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Evolutionary emergence of Hairless as a novel component of the Notch signaling pathway

  1. Steven W Miller
  2. Artem Movsesyan
  3. Sui Zhang
  4. Rosa Fernández
  5. James W Posakony  Is a corresponding author
  1. University of California, San Diego, United States
  2. Center for Genomic Regulation, Spain
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Cite this article as: eLife 2019;8:e48115 doi: 10.7554/eLife.48115

Abstract

Suppressor of Hairless [Su(H)], the transcription factor at the end of the Notch pathway in Drosophila, utilizes the Hairless protein to recruit two co-repressors, Groucho (Gro) and C-terminal Binding Protein (CtBP), indirectly. Hairless is present only in the Pancrustacea, raising the question of how Su(H) in other protostomes gains repressive function. We show that Su(H) from a wide array of arthropods, molluscs, and annelids includes motifs that directly bind Gro and CtBP; thus, direct co-repressor recruitment is ancestral in the protostomes. How did Hairless come to replace this ancestral paradigm? Our discovery of a protein (S-CAP) in Myriapods and Chelicerates that contains a motif similar to the Su(H)-binding domain in Hairless has revealed a likely evolutionary connection between Hairless and Metastasis-associated (MTA) protein, a component of the NuRD complex. Sequence comparison and widely conserved microsynteny suggest that S-CAP and Hairless arose from a tandem duplication of an ancestral MTA gene.

https://doi.org/10.7554/eLife.48115.001

Introduction

A very common paradigm in the regulation of animal development is that DNA-binding transcriptional repressors bear defined amino acid sequence motifs that permit them to recruit, by direct interaction, one or more common co-repressor proteins that are responsible for conferring repressive activity. Two such universal co-repressors are Groucho (Gro) and C-terminal Binding Protein (CtBP).

The ancient and highly conserved transcription factor Suppressor of Hairless [Su(H)] functions at the terminus of the widely utilized Notch cell-cell signaling pathway. Su(H) is converted into an activator by signaling through the Notch receptor, but in the absence of signaling it functions as a repressor. Earlier studies have revealed that in many settings in Drosophila, Su(H)’s repressive activity depends on binding to the Hairless protein (Figure 1). Hairless includes separate Gro- and CtBP-binding motifs, which permit it to function as an adaptor to bring these two corepressors to Su(H) (Figure 1B) (Barolo et al., 2002a). Thus, the Su(H)/H partnership in the fly represents a notable exception to the rule of direct co-repressor recruitment.

Figure 1 with 1 supplement see all
Hairless mediates indirect recruitment of co-repressor proteins to Su(H).

(A) Diagram denoting locations of conserved domains and motifs within Hairless, and illustrating extreme size differences of the protein in different species. Shown is Hairless from the carpenter bee Ceratina calcarata and the blowfly Protophormia terraenovae (Hase et al., 2017), with scale and protein sizes indicated. (B) Summary of Hairless’s known mode of action (Lai, 2002; Maier, 2006) as an adaptor protein that recruits the global co-repressors C-terminal Binding Protein (CtBP) and Groucho (Gro) to Suppressor of Hairless [Su(H)], the transducing transcription factor for the Notch (N) cell-cell signaling pathway; adapted from Figure 6 of Barolo et al. (2002a). In the absence of signaling through the Notch receptor (left), Su(H) acts as a repressor of Notch target genes, despite the presence of transcriptional activator proteins (orange oval). Upon activation of the Notch receptor (middle), Su(H), in a complex with the receptor’s intracellular domain (NICD) and the co-activator Mastermind (Mam), functions to activate transcription of pathway target genes in cooperation with other transcriptional activators. In the absence of Hairless and hence in the absence of Su(H)’s repressive activity (right), the partner transcription factors are often sufficient to activate expression of target genes in a signal-independent manner (Barolo and Posakony, 2002b).

https://doi.org/10.7554/eLife.48115.002

As genome and transcriptome sequences have become available for more and more insects and other arthropods, we have searched for possible Hairless orthologs in a wide variety of species, in an attempt to determine the protein’s phylogenetic distribution. We have found that Hairless is confined to the Pancrustacea (or Tetraconata), a clade of arthropods that includes the Crustacea and Hexapoda (Misof et al., 2014; Kjer et al., 2016). While this indicates that Hairless was gained at least 500 Mya, it also raises the question of how Su(H) in other protostomes acquires repressive activity.

Here we present evidence that direct co-repressor recruitment by Su(H) is likely to be ancestral in the protostomes. We show that Su(H) in a broad range of protostomes, including arthropods, molluscs, and annelids, bears both a short linear motif that mediates binding of CtBP and a novel motif for direct recruitment of Gro. Thus, the evolutionary appearance of Hairless has permitted the replacement of an ancient and predominant regulatory mechanism (direct co-repressor recruitment) with a novel one (indirect recruitment).

What can we learn about the evolutionary history of Hairless? While Hairless itself is found only in the Pancrustacea, we show that the genomes of Myriapods and Chelicerates encode a protein with clear sequence and functional similarities to Hairless. These proteins include a motif that strongly resembles the Su(H)-binding domain of Hairless, and we demonstrate that this motif from the house spider Parasteatoda tepidariorum does indeed bind Su(H). In addition, these Myriapod and Chelicerate proteins also include one or more canonical motifs for recruitment of CtBP. Accordingly, we designate these factors as ‘Su(H)-Co-repressor Adaptor Proteins’ (S-CAPs).

