Membrane-Anchored Hairless Protein Restrains Notch Signaling Activity

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 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.

OptIC Notch reveals mechanism that regulates receptor interactions with CSL

“…Although predominantly nuclear in most cell-types examined, CSL does not contain a recognizable nuclear localization signal and it likely requires other factors to chaperone it into the nucleus. In overexpression experiments, NICD and several co-repressors, such as Hairless and SMRT, can fulfil this role and promote nuclear accumulation of CSL (Fortini and Artavanis-Tsakonas, 1994; Gho et al, 1996; Gomez-Lamarca et al, 2018; Maier, 2020; Tabaja et al, 2017; Wacker et al, 2011; Wolf et al, 2019, 2021; Zhou and Hayward, 2001). Although it has been widely assumed that NICD first meets CSL in the nucleus, there is no direct evidence to support this and it remains unclear whether the two proteins could interact prior to nuclear entry.…”

OptIC-Notch reveals mechanism that regulates receptor interactions with CSL

“…Although our experiments are in Drosophila , the ΦWΦP motif and its interaction with CSL is highly conserved ( Choi et al, 2012 ; Contreras et al, 2015 ; Hall and Kovall, 2019 ; Johnson et al, 2010 ; Nam et al, 2003 ; Wilson and Kovall, 2006 ). Likewise, shuttling of CSL between nuclear and cytoplasmic compartments has been observed in several contexts, where its regulation appears important for fine-tuning Notch-dependent transcription ( Fechner et al, 2022 ; Gho et al, 1996 ; Maier, 2020 ; Maier et al, 2013 ; Tabaja et al, 2017 ; Wacker et al, 2011 ; Wolf et al, 2019 , 2021 ; Zhou and Hayward, 2001 ). Whether these impacts on signalling are because of consequences on nuclear CSL pools, which has largely been the inference, or whether effects on cytoplasmic pools are also important, as implied here, remains to be established.…”

“…In all species, CSL is predominantly nuclear even though it lacks a recognisable nuclear localisation signal, making it likely that other factors chaperone CSL into the nucleus. NICD and several co-repressors, such as Hairless and SMRT (NCOR2), can fulfil this role and promote nuclear accumulation of CSL ( Fortini and Artavanis-Tsakonas, 1994 ; Gho et al, 1996 ; Maier, 2020 ; Maier et al, 2013 ; Tabaja et al, 2017 ; Wacker et al, 2011 ; Wolf et al, 2019 , 2021 ; Zhou and Hayward, 2001 ). However, it has been widely assumed that NICD first meets CSL in the nucleus, although there is no direct evidence to support this, and it remains possible that the two proteins could interact prior to nuclear entry.…”

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

“…Only the SEM pictures of female flies were used for the quantification of micro-and macrochaetae, respectively. The numbers of bristles were determined as described before [57][58][59][60]. Statistical analysis was conducted by ANOVA two-tailed test for multiple comparisons using Tukey-Kramer's or Dunnett's approach, as indicated: *** p ≤ 0.001 highly significant; ** p ≤ 0.01 very significant; * p ≤ 0.05 significant; not significant (n.s.)…”