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12 pages, 4118 KiB

Open AccessArticle

Su(H) Modulates Enhancer Transcriptional Bursting in Prelude to Gastrulation

by Kelli D. Fenelon, Priyanshi Borad, Biraaj Rout, Parisa Boodaghi Malidarreh, Mohammad Sadegh Nasr, Jacob M. Luber and Theodora Koromila

Cells 2024, 13(21), 1759; https://doi.org/10.3390/cells13211759 - 24 Oct 2024

Viewed by 1869

Abstract

Transcriptional regulation, orchestrated by the interplay between transcription factors (TFs) and enhancers, governs gene expression dynamics crucial for cellular processes. While gross qualitative fluctuations in transcription factor-dependent gene expression patterning have a long history of characterization, the roles of these factors in the [...] Read more.

Transcriptional regulation, orchestrated by the interplay between transcription factors (TFs) and enhancers, governs gene expression dynamics crucial for cellular processes. While gross qualitative fluctuations in transcription factor-dependent gene expression patterning have a long history of characterization, the roles of these factors in the nuclei retaining expression in the presence or absence of these factors are now observable using modern techniques. Our study investigates the impact of Suppressor of Hairless (Su(H)), a broadly expressed transcription factor, on enhancer-driven transcriptional modulation using Drosophila early embryos as a model system. Building upon previous findings, we employ super-resolution microscopy to dissect Su(H)’s influence on sog-Distal (sogD) enhancer activity specifically in nuclei with preserved sogD-driven expression in the absence of Su(H) binding. We demonstrate that Su(H) occupancy perturbations alter expression levels and bursting dynamics. Notably, Su(H) absence during embryonic development exhibits region-specific effects, inhibiting expression dorsally and stabilizing expression ventrally, implying a nuanced role in enhancer regulation. Our findings shed light on the intricate mechanisms that govern transcriptional dynamics and suggest a critical patterning role for Notch/Hairless signaling in sog expression as embryos transition to gastrulation. Full article

(This article belongs to the Special Issue Advanced Technology for Cellular Imaging)

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26 pages, 12138 KiB

Open AccessArticle

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

by Tanja C. Mönch, Thomas K. Smylla, Franziska Brändle, Anette Preiss and Anja C. Nagel

Genes 2024, 15(5), 552; https://doi.org/10.3390/genes15050552 - 26 Apr 2024

Viewed by 1916

Abstract

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, [...] Read more.

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, H^FA, H^LLAA and H^WA were generated and a phenotypic series was established by several parameters, reflecting the residual H-Su(H) binding capacity. Occasionally, homozygous H^WA flies develop to adulthood. They were compared with the likewise semi-viable H^NN 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 H^WA and H^NN homozygous alleles displayed strong defects in wing venation and mechano-sensory bristle development. Strikingly, however, H^WA displayed only a loss of bristles, whereas bristle organs of H^NN flies showed a complete shaft-to-socket transformation. Apparently, the impact of H^WA is restricted to lateral inhibition, whereas that of H^NN also affects the respective cell type specification. Notably, reduction in Su(H) gene dosage only suppressed the H^NN bristle phenotype, but amplified that of H^WA. We interpret these differences as to the role of H regarding Su(H) stability and availability. Full article

(This article belongs to the Special Issue Gene Editing in Drosophila to Study Gene Function and Developmental Processes)

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24 pages, 36929 KiB

Open AccessArticle

Genetic and Molecular Interactions between H^ΔCT, a Novel Allele of the Notch Antagonist Hairless, and the Histone Chaperone Asf1 in Drosophila melanogaster

by Dieter Maier, Milena Bauer, Mike Boger, Anna Sanchez Jimenez, Zhenyu Yuan, Johannes Fechner, Janika Scharpf, Rhett A. Kovall, Anette Preiss and Anja C. Nagel

Genes 2023, 14(1), 205; https://doi.org/10.3390/genes14010205 - 13 Jan 2023

Viewed by 2849

Abstract

Cellular differentiation relies on the highly conserved Notch signaling pathway. Notch activity induces gene expression changes that are highly sensitive to chromatin landscape. We address Notch gene regulation using Drosophila as a model, focusing on the genetic and molecular interactions between the Notch [...] Read more.