Finally, further sequence analyses, along with the discovery of conserved microsynteny, have provided substantial evidence that Hairless and the S-CAPs are likely to be homologous and that they arose from a duplication of the gene encoding Metastasis-associated (MTA) protein, a component of the nucleosome remodeling and deacetylase (NuRD) complex.

An intriguing question in evolutionary biology concerns the path by which a particular clade has escaped a strongly selected character that has been conserved for hundreds of millions of years. We believe that our study has yielded valuable insight into both the emergence of an evolutionary novelty and its replacement of an ancestral paradigm.

Results

Hairless is present only in the Pancrustacea

We have conducted extensive BLAST searches of genome and transcriptome sequence data for a wide variety of metazoa in an attempt to define the phylogenetic distribution of Hairless. We find that Hairless as originally described (Bang and Posakony, 1992; Maier et al., 1992; Maier et al., 2008) is confined to the Pancrustacea (or Tetraconata), and occurs widely within this clade, including the Hexapoda, Vericrustacea, and Oligostraca (Figure 2A). By contrast, no evidence for a true Hairless gene has been detected in either Myriapods or Chelicerates, even in cases where substantially complete genome sequence assemblies are available.

Phylogenetic distribution of Hairless and related S-CAP proteins.

(A) Based on extensive BLAST searches of available genome and transcriptome assemblies, orthologs of the canonical Hairless gene are found only in the Pancrustacea (blue bar), while orthologs of a gene that encodes the related S-CAP protein are found in the Myriapods (mS-CAP, red bar) and Chelicerates (cS-CAP, pink bar). We suggest S-CAP as a suitable umbrella nomenclature for this gene family (black bracket). Tree adapted from Figure 2 of Regier et al. (2010). (B) Consistent with the presence of Hairless as an adaptor protein, Su(H) in most insect orders (the Neoptera clade) has lost the ancestral short linear motifs that mediate direct recruitment of the CtBP and Gro co-repressor proteins (red bar). However, in the Crustacea, Collembola, Diplura, and a subset of Insecta, the ancestral recruitment motifs have been retained in Su(H), despite the presence of Hairless (see Table 1 and Supplementary file 3). Tree adapted from Misof et al. (2014) and Kjer et al. (2016).

https://doi.org/10.7554/eLife.48115.004

The enormous variation in the size of the Hairless protein in various Pancrustacean clades is worthy of note (Figure 1A). The known extremes are represented by the Diplostracan (shrimp) Eulimnadia texana (343 aa) (Baldwin-Brown et al., 2018) and the Dipteran (fly) Protophormia terraenovae (1614 aa) (Hase et al., 2017), a 4.7-fold difference. 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 typically 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).

A known CtBP-binding motif is present in the non-conserved N-terminal region of Su(H) in a wide variety of protostomes

The apparent confinement of the Hairless co-repressor adaptor protein to the Pancrustacea raises the question of the mechanism(s) by which Su(H) in other protostomes might recruit co-repressor proteins to mediate its repressor function. Of course, other protostomes need not utilize the Gro and CtBP co-repressors for this purpose; different co-repressors might substitute. Nevertheless, we first sought to identify known binding motifs for Gro and CtBP in Su(H) from arthropods lacking Hairless. As shown in Table 1, we found a canonical CtBP recruitment motif of the form PϕDϕS (where ϕ = I, L, M, or V) in predicted Su(H) proteins from a variety of Myriapods and Chelicerates, including the centipede Strigamia maritima, the tick Ixodes scapularis, the spider Parasteatoda tepidariorum, the horseshoe crab Limulus polyphemus, and the scorpion Centruroides sculpturatus. These motifs are all located in the non-conserved N-terminal region of Su(H) (Supplementary file 3).

