In vivo analysis of internal ribosome entry at the Hairless locus by genome engineering in Drosophila

Smylla, Thomas K.; Preiss, Anette; Maier, Dieter

doi:10.1038/srep34881

Cited by 5 publications

(3 citation statements)

References 52 publications

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“…The alleles H FA , H LLAA and H WA were generated through genome engineering as previously described [30,35]. Mutant H cDNAs [32] were cloned into the pGE-attB GMR vector [35], to be integrated using the H attP allele as a platform by site-specific recombination as outlined earlier [30,31]. Genotypes were confirmed by PCR and sequencing where applicable.…”

Section: Fly Workmentioning

confidence: 99%

“…1 By way of genome engineering in D. melanogaster, we generated several specific alleles of Su(H) as well as of H to study the structure-function relationship of the repressor complex [28,30]. Mutations in the H gene were created to show, for example, that two characteristic protein isoforms of H are derived from differential translation initiation [31]. Moreover, several amino acid replacements allowed us to destroy H's essential nuclear localization signal NLS3, thereby demonstrating its requirement for H as well as for Su(H) nuclear entry [27].…”

Section: Introductionmentioning

confidence: 99%

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Novel Genome-Engineered H Alleles Differentially Affect Lateral Inhibition and Cell Dichotomy Processes during Bristle Organ Development

Mönch,

Smylla,

Brändle

et al. 2024

Genes

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Hairless (H) encodes the major antagonist in the Notch signaling pathway, which governs cellular differentiation of various tissues in Drosophila. By binding to the Notch signal transducer Suppressor of Hairless (Su(H)), H assembles repressor complexes onto Notch target genes. Using genome engineering, three new H alleles, HFA, HLLAAand HWA were generated and a phenotypic series was established by several parameters, reflecting the residual H-Su(H) binding capacity. Occasionally, homozygous HWA flies develop to adulthood. They were compared with the likewise semi-viable HNN allele affecting H-Su(H) nuclear entry. The H homozygotes were short-lived, sterile and flightless, yet showed largely normal expression of several mitochondrial genes. Typical for H mutants, both HWA and HNN homozygous alleles displayed strong defects in wing venation and mechano-sensory bristle development. Strikingly, however, HWA displayed only a loss of bristles, whereas bristle organs of HNN flies showed a complete shaft-to-socket transformation. Apparently, the impact of HWA is restricted to lateral inhibition, whereas that of HNN also affects the respective cell type specification. Notably, reduction in Su(H) gene dosage only suppressed the HNN bristle phenotype, but amplified that of HWA. We interpret these differences as to the role of H regarding Su(H) stability and availability.

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Section: Fly Workmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Novel Genome-Engineered H Alleles Differentially Affect Lateral Inhibition and Cell Dichotomy Processes during Bristle Organ Development

Mönch,

Smylla,

Brändle

et al. 2024

Genes

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“…Hairless possesses an internal ribosomal entry site (IRES) within the coding region, which leads to two main Hairless protein isoforms starting with the second and third Methionine, M2 and M3, respectively ( Figure S1) [42,43]. Both isoforms contain all known functional Hairless domains: Suppressor of Hairless binding domain (SBD), Groucho binding domain (GBD), and C-terminal protein binding domain (CBD) [23][24][25], as well as the known nuclear import and export signals [29].…”

Section: Cloning Strategymentioning

confidence: 99%

Membrane-Anchored Hairless Protein Restrains Notch Signaling Activity

Maier

2020

Genes

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The Notch signaling pathway governs cell-to-cell communication in higher eukaryotes. In Drosophila, after cleavage of the transmembrane receptor Notch, the intracellular domain of Notch (ICN) binds to the transducer Suppressor of Hairless (Su(H)) and shuttles into the nucleus to activate Notch target genes. Similarly, the Notch antagonist Hairless transfers Su(H) into the nucleus to repress Notch target genes. With the aim to prevent Su(H) nuclear translocation, Hairless was fused to a transmembrane domain to anchor the protein at membranes. Indeed, endogenous Su(H) co-localized with membrane-anchored Hairless, demonstrating their binding in the cytoplasm. Moreover, adult phenotypes uncovered a loss of Notch activity, in support of membrane-anchored Hairless sequestering Su(H) in the cytosol. A combined overexpression of membrane-anchored Hairless with Su(H) lead to tissue proliferation, which is in contrast to the observed apoptosis after ectopic co-overexpression of the wild-type genes, indicating a shift to a gain of Notch activity. A mixed response, general de-repression of Notch signaling output, plus inhibition at places of highest Notch activity, perhaps reflects Su(H)’s role as activator and repressor, supported by results obtained with the Hairless-binding deficient Su(H)LLL mutant, inducing activation only. Overall, the results strengthen the idea of Su(H) and Hairless complex formation within the cytosolic compartment.

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Conservation of the Notch antagonist Hairless in arthropods: functional analysis of the crustacean Daphnia pulex Hairless gene

et al. 2017

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The Notch signaling pathway is highly conserved in all animal metazoa: upon Notch receptor activation, transcription of Notch target genes is turned on by an activator complex that centers on the transcription factor CSL. In the absence of signal, CSL assembles transcriptional repression complexes that display remarkable evolutionary diversity. The major antagonist of Notch signaling in insects named Hairless was originally identified in Drosophila melanogaster. It binds to the Drosophila CSL homologue Suppressor of Hairless [Su(H)] and recruits the two general co-repressors, Groucho and C-terminal binding protein. Whereas the majority of Notch signaling components is conserved between insects and vertebrates, Hairless is found only in insects. Here, we present the analysis of the Hairless gene from Daphnia pulex and, hence, for the first time from a crustacean. Daphnia and Drosophila Hairless protein sequences are highly diverged. Known functional domains, however, the Su(H), Groucho and the C-terminal binding protein interactions domains, are well conserved. Moreover, direct binding of the Daphnia Hairless protein and the respective Drosophila interaction partners was detected, demonstrating the conservation at the molecular level. In addition, interaction between Daphnia Hairless and Drosophila Su(H) was demonstrated in vivo, as co-overexpression of the respective genes during Drosophila development resulted in the expected downregulation of Notch activity in the fly. Structural models show that the Hairless-Su(H) repressor complexes from Daphnia and Drosophila are almost indistinguishable from one another. Amino acid residues in direct contact within the Hairless-Su(H) complex are at absolutely identical positions in the two homologues.

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In vivo analysis of internal ribosome entry at the Hairless locus by genome engineering in Drosophila

Cited by 5 publications

References 52 publications

Novel Genome-Engineered H Alleles Differentially Affect Lateral Inhibition and Cell Dichotomy Processes during Bristle Organ Development

Novel Genome-Engineered H Alleles Differentially Affect Lateral Inhibition and Cell Dichotomy Processes during Bristle Organ Development

Membrane-Anchored Hairless Protein Restrains Notch Signaling Activity

Conservation of the Notch antagonist Hairless in arthropods: functional analysis of the crustacean Daphnia pulex Hairless gene

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