Nucleo-cytoplasmic shuttling of Drosophila Hairless/Su(H) heterodimer as a means of regulating Notch dependent transcription

Wolf, Dorina; Smylla, Thomas K.; Reichmuth, Jan; Hoffmeister, Philipp; Kober, Ludmilla; Zimmermann, Mirjam; Turkiewicz, Aleksandra; Borggrefe, Tilman; Nagel, Anja C.; Oswald, Franz; Preiss, Anette; Maier, Dieter

doi:10.1016/j.bbamcr.2019.07.008

Cited by 19 publications

(58 citation statements)

References 69 publications

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“…As shown earlier, Su(H) relies on Hairless for nuclear entry, closely following H protein distribution in wild-type and experimental settings [28,29]. Therefore, in co-overexpression experiments of wild-type Hairless with Su(H), both proteins are almost completely nuclear (Figure 6a).…”

Section: Co-localization Of Sptm-h Myc and Su(h) In Salivary Glandssupporting

confidence: 71%

“…Therefore, expression of SPTM-H myc was first induced in salivary glands of third instar larvae that contain giant cells and nuclei, using the driver line sd-Gal4. Since endogenous Hairless protein is primarily detected in the nucleus (Figure 2a) [28][29][30], the nuclear protein Putzig (Pzg) was used as a marker [40]. As expected, the overexpressed SPTM-GFP control protein was not nuclear but mainly associated with the cell membrane, however, was also seen in a meshwork throughout the cytosol (Figure 2b).…”

Section: Subcellular Localization Of Overexpressed Sptm-h Myc Proteinmentioning

confidence: 67%

“…A Myc-tag allowed the specific detection of the subcellular localization of SPTM-H myc protein, clearly distinguishable from the endogenous Hairless protein. In contrast to the wild-type protein, SPTM-H myc is not nuclear [28][29][30]. Instead, it primarily accumulates at the periphery of the nucleus along the nuclear envelope.…”

Section: Discussionmentioning

confidence: 86%

“…Instead, Su(H) LLL protein was evenly distributed in the cytoplasm of salivary glands cells, whereas the punctuate-pattern typifying SPTM-H myc protein was present in the nuclear periphery (Figure 6c). In contrast to Su(H), the mutant Su(H) LLL protein was also weakly detected in the nucleus (asterisk in Figure 6c), which is attributed to Notch-mediated nuclear import [2,29].…”

Section: Downregulation Of Notch Target Gene Expression Is a Clear Rementioning

confidence: 97%

“…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]. To avoid translation of a Hairless protein isoform without transmembrane domain by internal ribosomal entry, only the Hairless portion downstream of the IRES was fused with the transmembrane domain ( Figure 1a and Figure S1).…”

Section: Cloning Strategymentioning

confidence: 99%

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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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Section: Co-localization Of Sptm-h Myc and Su(h) In Salivary Glandssupporting

confidence: 71%

Section: Subcellular Localization Of Overexpressed Sptm-h Myc Proteinmentioning

confidence: 67%

Section: Discussionmentioning

confidence: 86%

Section: Downregulation Of Notch Target Gene Expression Is a Clear Rementioning

confidence: 97%

Section: Cloning Strategymentioning

confidence: 99%

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Membrane-Anchored Hairless Protein Restrains Notch Signaling Activity

Maier

2020

Genes

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A Drosophila Su(H) Model of Adams-Oliver Syndrome Reveals Notch Cofactor Titration as a Mechanism Underlying Developmental Defects

Gagliani

Gutzwiller

Kuang

et al. 2021

Preprint

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Notch signaling is a conserved pathway that converts extracellular receptor-ligand interactions into changes in gene expression via a single transcription factor (CBF1/RBPJ in mammals; Su(H) in Drosophila). In humans, RBPJ variants have been linked to Adams-Oliver syndrome (AOS), a rare autosomal dominant disorder characterized by scalp, cranium, and limb defects. Here, we found that a previously described Drosophila Su(H) allele encodes a missense mutation that alters an analogous residue found in an AOS-associated RBPJ variant. Importantly, genetic studies support a model that Drosophila with a single copy of the AOS-like Su(H) allele behave in an opposing manner as flies with a Su(H) null allele due to a dominant activity of sequestering either the Notch co-activator or the antagonistic Hairless co-repressor. Consistent with this model, AOS-like Su(H) and Rbpj variants decrease DNA binding activity compared to wild type proteins, but these variants do not significantly alter protein binding to the Notch co-activator or the fly and mammalian co-repressors, respectively. Taken together, these data suggest a cofactor sequestration mechanism underlies AOS phenotypes associated with RBPJ variants, whereby a single RBPJ allele encodes a protein with compromised DNA binding activity that retains cofactor binding, resulting in Notch target gene dysregulation.

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OptIC Notch reveals mechanism that regulates receptor interactions with CSL

Townson

Gomez-Lamarca

Bray

2023

Preprint

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Active Notch signalling is brought about through receptor-ligand interactions that result in the release of the Notch intracellular domain (NICD) which translocates to the nucleus. NICD activates transcription at target sites in a tripartite complex with the DNA binding transcription factor CSL (CBF1/Su(H)/Lag-1) and co-activator Mastermind. Despite this, CSL lacks its own nuclear localisation sequence, and it remains unclear when and how the tripartite complex is formed. To probe the mechanisms involved, we designed an optogenetic approach to control NICD release (OptIC-Notch) and monitor consequences on complex formation and target gene activation. Strikingly we observed that, when uncleaved, OptIC-Notch sequesters CSL in the cytoplasm. Hypothesising that exposure of a juxta membrane ΦWΦP motif is key to sequestration, we masked this motif with a second light sensitive domain in OptIC-Notch{ω}, which was sufficient to prevent CSL sequestration. Furthermore, NICD produced by light induced cleavage of OptIC-Notch or OptIC-Notch{ω} chaperoned CSL into the nucleus and induced target gene activity, showing efficient light controlled Notch activation. Our results demonstrate that exposure of the ΦWΦP motif leads to recruitment of CSL and suggests that this is an important regulatory step in Notch signalling that can occur in the cytoplasm prior to nuclear entry.

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Nucleo-cytoplasmic shuttling of Drosophila Hairless/Su(H) heterodimer as a means of regulating Notch dependent transcription

Cited by 19 publications

References 69 publications

Membrane-Anchored Hairless Protein Restrains Notch Signaling Activity

Membrane-Anchored Hairless Protein Restrains Notch Signaling Activity

A Drosophila Su(H) Model of Adams-Oliver Syndrome Reveals Notch Cofactor Titration as a Mechanism Underlying Developmental Defects

OptIC Notch reveals mechanism that regulates receptor interactions with CSL

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