Table 1
Co-repressor recruitment motifs in protostome Su(H) proteins.
https://doi.org/10.7554/eLife.48115.005
SpeciesCtBP motifGro motifSource
Ecdyonurus insignisYPDNHPVDLSSPRPHAPMIPGSLTPPDKMNGEHPHHGGCCL01029953.1 (Simon et al., 2018)
Calopteryx splendensYTDNHPVDLSSPRPPHHMIPGSLTPPDKMNGEHPAMHLYUA01002621.1 (Ioannidis et al., 2017)
Atelura formicariaYPDNHPVDLSSPRPQPHMIPGSLTPPDKMNGEHPHHSGAYJ02050375.1 (Misof et al., 2014)
Machilis hrabeiYPDNHPVDLSSPRPHPHMLPGSLTPPDKMNGEHPHHGScaffold 1 (i5K Consortium, 2013)
Catajapyx aquilonarisSTANNPVDLSSPRGSAPMIPGSLTPPDKVNGEHHSHHJYFJ02000853.1 (i5K Consortium, 2013)
Holacanthella duospinosaVPNSNPVDLSNPSPSSNFVPGSLSPPERMNGNDPSLLNIPM01000059.1 (Wu et al., 2017)
Pollicipes pollicipesYPDNHPVDLSSPRPEGPLIAGSLTPPDKLGAELGLHAGGJN01104381.1 (unpublished)
Hyalella aztecaSLGHRPVDLSQAPSPAAMLAGSLTPPDKLNSDPQQQQNW_017238139.1 (i5K Consortium, 2013)
Eurytemora affinisSETSAPVDLSAPRPNYGMLPGSLTPPDKLNGDHCSPGNW_019396104.1 (i5K Consortium, 2013)
Triops cancriformisHPEARPVDLSSSRLLYHSSSLTLTPPDKVNVDGSNSQBAYF01001879.1 (Ikeda et al., 2015)
Argulus siamensisYPENNPVDLSNSRTGSPMIPGSLTPPDKMNGEHHPGHJW959185.1 (Sahoo et al., 2013)
Strigamia maritimaFADNHPVDLSNSHRGSHMIAGSLTPPDKVNGEHGHQLJH430541.1 (Chipman et al., 2014)
Sigmoria latior mundaTNENHPVDLSSSHRSSHMIPGSLTPPDKGNAEHSHSH(Rodriguez et al., 2018)
Metaseiulus occidentalisGADRKPLDMSAAHRSNW_003805473.1 (Hoy et al., 2016)
Ixodes scapularisQAAGAPVDMSSHPARNW_002722632.1 (Gulia-Nuss et al., 2016)
Parasteatoda tepidariorum 1VIDSHPVDLSSPKPSNW_018383625.1 (Schwager et al., 2017)
Parasteatoda tepidariorum 2RYEGRPVDLSSPRPNNW_018370942.1 (Schwager et al., 2017)
Limulus polyphemus 1PYDGHPVDLSNQRPDNW_013671976.1 (Battelle et al., 2016)
Limulus polyphemus 2TYESHPVDLSNQRPDNW_013676581.1 (Battelle et al., 2016)
Centruroides sculpturatusGYESSPVDLSSHRSVMQLISGSMTSHDKVNGDQHSLGNW_019384406.1 (Schwager et al., 2017)
Euperipatoides kanangrensisNSYDNPVDLSSHRSSQQILPGSLGPSDKVNGDLVSLALN881712.1 (unpublished)
Naineris dendriticaDPNGHPVDLSHSRHIPHMIHGSLTPPDRVNGEPGSGL(Andrade et al., 2015)
Platynereis dumeriliiMASENPVDLSSRHVGGNHFPGTLTPPDKLNGDHNAHHKP293861.1 (Gazave et al., 2017)
Nephasoma pellucidumAGYETPVDLSSPRPCSHLIPGSLTPPDKINGEGITTS(Lemer et al., 2015)
Owenia sp.QPYENPVDLSRRHIKAHLIPGSLTPPDKINGDMVTMA(Andrade et al., 2015)
Octopus bimaculoidesNGFDNPMDLSNGKVVHLMPAGSLTPPDKISGDSISMANW_014678436.1 (Albertin et al., 2015)
Crassostrea gigasGGYENPMDLSSNKPGSHIVAGSLTPPEKINGDPGAMANW_011936122.1 (Zhang et al., 2012)
Lottia giganteaAGVENPVDLSNGRISSHLFTGSLTPPEKPNGDLVPMSNW_008708401.1 (Simakov et al., 2013)
Notospermus geniculatusVQYDNPIDLSNRLEGNHMIPGSLTPPDKVNGDMVPLPGFRY01035878.1 (Luo et al., 2018)
Malacobdella grossaLHYDNPLDLTNRLDEGSGIAGSMTPPDGGKGNDLDLQ(Whelan et al., 2014)
Lingula anatinaGGYENPMDLSRRTEMAHMIPGNLTPPDKVNGEMVPMAGDJY01029776.1 (Luo et al., 2015)
Phoronis australisQHDNRPMDLSSRGQHSHLIAGSLTPPDKVNGDVVSMAGFSC01078935.1 (Luo et al., 2018)
Procotyla fluviatilisETLFEPLDLRSPIGVGAKZ01044347.1 (unpublished)
Brachionus koreanusAKDETPIDLSSKKSKGBXV02009219.1 (Lee et al., 2015)
Xenoturbella bockiKRYSAPLNLTVHDKCDVRVLGRLTPPDKQHVNNDVGA(Brauchle et al., 2018)
  1. Shown are alignments of short linear amino acid motifs (bold) in the N-terminal region of Su(H) proteins that mediate direct recruitment of the co-repressors CtBP and Gro. Column at right shows the source of the corresponding sequence data, with accession numbers and publication citations indicated.

Extending this sequence analysis to other protostome phyla led to the finding that a similar PϕDϕS motif occurs in the N-terminal region of Su(H) from a large number of molluscs and annelids, as well as from multiple Nemertea, Brachiopoda, Phoronida, and monogonont rotifers, and also from some flatworms (Table 1). It is notable, by contrast, that we do not find CtBP-binding motifs present in Su(H) from nematodes. Nevertheless, given the broad phylogenetic distribution of the PϕDϕS motif in Su(H) from both Ecdysozoa and Lophotrochozoa, our observations strongly suggest that direct recruitment of CtBP by Su(H) is ancestral in the protostomes.

To verify that the shared PϕDϕS motif in protostome Su(H) proteins can indeed mediate direct recruitment of CtBP, we carried out an in vitro pulldown assay using GST-tagged Drosophila CtBP (bound to Glutathione Sepharose beads) and a His-tagged fragment of Strigamia maritima Su(H) (Figure 3A). We found that the two proteins do interact directly and robustly, in a manner that is dependent on the integrity of the PVDLS motif in Strigamia Su(H).

Direct binding of co-repressor proteins by Su(H) from the centipede Strigamia maritima.

