is indispensable for full transcriptional activation. This transactivation domain is localized adjacent to the ankyrin repeats of Notch-1-IC. In cotransfection experiments, Notch-1-IC-mediated transcriptional activation was inhibited by E1A12S and p53, two proteins, which interfere with the function of the common coactivator p300. Protein-protein interaction assays demonstrated the association of Notch-1-IC and the CH3 region of p300. In addition, the interaction of mammalian Notch-1-IC with p300 was destabilized after deletion of the EP domain of Notch-1-IC. Based on physical interaction with Notch-1-IC and coactivator functions of p300, we propose a model for Notch-1-mediated gene regulation via p300.

SHARP is a novel component of the Notch/RBP-Jκ signalling pathway

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.

Fluorescent proteins for live cell imaging: Opportunities, limitations, and challenges

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria can be used as a genetically encoded fluorescence marker due to its autocatalytic formation of the chromophore. In recent years, numerous GFP‐like proteins with emission colors ranging from cyan to red were discovered in marine organisms. Their diverse molecular properties enabled novel approaches in live cell imaging but also impose certain limitations on their applicability as markers. In this review, we give an overview of key structural and functional properties of fluorescent proteins that should be considered when selecting a marker protein for a particular application and also discuss challenges that lie ahead in the further optimization of the glowing probes.

RBP-Jκ/SHARP Recruits CtIP/CtBP Corepressors To Silence Notch Target Genes

ABSTRACT Notch is a transmembrane receptor that determines cell fates and pattern formation in all animal species. After ligand binding, proteolytic cleavage steps occur and the intracellular part of Notch translocates to the nucleus, where it targets the DNA-binding protein RBP-Jκ/CBF1. In the absence of Notch, RBP-Jκ represses Notch target genes through the recruitment of a corepressor complex. We and others have identified SHARP as a component of this complex. Here, we functionally demonstrate that the SHARP repression domain is necessary and sufficient to repress transcription and that the absence of this domain causes a dominant negative Notch-like phenotype. We identify the CtIP and CtBP corepressors as novel components of the human RBP-Jκ/SHARP-corepressor complex and show that CtIP binds directly to the SHARP repression domain. Functionally, CtIP and CtBP augment SHARP-mediated repression. Transcriptional repression of the Notch target gene Hey1 is abolished in CtBP-deficient cells or after the functional knockout of CtBP. Furthermore, the endogenous Hey1 promoter is derepressed in CtBP-deficient cells. We propose that a corepressor complex containing CtIP/CtBP facilitates RBP-Jκ/SHARP-mediated repression of Notch target genes.

mRuby, a Bright Monomeric Red Fluorescent Protein for Labeling of Subcellular Structures

A monomeric variant of the red fluorescent protein eqFP611, mRuby, is described. With excitation and emission maxima at 558 nm and 605 nm, respectively, and a large Stokes shift of 47 nm, mRuby appears particularly useful for imaging applications. The protein shows an exceptional resistance to denaturation at pH extremes. Moreover, mRuby is about ten-fold brighter compared to EGFP when being targeted to the endoplasmic reticulum. The engineering process of eqFP611 revealed that the C-terminal tail of the protein acts as a natural peroxisomal targeting signal (PTS). Using an mRuby variant carrying the eqFP611-PTS, we discovered that ordered inheritance of peroxisomes is widespread during mitosis of different mammalian cell types. The ordered partitioning is realized by the formation of peroxisome clusters around the poles of the mitotic spindle and ensures that equal numbers of the organelle are inherited by the daughter cells. The unique spectral properties make mRuby the marker of choice for a multitude of cell biological applications. Moreover, the use of mRuby has allowed novel insights in the biology of organelles responsible for severe human diseases.

Histone demethylase KDM5A is an integral part of the core Notch–RBP-J repressor complex

Timely acquisition of cell fates and the elaborate control of growth in numerous organs depend on Notch signaling. Upon ligand binding, the core transcription factor RBP-J activates transcription of Notch target genes. In the absence of signaling, RBP-J switches off target gene expression, assuring the tight spatiotemporal control of the response by a mechanism incompletely understood. Here we show that the histone demethylase KDM5A is an integral, conserved component of Notch/RBP-J gene silencing. Methylation of histone H3 Lys 4 is dynamically erased and re-established at RBP-J sites upon inhibition and reactivation of Notch signaling. KDM5A interacts physically with RBP-J; this interaction is conserved in Drosophila and is crucial for Notch-induced growth and tumorigenesis responses.

Photoconvertible Fluorescent Protein EosFP: Biophysical Properties and Cell Biology Applications

Abstract EosFP is a fluorescent protein from the coral Lobophyllia hemprichii that changes its fluorescence emission from green to red upon irradiation with near-UV light. Here we present the spectroscopic properties of wild-type EosFP and a variety of monomeric and dimeric mutants and provide a structural interpretation of its oligomerization and photoconversion, which is based on X-ray structure analysis of the green and red species that we reported recently. Because functional expression of the monomeric EosFP variant is limited to temperatures of 30°C, we have developed a tandem dimer. This construct, in which two EosFP subunits are connected by a flexible 12 amino acid linker, expresses well after fusion with the androgen and endothelin A receptors at 37°C. A variety of applications in cellular imaging, developmental biology and automated high-content screening applications are presented, which demonstrate that EosFP is a powerful tool for in vivo monitoring of cellular processes.

CD147 silencing inhibits lactate transport and reduces malignant potential of pancreatic cancer cells in in vivo and in vitro models

Background: CD147 (basigin, EMMPRIN) is a multifunctional, highly conserved glycoprotein enriched in pancreatic ductal adenocarcinomas (PDACs) which is associated with poor prognosis in many malignancies. The role of CD147 in pancreatic cancer, however, remains elusive. Methods and Results: Silencing of CD147 by RNA interference (RNAi) reduced the proliferation rate of MiaPaCa2 and Panc1 cells. CD147 is required for the function and expression of the monocarboxylate transporters MCT1 and MCT4 that are expressed in human PDAC cells as demonstrated by real-time reverse transcription-PCR (RT-PCR) as well as immunohistology. MCT1 and MCT4 are the natural transporters of lactate, and MiaPaCa2 cells exhibited a high rate of lactate production, which is characteristic for the Warburg effect, an early hallmark of cancer that confers a significant growth advantage. Further induction of lactate production by sodium azide in MiaPaCa2 cells increased MCT1 as well as MCT4 expression. CD147 silencing inhibited the expression and function of MCT1 and MCT4 and resulted in an increased intracellular lactate concentration. Addition of exogenous lactate inhibited cancer cell growth in a dose-dependent fashion. In vivo, knock-down of CD147 in MiaPaCa2 cells by inducible short hairpin RNA (shRNA)-mediated CD147 silencing reduced invasiveness through the chorioallantoic membrane of chick embryos (CAM assay) and inhibited tumourigenicity in a xenograft model in nude mice. Conclusion : The function of CD147 as an ancillary protein that is required to sustain the expression and function of MCT1 and MCT4 is involved in the association of CD147 expression with the malignant potential of pancreatic cancer cells exhibiting the Warburg effect.