Cellular differentiation relies on the highly conserved Notch signaling pathway. Notch activity induces gene expression changes that are highly sensitive to chromatin landscape. We address Notch gene regulation using Drosophila as a model, focusing on the genetic and molecular interactions between the Notch antagonist Hairless and the histone chaperone Asf1. Earlier work implied that Asf1 promotes the silencing of Notch target genes via Hairless (H). Here, we generate a novel H^ΔCT allele by genome engineering. Phenotypically, H^ΔCT behaves as a Hairless gain of function allele in several developmental contexts, indicating that the conserved CT domain of H has an attenuator role under native biological contexts. Using several independent methods to assay protein–protein interactions, we define the sequences of the CT domain that are involved in Hairless–Asf1 binding. Based on previous models, where Asf1 promotes Notch repression via Hairless, a loss of Asf1 binding should reduce Hairless repressive activity. However, tissue-specific Asf1 overexpression phenotypes are increased, not rescued, in the H^ΔCT background. Counterintuitively, Hairless protein binding mitigates the repressive activity of Asf1 in the context of eye development. These findings highlight the complex connections of Notch repressors and chromatin modulators during Notch target-gene regulation and open the avenue for further investigations. Full article

(This article belongs to the Section Molecular Genetics and Genomics)

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20 pages, 3926 KiB

Open AccessArticle

The Binding of CSL Proteins to Either Co-Activators or Co-Repressors Protects from Proteasomal Degradation Induced by MAPK-Dependent Phosphorylation

by Johannes Fechner, Manuela Ketelhut, Dieter Maier, Anette Preiss and Anja C. Nagel

Int. J. Mol. Sci. 2022, 23(20), 12336; https://doi.org/10.3390/ijms232012336 - 15 Oct 2022

Cited by 5 | Viewed by 3966

Abstract

The primary role of Notch is to specify cellular identities, whereby the cells respond to amazingly small changes in Notch signalling activity. Hence, dosage of Notch components is crucial to regulation. Central to Notch signal transduction are CSL proteins: together with respective cofactors, [...] Read more.

The primary role of Notch is to specify cellular identities, whereby the cells respond to amazingly small changes in Notch signalling activity. Hence, dosage of Notch components is crucial to regulation. Central to Notch signal transduction are CSL proteins: together with respective cofactors, they mediate the activation or the silencing of Notch target genes. CSL proteins are extremely similar amongst species regarding sequence and structure. We noticed that the fly homologue suppressor of hairless (Su(H)) is stabilised in transcription complexes. Using specific transgenic fly lines and HeLa RBPJ^KO cells we provide evidence that Su(H) is subjected to proteasomal degradation with a half-life of about two hours if not protected by binding to co-repressor hairless or co-activator Notch. Moreover, Su(H) stability is controlled by MAPK-dependent phosphorylation, matching earlier data for RBPJ in human cells. The homologous murine and human RBPJ proteins, however, are largely resistant to degradation in our system. Mutating presumptive protein contact sites, however, sensitised RBPJ for proteolysis. Overall, our data highlight the similarities in the regulation of CSL protein stability across species and imply that turnover of CSL proteins may be a conserved means of regulating Notch signalling output directly at the level of transcription. Full article

(This article belongs to the Special Issue Notch Signaling in Health and Disease)

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20 pages, 7047 KiB

Open AccessArticle

The Membrane-Bound Notch Regulator Mnr Supports Notch Cleavage and Signaling Activity in Drosophila melanogaster

by Anja C. Nagel, Dominik Müller, Mirjam Zimmermann and Anette Preiss

Biomolecules 2021, 11(11), 1672; https://doi.org/10.3390/biom11111672 - 10 Nov 2021

Cited by 2 | Viewed by 3222

Abstract

The Notch signaling pathway is pivotal to cellular differentiation. Activation of this pathway involves proteolysis of the Notch receptor and the release of the biologically active Notch intracellular domain, acting as a transcriptional co-activator of Notch target genes. While the regulation of Notch [...] Read more.

The Notch signaling pathway is pivotal to cellular differentiation. Activation of this pathway involves proteolysis of the Notch receptor and the release of the biologically active Notch intracellular domain, acting as a transcriptional co-activator of Notch target genes. While the regulation of Notch signaling dynamics at the level of ligand–receptor interaction, endocytosis, and transcriptional regulation has been well studied, little is known about factors influencing Notch cleavage. We identified EP555 as a suppressor of the Notch antagonist Hairless (H). EP555 drives expression of CG32521 encoding membrane-bound proteins, which we accordingly rename membrane-bound Notch regulator (mnr). Within the signal-receiving cell, upregulation of Mnr stimulates Notch receptor activation, whereas a knockdown reduces it, without apparent influence on ligand–receptor interaction. We provide evidence that Mnr plays a role in γ-secretase-mediated intramembrane cleavage of the Notch receptor. As revealed by a fly-eye-based reporter system, γ-secretase activity is stimulated by the overexpression of Mnr, and is inhibited by its knockdown. We conclude that Mnr proteins support Notch signaling activity by fostering the cleavage of the Notch receptor. With Mnr, we identified a membrane-bound factor directly augmenting Notch intra-membrane processing, thereby acting as a positive regulator of Notch signaling activity. Full article