(A) The PVDLS motif in the N-terminal region of Su(H) from the centipede Strigamia maritima directly binds Drosophila CtBP. A His-tagged 116-aa segment of the Strigamia Su(H) protein, bearing a PVDLS recruitment motif for CtBP, binds strongly to GST-dCtBP (WT, lane 2). Mutation of the motif to alanines (AAAAA) abolishes this interaction (PVDLSmut, lane 1). The results shown in this panel have been replicated in eight additional experiments, utilizing three independent isolations of GST-CtBP protein from bacterial cultures. (B) The conserved GSLTPPDKV motif in the N-terminal region of Strigamia Su(H) directly binds Drosophila Gro. His-tagged E(spl)mβ-HLH protein, which bears a C-terminal WRPW motif that recruits Gro, is used as a binding control. Wild-type (WT) HLHmβ binds GST-Gro (lane 2), while a truncated version of the protein lacking the WRPW motif (lane 1) fails to bind. A synthetic version of HLHmβ in which the WRPW motif has been replaced by the wild-type GSLTPPDKV motif also binds GST-Gro efficiently (lane 4), while a mutant version in which GSLTPPDKV is replaced by alanines (AAAAAAAAA) shows extremely weak binding (lane 3). No binding of any of the His-tagged proteins to GST alone is observed, even with substantially greater amounts of GST compared to GST-Gro. The results shown in this panel have been replicated in seven additional experiments, utilizing five independent isolations of GST-Gro protein from bacterial cultures.

https://doi.org/10.7554/eLife.48115.006

A novel conserved motif in protostome Su(H) binds the Gro co-repressor

In addition to a PϕDϕS CtBP-binding motif, we have found that Su(H) from a wide variety of protostomes includes a novel motif similar to GSLTPPDKV (Table 1). Where present, this sequence typically lies a short (but variable) distance C-terminal to the PϕDϕS motif, also within the non-conserved N-terminal region of the protein (Supplementary file 3). The GSLTPPDKV motif is particularly prevalent in Su(H) from the Trochozoa, which includes annelids, sipunculans, molluscs, nemerteans, brachiopods, and phoronids (Kocot et al., 2017). Among the Ecdysozoa, it appears consistently in Su(H) from Crustacea and Myriapoda, and in small subsets of both Hexapoda (Ephemeroptera, Odonata, Zygentoma, Archaeognatha, Diplura, and Collembola) and Chelicerata [harvestmen (Opiliones) and Scorpiones]. The motif is absent from Su(H) in all other insect orders, and we have not found it so far in Su(H) from nematodes, flatworms, rotifers, or tardigrades; it is, however, found in the onychophoran Euperipatoides kanangrensis (Table 1). Perhaps surprisingly, the motif is present in Su(H) from the acorn worms Saccoglossus kowalevskii and Ptychodera flava (Simakov et al., 2015), which are hemichordates (deuterostomes).

Using an in vitro pulldown assay, we tested the possibility that the GSLTPPDKV motif mediates binding of the Gro co-repressor (Figure 3B). Indeed, we find that GST-tagged Gro protein interacts strongly with a His-tagged protein bearing this motif at its C-terminus, and that this binding is abolished when the motif is replaced by alanine residues. We conclude that Su(H) from a broad range of protostomes is capable of directly recruiting both CtBP and Gro (Table 1), and that this capacity is hence very likely to be ancestral in this clade.

Retention of the hybrid state: Species that have both Hairless and the co-repressor-binding motifs in Su(H)

The evolutionary emergence of Hairless as an adaptor protein capable of mediating the indirect recruitment of both Gro and CtBP to Su(H) might be expected to relieve a selective pressure to retain the ancestral Gro- and CtBP-binding motifs in Su(H) itself. And indeed, we find that Su(H) from multiple insect orders comprising the Neoptera lacks both of these sequences (Figure 2B). Strikingly, however, we have observed that Crustacea and a small group of Hexapoda retain both traits (Figure 2B). Thus, multiple representatives of the Branchiopoda, Malacostraca, and Copepoda, along with Ephemeroptera, Odonata, Zygentoma, Archaeognatha, Diplura, and Collembola, have both a canonical Hairless protein (including its Gro- and CtBP-binding motifs) and Gro- and CtBP-binding motifs within Su(H). These clades, then, appear to have retained a ‘hybrid intermediate’ state (Baker et al., 2012) characterized by the presence of both co-repressor recruitment mechanisms.

Myriapods and Chelicerates encode a protein with similarity to Hairless

While canonical Hairless proteins are confined to the Pancrustacea, we have discovered that the genomes of Myriapods and Chelicerates nevertheless encode a protein with intriguing similarities to Hairless. Most notable is the presence of a motif that strongly resembles the ‘Su(H)-binding domain’ (SBD) of Hairless, which mediates its high-affinity direct interaction with Su(H) (Figure 1; Figure 4A). We will refer to these proteins as ‘S-CAPs’; the basis for this designation will be made clear in forthcoming figures. We note that the occurrence of this protein in the centipede Strigamia maritima has also recently been reported by Maier (2019). In the Pancrustacea, the N-terminal and C-terminal halves of the Hairless SBD are encoded by separate exons (Figure 4B). Strikingly, the related motif in Myriapod and Chelicerate S-CAPs is likewise encoded by separate exons, with exactly the same splice junction as in Hairless (Figure 4B). We believe that this is highly unlikely to be coincidental, and is instead strongly suggestive of an evolutionary relationship between Hairless and S-CAPs.

S-CAP proteins in Myriapods and Chelicerates contain a Hairless-like domain that binds Su(H).

