Anette Preiss

Transcriptional repression by Suppressor of Hairless involves the binding of a Hairless-dCtBP complex in Drosophila

Notch is the receptor for a conserved signaling pathway that regulates numerous cell fate decisions during development [1]. Signal transduction involves the presenilin-dependent intracellular processing of Notch and the nuclear translocation of the intracellular domain of Notch, NICD [2-6]. NICD associates with Suppressor of Hairless [Su(H)], a DNA binding protein, and Mastermind (Mam), a transcriptional coactivator [7-9]. In the absence of Notch signaling, Su(H) acts as a transcriptional repressor [10, 11]. Repression by Su(H) is relieved by the activation of Notch [12-16]. In the Drosophila embryo, this transcriptional switch from repression to activation is important for patterning the expression of the single-minded (sim) gene along the dorsoventral axis [12]. Here, we investigate the mechanisms by which Su(H) inhibits the expression of Notch target genes in Drosophila. We show that Hairless, an antagonist of Notch signaling [17-19], is required to repress the transcription of the sim gene. Hairless forms a DNA-bound complex with Su(H). Furthermore, it directly binds the Drosophila C-terminal Binding Protein (dCtBP), which acts as a transcriptional corepressor. The dCtBP binding motif of Hairless is essential for the function of Hairless in vivo. We propose that Hairless mediates transcriptional repression by Su(H) via the recruitment of dCtBP.

Hairless-Mediated Repression of Notch Target Genes Requires the Combined Activity of Groucho and CtBP Corepressors

ABSTRACT Notch signal transduction centers on a conserved DNA-binding protein called Suppressor of Hairless [Su(H)] in Drosophila species. In the absence of Notch activation, target genes are repressed by Su(H) acting in conjunction with a partner, Hairless, which contains binding motifs for two global corepressors, CtBP and Groucho (Gro). Usually these corepressors are thought to act via different mechanisms; complexed with other transcriptional regulators, they function independently and/or redundantly. Here we have investigated the requirement for Gro and CtBP in Hairless-mediated repression. Unexpectedly, we find that mutations inactivating one or the other binding motif can have detrimental effects on Hairless similar to those of mutations that inactivate both motifs. These results argue that recruitment of one or the other corepressor is not sufficient to confer repression in the context of the Hairless-Su(H) complex; Gro and CtBP need to function in combination. In addition, we demonstrate that Hairless has a second mode of repression that antagonizes Notch intracellular domain and is independent of Gro or CtBP binding.

The gooseberry-zipper region of Drosophila: five genes encode different spatially restricted transcripts in the embryo.

Genetic analysis of the Drosophila chromosome region 60 E9‐F1 identified two functions affecting embryonic development; gooseberry (gsb), a segment polarity gene, and zipper (zip), an unclassified gene which affects cuticle formation severely. By contrast, molecular analysis revealed five genes with different temporal and spatial patterns of expression in the embryo. Candidate genes for gsb and zip functions were identified. Two adjacent genes are eventually expressed in regular stripes within the posterior region of each segment. One of them is expressed initially in a pair‐rule mode; the second gene expresses reduced levels of transcripts in a mutant which leaves the transcribed region and the sequences up to the second gene intact. This observation, the patterns of transcripts in the embryo and the genetic data suggest that both genes are involved in gooseberry segmentation function. zip is expressed in neural tissue and not in epidermal anlagen. Embryos lacking zip activity also develop abnormal neural tissue consistent with the argument that the zip cuticle phenotype is a secondary effect. Additional newly identified genes are expressed in specific domains of the embryo, covering mesoderm anlagen and the dorsal region of embryos at blastoderm stage, respectively.

The Enhancer of split complex of Drosophila melanogaster harbors three classes of Notch responsive genes.

Many cell fate decisions in higher animals are based on intercellular communication governed by the Notch signaling pathway. Developmental signals received by the Notch receptor cause Suppressor of Hairless (Su(H)) mediated transcription of target genes. In Drosophila, the majority of Notch target genes known so far is located in the Enhancer of split complex (E(spl)-C), encoding small basic helix-loop-helix (bHLH) proteins that presumably act as transcriptional repressors. Here we show that the E(spl)-C contains three additional Notch responsive, non-bHLH genes: m4 and ma are structurally related, whilst m2 encodes a novel protein. All three genes depend on Su(H) for initiation and/or maintenance of transcription. The two other non-bHLH genes within the locus, m1 and m6, are unrelated to the Notch pathway: m1 might code for a protease inhibitor of the Kazal family, and m6 for a novel peptide.