(This article belongs to the Special Issue Notch Signalling and Cell Fate)

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23 pages, 20727 KiB

Open AccessArticle

Membrane-Anchored Hairless Protein Restrains Notch Signaling Activity

by Dieter Maier

Genes 2020, 11(11), 1315; https://doi.org/10.3390/genes11111315 - 6 Nov 2020

Cited by 2 | Viewed by 3019

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Regulating Gene Activity By Sequestering Transcriptional Regulators)

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17 pages, 3145 KiB

Open AccessArticle

Limited Availability of General Co-Repressors Uncovered in an Overexpression Context during Wing Venation in Drosophila melanogaster

by Anja C. Nagel, Dieter Maier, Janika Scharpf, Manuela Ketelhut and Anette Preiss

Genes 2020, 11(10), 1141; https://doi.org/10.3390/genes11101141 - 28 Sep 2020

Cited by 2 | Viewed by 3450

Abstract

Cell fate is determined by the coordinated activity of different pathways, including the conserved Notch pathway. Activation of Notch results in the transcription of Notch targets that are otherwise silenced by repressor complexes. In Drosophila, the repressor complex comprises the transcription factor [...] Read more.

Cell fate is determined by the coordinated activity of different pathways, including the conserved Notch pathway. Activation of Notch results in the transcription of Notch targets that are otherwise silenced by repressor complexes. In Drosophila, the repressor complex comprises the transcription factor Suppressor of Hairless (Su(H)) bound to the Notch antagonist Hairless (H) and the general co-repressors Groucho (Gro) and C-terminal binding protein (CtBP). The latter two are shared by different repressors from numerous pathways, raising the possibility that they are rate-limiting. We noted that the overexpression during wing development of H mutants H^dNT and H^LD compromised in Su(H)-binding induced ectopic veins. On the basis of the role of H as Notch antagonist, overexpression of Su(H)-binding defective H isoforms should be without consequence, implying different mechanisms but repression of Notch signaling activity. Perhaps excess H protein curbs general co-repressor availability. Supporting this model, nearly normal wings developed upon overexpression of H mutant isoforms that bound neither Su(H) nor co-repressor Gro and CtBP. Excessive H protein appeared to sequester general co-repressors, resulting in specific vein defects, indicating their limited availability during wing vein development. In conclusion, interpretation of overexpression phenotypes requires careful consideration of possible dominant negative effects from interception of limiting factors. Full article

(This article belongs to the Special Issue Regulating Gene Activity By Sequestering Transcriptional Regulators)

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20 pages, 3642 KiB

Open AccessArticle

An RBPJ-Drosophila Model Reveals Dependence of RBPJ Protein Stability on the Formation of Transcription–Regulator Complexes

by Bernd M. Gahr, Franziska Brändle, Mirjam Zimmermann and Anja C. Nagel

Cells 2019, 8(10), 1252; https://doi.org/10.3390/cells8101252 - 14 Oct 2019

Cited by 5 | Viewed by 3680

Abstract

Notch signaling activity governs widespread cellular differentiation in higher animals, including humans, and is involved in several congenital diseases and different forms of cancer. Notch signals are mediated by the transcriptional regulator RBPJ in a complex with activated Notch (NICD). Analysis of Notch [...] Read more.

Notch signaling activity governs widespread cellular differentiation in higher animals, including humans, and is involved in several congenital diseases and different forms of cancer. Notch signals are mediated by the transcriptional regulator RBPJ in a complex with activated Notch (NICD). Analysis of Notch pathway regulation in humans is hampered by a partial redundancy of the four Notch receptor copies, yet RBPJ is solitary, allowing its study in model systems. In Drosophila melanogaster, the RBPJ orthologue is encoded by Suppressor of Hairless [Su(H)]. Using genome engineering, we replaced Su(H) by murine RBPJ in order to study its function in the fly. In fact, RBPJ largely substitutes for Su(H)’s function, yet subtle phenotypes reflect increased Notch signaling activity. Accordingly, the binding of RBPJ to Hairless (H) protein, the general Notch antagonist in Drosophila, was considerably reduced compared to that of Su(H). An H-binding defective RBPJ^LLL mutant matched the respective Su(H)^LLL allele: homozygotes were lethal due to extensive Notch hyperactivity. Moreover, RBPJ^LLL protein accumulated at lower levels than wild type RBPJ, except in the presence of NICD. Apparently, RBPJ protein stability depends on protein complex formation with either H or NICD, similar to Su(H), demonstrating that the murine homologue underlies the same regulatory mechanisms as Su(H) in Drosophila. These results underscore the importance of regulating the availability of RBPJ protein to correctly mediate Notch signaling activity in the fly. Full article

(This article belongs to the Section Cell Signaling)

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