(A) Alignment of the Suppressor of Hairless Binding Domain (SBD) in Drosophila melanogaster (Dmel) Hairless with the related motif in the S-CAP proteins from a representative set of Myriapods and Chelicerates. Numbers flanking each sequence segment represent amino acid positions within the protein. The contiguous SBD motif is highlighted in bold. Pairwise amino acid sequence identities within the motifs are indicated by vertical lines; conservative substitutions are indicated by + signs. Amino acids in Hairless that have been shown to make direct contact with Su(H) [including the non-contiguous tryptophan (W) residue] (Yuan et al., 2016) are highlighted in red. Hydrophobic residues nearly always found immediately adjacent to the W are underlined. Species names are as follows: Smar (Strigamia maritima); Hsub (Hydroschendyla submarina) (Fernández et al., 2016); Agir (Anopsobius giribeti) (Fernández et al., 2016); Onosp (Onomeris sp.) (Rodriguez et al., 2018); Slm (Sigmoria latior munda) (Rodriguez et al., 2018); Ptep (Parasteatoda tepidariorum); Hruf (Hypochthonius rufulus) (Bast et al., 2016); Ssca (Sarcoptes scabiei); Vjac (Varroa jacobsoni) (Techer et al., 2019); Iper (Ixodes persulcatus); Mmar (Mesobuthus martensii) (Cao et al., 2013); Lpol (Limulus polyphemus). We note that Maier (2019) has previously described the presence of the SBD-like element in the Strigamia maritima sequence. (B) SBD motifs in both Hairless and S-CAP proteins (red) are encoded in two exons with the same splice junction (indicated by /; see gray highlight). Pairwise amino acid sequence identities within the motifs are indicated by vertical lines; conservative substitutions are indicated by + signs. Species names as in A, except for Isca (Ixodes scapularis). (C) Spider S-CAP protein binds directly to Drosophila Su(H) in vitro. In all panels, lanes 1–4 represent the indicated His-tagged segments of wild-type Drosophila (Dmel) Hairless (lane 1); Dmel Hairless bearing alanine substitutions for each of five SBD residues shown to contact Su(H) (lane 2); wild-type S-CAP from the spider Parasteatoda tepidariorum (Ptep) (lane 3); Ptep S-CAP bearing the same five alanine substitutions (lane 4). Input levels of these His-tagged proteins for each experiment are shown in the respective ‘input’ panels. Remaining two panels show the results of pulldown assays using Sepharose beads bearing only GST (left side) or GST-Su(H) (right side). Left: No binding of the His-tagged proteins to GST alone is observed. Right: Wild-type Dmel Hairless binds efficiently to GST-Su(H) (lane 1); this interaction is severely reduced by the introduction of the five alanine substitutions (lane 2). Wild-type Ptep S-CAP likewise binds to GST-Su(H) (lane 3), while no binding is observed with the alanine-substitution mutant (lane 4); the same result is obtained even when the amount of input Ptep S-CAPs (wild-type and mutant) is increased by a factor of 10 (lanes 3 and 4, 10X). Amounts of GST and GST-Su(H) on the beads are shown in the Coomassie stains below the corresponding pulldown lanes. The results shown in this panel have been replicated in two additional experiments, including one utilizing new isolations of GST-Su(H) and His-tagged proteins.

https://doi.org/10.7554/eLife.48115.007

A recent structural analysis of the Su(H)-Hairless protein complex identified several residues in the Hairless SBD that are involved in binding to the C-terminal domain (CTD) of Su(H) (Yuan et al., 2016) (Figure 4A). These include four hydrophobic amino acids in the main body of the SBD (L235, F237, L245, and L247; these are highlighted in red in Figure 4A). Note that the Myriapod and Chelicerate S-CAP motifs share these same residues. In addition, a tryptophan (W258) C-terminal to the main body of the Hairless SBD also participates in binding to Su(H) (Figure 4A). Myriapod and Chelicerate S-CAPs all include a tryptophan residue at a similar position C-terminal to the main SBD-like domain (Figure 4A). Moreover, this particular W residue in both Hairless and the S-CAPs is followed by a hydrophobic residue, typically V or I. These sequence features, we suggest, is further strong evidence of a common ancestry for the respective segments of Hairless and S-CAPs.

A third structural similarity between Hairless and S-CAPs is the presence in the latter of one or more short linear motifs capable of binding the CtBP co-repressor (Figure 5A). These motifs typically reside in the C-terminal half of the S-CAPs, superficially resembling the C-terminal location of Hairless's CtBP recruitment motif.

Figure 5 with 1 supplement see all
Sequence characteristics of S-CAP proteins in Myriapods and Chelicerates.

(A) Diagrams of representative examples of Myriapod and Chelicerate S-CAP proteins, denoting locations of SBD motifs and CtBP recruitment motifs. Scale and protein sizes are indicated. (B) Chelicerate, but not Myriapod, S-CAP proteins share N-terminal ELM2 and SANT domains with an MTA zinc-finger protein from the same species. Scale and protein sizes are indicated. (C) Shared ELM2 and SANT domains in Chelicerate MTA and S-CAP proteins are encoded in two exons with the same splice junction (indicated by /; red arrows). Pairwise amino acid sequence identities within the motifs are indicated by vertical lines; conservative substitutions are indicated by + signs. Species names as in Figure 4A.

https://doi.org/10.7554/eLife.48115.008

A table listing representative examples of Myriapod and Chelicerate S-CAPs is provided as Supplementary file 4, and an annotated FASTA file of their amino acid sequences is included as Supplementary file 5. It is important to note that we have not found non-Hairless S-CAPs in the Pancrustacea.

