The tiny Hairless protein from Apis mellifera: a potent antagonist of Notch signaling in Drosophila melanogaster

Abstract

BackgroundThe Notch signaling pathway is fundamental to the regulation of many cell fate decisions in eumetazoans. Not surprisingly, members of this pathway are highly conserved even between vertebrates and invertebrates. There is one notable exception, Hairless, which acts as a general Notch antagonist in Drosophila. Hairless silences Notch target genes by assembling a repressor complex together with Suppressor of Hairless [Su(H)] and the co-repressors Groucho (Gro) and C-terminal binding protein (CtBP). Now with the availability of genomic databases, presumptive Hairless homologues are predicted, however only in insect species. To further our understanding of Hairless structure and function, we have cloned the Hairless gene from Apis mellifera (A.m.H) and characterized its functional conservation in Drosophila.ResultsThe Apis Hairless protein is only one third of the size of the Drosophila orthologue. Interestingly, the defined Suppressor of Hairless binding domain is interrupted by a nonconserved spacer sequence and the N-terminal motif is sufficient for binding. In contrast to Apis Hairless, the Drosophila orthologue contains a large acidic domain and we provide experimental evidence that this acidic domain is necessary to silence Hairless activity in vivo. Despite the dramatic size differences, Apis Hairless binds to the Drosophila Hairless interactors Su(H), Gro, CtBP and Pros26.4. Hence, Apis Hairless assembles a repressor complex with Drosophila components that may have a different topology. Nevertheless, Apis Hairless is sufficient to repress the Notch target gene vestigial in Drosophila. Moreover, it is able to rescue Hairless mutant phenotypes, providing in vivo evidence for its function as a bona fide Notch antagonist.ConclusionThis is the first interspecies-complementation analysis of the Hairless gene. Guided by evolutionary comparisons, we hope to eventually identify all the relevant structural domains and cofactors of Hairless, thereby opening an avenue for further insights into the repressor-complexes that down-regulate Notch signaling also in other, higher eukaryotes.

Hairless as a novel component of the Notch signaling pathway

“…The enormous variation in the size of the Hairless protein in various Pancrustacean clades is worthy of note ( Figure 1A There is a broad tendency for the size of the protein to be relatively stable within an order (Supplementary file 1). Thus, as noted previously (Maier et al 2008), the Hymenoptera generally have a small Hairless (of the order of 400 aa; see Figure 1A), while the Diptera typically have a much larger version (of the order of 1000 aa or more).…”

“…Notable exceptions to this pattern of uniformity are aphids, where Hairless is typically ~900 aa compared to ~400 aa in other Hemiptera, and chalcid wasps, where the protein is over 500 aa instead of the Hymenoptera-typical ~400 aa noted above (Supplementary file 1). Smaller Hairless proteins retain all five conserved motifs/domains characteristic of this factor (Maier et al 2008), while the regions that flank and lie between these sequences are reduced in size ( Figure 1A; Supplementary file 2).…”

Insensitive is a corepressor for Suppressor of Hairless and regulates Notch signalling during neural development

“…The major corepressor for the Drosophila CSL protein Su(H) is Hairless, an adaptor protein that recruits both CtBP and Groucho repressor complexes (Morel et al , 2001; Barolo et al , 2002). Mammalian Hairless proteins have not been identified; however, it should be noted that Hairless is extremely rapidly evolving and not trivial to identify even in other insects (Maier, 2006; Maier et al , 2008). Therefore, the absence of mammalian proteins aligning to Hairless is not necessarily conclusive.…”

Structural analysis of point mutations in the Hairless gene and their association with the activity of the Hairless protein

“…It is remarkable that even though all members of the NSP are highly conserved in metazoans, H protein has been identified and characterized only in insect species: practically in all members of the Drosophilidae family as well as in some members of the Culicidae family and, Lepidoptera, Hymenoptera and Coleoptera orders [27][28][29]. For this reason, it is important to determinate the positions of the important amino acids in the different H protein domains.…”

“…In the case of the H 107 and H 133 mutant genes, is not so clear to define if the characterized mutations are responsible of the reported phenotypes, especially in the last one because the mutation described was found in the first intron of the Hairless gene, and because in the H 107 mutant, the substitution of one threonine by an alanine is present in almost all the H orthologues from Drosophilids [29]. All these together suggest that the mutations responsible for the miss function of those mutant proteins could be located at another site in the primary structure.…”