Two isoforms of the Notch antagonist Hairless are produced by differential translation initiation

The Notch-signaling pathway controls cellular differentiation, including proliferation and cell death in all higher metazoans (including flies and men). Signal transduction through activated Notch involves the CSL group of transcriptional regulators. Notch signals need to be tightly regulated, and in Drosophila they are antagonized by the Hairless (H) protein. H silences the activity of Notch target genes by transforming the Drosophila CSL protein, Suppressor of Hairless [Su(H)], from a transcriptional activator into a repressor while recruiting one of the corepressors dCtBP or Groucho. The H protein has a calculated molecular mass of ≈110 kDa and contains several functional domains apart from the two small corepressor-binding domains. However, although there is no indication for alternative splicing, two Hairless protein isoforms, Hp120 and Hp150, are observed throughout development. Here, we show that the smaller isoform derives from an internal ribosome entry site (IRES) within the ORF. The IRES is active in a heterologous assay and contains an essential, conserved structural element. The two Hairless isoforms have residual activity in vivo which is, however, reduced compared to a combination of both, which implies that both protein isoforms are necessary for WT function. In larval tissues, translation of the two isoforms is cell-cycle regulated: whereas the Hp150 isoform is translated during interphase, Hp120 is enriched during mitosis. Thus, the presence of either H isoform throughout the cell cycle allows efficient inhibition of Notch-regulated cell proliferation.

Subcellular localization of Hairless protein shows a major focus of activity within the nucleus

Hairless, a major antagonist of the Notch signaling-pathway in Drosophila (Bang and Posakony, 1992; Maier et al., 1992), associates with Suppressor of Hairless [Su(H)], thereby inhibiting trans-activation of Notch target genes (Brou et al., 1994). These molecular interactions could occur either at the step of signal transduction in the cytoplasm or during implementation of the signal within the nucleus. We examined the subcellular distribution of Hairless, showing that it is a low abundant, ubiquitous protein that is cytosolic as well as nuclear. High levels of Hairless cause nuclear retention of Su(H), loss of Hairless reduces the amount of Su(H) in the nucleus.

Genetic Modifier Screens on Hairless Gain-of-Function Phenotypes Reveal Genes Involved in Cell Differentiation, Cell Growth and Apoptosis in Drosophila melanogaster

Overexpression of Hairless (H) causes a remarkable degree of tissue loss and apoptosis during imaginal development. H functions as antagonist in the Notch-signaling pathway in Drosophila, and the link to growth and apoptosis is poorly understood. To further our insight into H-mediated apoptosis, we performed two large-scale screens for modifiers of a small rough eye phenotype caused by H overexpression. Both loss- and gain-of-function screens revealed known and new genetic interactors representing diverse cellular functions. Many of them did not cause eye phenotypes on their own, emphasizing a specific genetic interaction with H. As expected, we also identified components of different signaling pathways supposed to be involved in the regulation of cell growth and cell death. Accordingly, some of them also acted as modifiers of proapoptotic genes, suggesting a more general involvement in the regulation of apoptosis. Overall, these screens highlight the importance of H and the Notch pathway in mediating cell death in response to developmental and environmental cues and emphasize their role in maintaining developmental cellular homeostasis.

Green fluorescent protein as a convenient and versatile marker for studies on functional genomics in Drosophila.

Abstract. Gene function can be deduced from lack or gain of activity. For the manipulation of gene doses or activity in Drosophila, a set of P-based vectors was constructed containing green fluorescent protein as marker. pBLUEi, pGREENi and pYELLi were designed for large insert transformation. Mosaicism was generated in vivo with pFlipG which is also ideal for targeted gene disruption. Tissue-specific gene silencing in vivo was performed with the vector set pHIBS and pUdsGFP. pHIBS allows easy cloning and shuttling of double-headed constructs. With pUdsGFP, double stranded RNA can be produced in defined patterns and the area of interference simultaneously visualized by green fluorescence. We demonstrate nearly complete silencing of a ubiquitously expressed gene in a tissue-specific manner.

Su(H)-independent activity of Hairless during mechano-sensory organ formation in Drosophila

Formation of mechano-sensory organs in Drosophila involves the selection of neural precursor cells (SOPs) mediated by the classical Notch pathway in the process of lateral inhibition. Here we show that the subsequent cell type specifications rely on distinct subsets of Notch signaling components. Whereas E(spl) bHLH genes implement SOP selection, they are not required for later decisions. Most remarkably, the Notch signal transducer Su(H) is essential to determine outer but not inner cell fates. In contrast, the Notch antagonist Hairless, thought to act upon Su(H), influences strongly the entire cell lineage demonstrating that it functions through targets other than Su(H) within the inner lineage. Thereby, Hairless and numb may have partly redundant activities. This suggests that Notch-dependent binary cell fate specifications involve different sets of mediators depending on the cell type considered.

Wing vein formation in Drosophila melanogaster: Hairless is involved in the cross-talk between Notch and EGF signaling pathways

Wing vein development in Drosophila is controlled by different morphogenetic pathways, including Notch. Hairless (H) antagonizes Notch target gene activation by binding to the Notch signal transducer Suppressor of Hairless [Su(H)]. Accordingly, overexpression of H phenocopies reduction of Notch activity. Deletion of the Su(H)-binding domain in H-C2 results in loss of H activity. However, overexpression of H-C2 induces formation of ectopic veins. In a screen for genetic modifiers of this phenotype, we have identified several genes involved in Notch and epidermal growth factor (EGF) signaling. Most notably veinlet, an activator of EGF signaling, acts downstream of H-C2. H-C2 positively regulates veinlet maybe through inhibition of inter-vein determinants in agreement with a model, whereby Notch and EGF signaling pathways cross-regulate vein pre-patterning.