Spider S-CAP binds to Drosophila Su(H)

Given the clear sequence similarity between the Hairless SBD and the SBD-like motif in Myriapod and Chelicerate S-CAPs, we investigated whether the latter motif is likewise capable of mediating direct binding to Su(H). As noted above, the Hairless SBD interacts specifically with the CTD of Su(H). Since this domain in Su(H) is very highly conserved throughout the Bilateria and Cnidaria, we thought it reasonable to utilize Drosophila Su(H) for this binding assay. As shown in Figure 4C, we find that a 200-amino-acid segment of S-CAP from the spider Parasteatoda tepidariorum binds directly to Drosophila Su(H) in vitro. This interaction depends strictly on the integrity of the five residues that in Hairless have been shown to contact the Su(H) CTD (highlighted in red in Figure 4A).

Given the presence of one or more CtBP recruitment motifs in the Myriapod and Chelicerate S-CAP proteins (Figure 5A), along with the ability of their SBD-like domains to bind Su(H) (Figure 4C), we have designated these as ‘Su(H)-Co-repressor Adaptor Proteins’ (S-CAPs).

Chelicerate S-CAP proteins are related to Metastasis-associated (MTA) proteins

In addition to their similarities to Hairless, the S-CAP proteins of Chelicerates include two regions with strong sequence homology to the Metastasis-associated (MTA) protein family, which is highly conserved among Metazoa. The MTA proteins play an important role in transcriptional regulation via their function as core components of the nucleosome remodeling and deacetylase (NuRD) complex (Allen et al., 2013; Burgold et al., 2019). The N-terminal half of MTAs includes four well-defined functional domains: BAH (Bromo-Adjacent Homology), ELM2 (Egl-27 and MTA1 homology), SANT (Swi3, Ada2, N-CoR, and TFIIIB), and GATA-like zinc finger (Millard et al., 2014) (Figure 5B). Of these, the ELM2 and SANT domains are retained at the N-terminal end of Chelicerate S-CAPs (Figure 5B; Figure 5—figure supplement 1A). This is highly likely to have functional significance, as the ELM2 and SANT domains of MTA proteins work together to recruit and activate the histone deacetylases HDAC1 and HDAC2 (Millard et al., 2013). Further suggesting homology between Chelicerate S-CAPs and MTAs is the observation that their shared ELM2 and SANT domains are each encoded by two exons with exactly the same splice junction (Figure 5C).

It is noteworthy that, despite sharing the SBD-like and CtBP recruitment motifs of Chelicerate S-CAPs, the available Myriapod S-CAP protein sequences lack the N-terminal ELM2 and SANT homologies with MTA proteins (Figure 5B). Consistent with this, the SBD motif in Myriapod S-CAPs lies much closer to the protein’s N terminus than the SBD motif in Chelicerate S-CAPs, suggesting that simple loss of the ELM2/SANT-encoding exons might underlie this difference between the two S-CAP clades. Likewise, Hairless proteins are devoid of clear similarities to MTAs.

In addition to their SBD and ELM2/SANT domains, Chelicerate S-CAPs share a third region of homology that lies between the ELM2 and SANT sequences (Figure 5—figure supplement 1A). This region is absent from both Hairless and the Myriapod S-CAPs. Conversely, Myriapod S-CAPs include a segment of sequence similarity that is not found in either Hairless or Chelicerate S-CAPs (Figure 5—figure supplement 1B).

Conserved microsynteny between MTA and S-CAP/Hairless genes

Our analysis of the genomic locations of genes encoding MTA proteins in Arthropoda, Hairless in Pancrustacea, and S-CAPs in Myriapods and Chelicerates has yielded the surprising finding that proximate or near-proximate linkage between MTA and Hairless genes or between MTA and S-CAP genes is broadly conserved among arthropods (Figure 6; Supplementary file 1; Supplementary file 4). Thus, in the centipede Strigamia maritima, the gene encoding S-CAP lies immediately upstream of that encoding MTA, in the same orientation (Figure 6; Supplementary file 4). A similar linkage relationship between S-CAP and MTA genes is seen in many arachnids, including the spiders Nephila clavipes (Supplementary file 4) and Parasteatoda tepidariorum (Figure 6; Supplementary file 4) and the mites Achipteria coleoptrata and Sarcoptes scabiei (Supplementary file 4). Likely due at least in part to its history of whole-genome duplication (Nossa et al., 2014; Kenny et al., 2016), the horseshoe crab Limulus polyphemus (representing the Merostomata/Xiphosura) has three paralogous copies of this same S-CAP-MTA linkage pairing (Supplementary file 4). Some exceptions to this pattern do exist. In the genomes of the mites Metaseiulus occidentalis (Supplementary file 4) and Varroa destructor (Techer et al., 2019), for example, the genes encoding S-CAP and MTA are far separated from each other.

Genes encoding both Hairless and S-CAP proteins are frequently located immediately adjacent to an MTA gene.

Separate scale for each diagram is shown at the left. Three examples are shown for Hairless: the carpenter bee Ceratina calcarata (Ccal), the wheat aphid Schizaphis graminum (Sgra) (QEWZ01001380.1), and the lady beetle Harmonia axyridis (Haxy). Note that microsynteny is often preserved even when gene locations and relative orientations are changed. One example each is shown for S-CAP in Myriapods [the centipede Strigamia maritima (Smar)] and Chelicerates [the house spider Parasteatoda tepidariorum (Ptep)]. See also Supplementary file 1 and Supplementary file 4.

https://doi.org/10.7554/eLife.48115.010

Close, typically adjacent, linkage between Hairless and MTA genes is likewise widely observed in the genomes of Pancrustacea. Among the Hexapoda, this pattern can be found in many different orders (Supplementary file 1), including Diptera, Lepidoptera, Coleoptera (Figure 6), Hymenoptera (Figure 6), Psocodea, Hemiptera (Figure 6), Thysanoptera, Blattodea, Orthoptera, Odonata, and Collembola. Among the Vericrustacea, adjacent linkage of Hairless and MTA is seen in the shrimp Triops cancriformis (Notostraca) (Supplementary file 1). Nevertheless, exceptions are readily found, even within the same orders as above (Supplementary file 1). Examples include Drosophila melanogaster, Ceratitis capitata, and Lucilia cuprina (Diptera; Supplementary file 1), Bicyclus anynana (Lepidoptera), Anoplophora glabripennis, Dendroctonus ponderosae, and Nicrophorus vespilloides (Coleoptera), and Cimex lectularius (Hemiptera; Supplementary file 1).

Interestingly, in some instances Hairless/MTA microsynteny is preserved, but the genes’ relative orientation is different (Figure 6; Supplementary file 1). Thus, in the aphids — in contrast to other Hemiptera — MTA lies downstream of Hairless, but in the opposite orientation (Figure 6). In the beetle Harmonia axyridis (Coleoptera), MTA lies upstream of Hairless (Figure 6).

Despite the multiple instances in which it has been lost, we believe that the most parsimonious interpretation of our analysis is that close linkage between MTA and S-CAP/Hairless genes is ancestral in the respective taxa (Myriapods/Chelicerates and Pancrustacea). We leave for the Discussion our proposed interpretation of the evolutionary significance of this adjacency.

Discussion

The evolution of Hairless represents a shift from the ancestral and dominant paradigm of direct co-repressor recruitment by Su(H)

Our analysis of sequences from a broad range of protostomes strongly suggests that direct recruitment of the CtBP and Gro co-repressors by Su(H) is ancestral in this clade. This is consonant with the fact that direct co-repressor recruitment by DNA-binding repressor proteins in general is a dominant paradigm among Metazoa. This evokes the intriguing question of what might have led to the loss of direct recruitment by Su(H) in the Neoptera (see Figure 1B) and its replacement by Hairless-mediated indirect recruitment? Does Hairless provide some advantageous functional capacity? Note that this is not intended to suggest that Hairless must be an evolutionary adaptation per se (Lynch, 2007); rather, we are asking: What capability might it have conferred that would lead to its retention and the subsequent loss of the recruitment motifs in Su(H)?

One appealing (but of course speculative) possibility is that Hairless may have permitted Su(H) for the first time to recruit both CtBP and Gro simultaneously to the same target genes. As we have noted, the apparently ancestral PϕDϕS and GSLTPPDKV motifs in protostome Su(H) typically lie quite close to each other in the protein’s linear sequence (Supplementary file 3). CtBP (~400 aa) and Gro (~700 aa) are both large proteins that engage in oligomerization as part of their functional mechanism (Song et al., 2004; Bhambhani et al., 2011). It is very unlikely that both could bind stably to DNA-bound Su(H) at the same time. In contrast, the Gro and CtBP recruitment motifs in Hairless are far apart in the linear sequence (Figure 1A) and are separated by a region predicted to be largely disordered (Figure 1—figure supplement 1). We suggest that this might be compatible with simultaneous recruitment of the two co-repressors.

Whatever may have been the selective forces that led to the loss of direct co-repressor recruitment by Su(H) in the Neoptera and its replacement by Hairless-mediated indirect recruitment, Hairless is a notable evolutionary novelty for having permitted the unusual abandonment of an ancestral and highly conserved paradigm. We suggest that this represents a striking example of ‘developmental system drift’ (True and Haag, 2001), in which a common output (widespread ‘default repression’ of Notch pathway target genes) is achieved via distinct molecular mechanisms in different species.

A possible evolutionary pathway for the appearance of Hairless

We have described here several findings that we believe have important implications for an attempt to reconstruct the history of Hairless as an evolutionary novelty. First, we observe that Hairless is apparently confined to the Pancrustacea, wherein it is widely distributed among diverse taxa (Figure 2A; Supplementary file 1). Second, we have discovered in the sister groups Myriapoda and Chelicerata a protein (S-CAP) with clear sequence homology to the Su(H)-binding domain (SBD) of Hairless (Figure 4A). Significantly, in both Hairless and the S-CAPs these motifs are encoded by contributions from two exons, with the associated splice junction in precisely the same location (Figure 4B; Supplementary file 4). Third, we find that S-CAPs in the Chelicerata include in their N-terminal region strong homology to the ELM2 and SANT domains of MTAs, which themselves are highly conserved among Metazoa, and therefore would have been present in the arthropod common ancestor (Figure 5B,C). Finally, our analysis indicates that close, usually adjacent, linkage of Hairless and MTA genes (in the Pancrustacea) and between S-CAP and MTA genes (in the Myriapoda and Chelicerata) is widespread (Figure 6; Supplementary file 1; Supplementary file 4), and hence very likely to be ancestral, in these taxa.

While any attempt to infer the sequence of evolutionary events that led to the appearance of Hairless is necessarily speculative, we believe that the above findings offer substantial support for the following hypothetical pathway. We propose that in a deep arthropod ancestor a tandem duplication of the MTA gene occurred. One copy retained the strong sequence conservation (and presumably ancestral function) of metazoan MTA genes, while the second copy diverged very substantially, eventually encoding a protein that had lost all but the ELM2 and SANT domains of the MTA ancestor. The extensive reconfiguration of this paralog also included the eventual acquisition of the SBD motif and the addition of one or more CtBP recruitment motifs (see Figure 7 for some possible sources of these components). In the Myriapod lineage, even the ELM2 and SANT domains were eventually lost. In the Pancrustacea, we suggest that this same divergent MTA paralog evolved to become Hairless. Beyond the alterations described for the Myriapoda, this would have involved the acquisition of sequences encoding additional now-conserved domains and motifs, including the Gro recruitment motif (Supplementary file 2). This radical evolutionary transformation resulted in a protein with little or no remaining homology to its MTA ancestor, and with an entirely novel regulatory function (Holland et al., 2017).

Speculative sources for key elements of the Hairless and S-CAP proteins.

Note that these are intended to be only illustrative examples; other sources are of course possible. (A) A C-terminal segment of the highly conserved Yippee-like protein (Roxström-Lindquist and Faye, 2001; Hosono et al., 2004) is closely related to the C-terminal half of Hairless and S-CAP SBDs. Upper diagram is a sequence alignment of the entire Yippee-like proteins from Drosophila melanogaster (Dm) and Homo sapiens (Hs). Aligned below are contiguous SBD motifs from Drosophila Hairless and five Myriapod and Chelicerate S-CAPs; their C-terminal halves are shown in bold. Two leucine (L) residues shown to make direct contact with Su(H) (Yuan et al., 2016) are highlighted in red. Amino acid sequence identities are indicated by vertical lines; conservative substitutions are indicated by + signs. Other species names as in Figure 4A. (B) As shown in the gene diagram at the bottom, the CtBP recruitment motif in Hairless is encoded by a very small exon located at the extreme 3’ end of the gene [example is from the Oriental fruit fly Bactrocera dorsalis (Bdor; JFBF01000273.1); scale indicated]. A pre-existing gene encoding a protein that utilizes the same PLNLS recruitment motif is a possible source of this exon. Example shown is a portion of the senseless gene from the red flour beetle Tribolium castaneum (Tribolium Genome Sequencing Consortium, 2008). Senseless directly recruits the CtBP co-repressor via the PLNLS motif (Miller et al., 2014). This portion of the protein is encoded in exon 2; splice junction is indicated by a red /. Aligned beneath it is the last exon of the Bdor Hairless gene, illustrating its splice junction in the same frame as senseless exon 2.

https://doi.org/10.7554/eLife.48115.011

In this context, it is of interest that the Drosophila Mi-2/Nurd complex — which includes the MTA protein — has recently been shown to engage in direct repression of multiple Notch pathway target genes, independent of both Su(H) and Hairless (Zacharioudaki et al., 2019). Whether this activity preceded the emergence of Hairless is unknown, but the possibility that it is in some way connected to Hairless’s evolutionary history is indeed intriguing.

Materials and methods

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Antibodyanti-HIS G antibody, mouse monoclonalInvitrogenCAT#46–1008 (now ThermoFisher CAT#R940-25), RRID:AB_25565571:5000 dilution
AntibodyGOAT anti-mouse HRP, polyclonalJackson Immuno-researchCAT#115-035-003, RRID:AB_100152891:10000 dilution
Recombinant DNA reagentGST-dCtBP pGEX-5X-3 cloneNibu et al., 1998Construct encoding GST-tagged Drosophila CtBP for expression in E. coli
Recombinant DNA reagentGST-Gro pGEX-KG cloneThis paperConstruct encoding GST-tagged Drosophila Groucho for expression in E. coli
Recombinant DNA reagentGST-Su(H) pGEX-KG cloneBailey and Posakony, 1995Construct encoding GST-tagged Drosophila Su(H) for expression in E. coli
Recombinant DNA reagentHIS-H192-389 WT pRSET-C cloneThis paperHIS-tagged expression construct encoding amino acids 192–389 of Drosophila Hairless, synthesized by GeneWiz, Inc, and codon-optimized for expression in E. coli
Recombinant DNA reagentHIS-H192-389 5AMUT pRSET-C cloneThis paperHIS-tagged expression construct encoding amino acids 192–389 of Drosophila Hairless with five alanine substitutions, synthesized by GeneWiz, Inc, and codon-optimized for expression in E. coli
Recombinant DNA reagentHIS-HLHmBetaSmar WT pRSET-C cloneThis paperHIS-tagged expression construct encoding Drosophila HLHmBeta with the last four amino acids (WRPW) replaced with the sequence GSLTPPDKV
Recombinant DNA reagentHIS-HLHmBetaSmar MUT pRSET-C cloneThis paperHIS-tagged expression construct encoding Drosophila HLHmBeta with the last four amino acids (WRPW) replaced with the sequence AAAAAAAAA
Recombinant DNA reagentHIS-HLHmBetaWT pRSET-C cloneThis paperHIS-tagged expression construct encoding full-length Drosophila HLHmBeta
Recombinant DNA reagentHIS-HLHmBetatrunc pRSET-C cloneThis paperHIS-tagged expression
construct encoding Drosophila HLHmBeta with the last four amino acids (WRPW) deleted
Recombinant DNA reagentHIS-PtepSCAP233-432 WT pRSET-C cloneThis paperHIS-tagged expression construct encoding amino acids 233–432 of Parasteatoda tepidariorum S-CAP, synthesized by GeneWiz, Inc, and codon-optimized for expression in E. coli
Recombinant DNA reagentHIS-PtepSCAP233-432 5AMUT pRSET-C cloneThis paperHIS-tagged expression construct encoding amino acids 233–432 of Parasteatoda tepidariorum S-CAP with five alanine substitutions, synthesized by GeneWiz, Inc, and codon-optimized for expression in E. coli
Recombinant DNA reagentHIS-SmarSu(H)ex2-3 WT pRSET-C cloneThis paperHIS-tagged expression construct containing exons 2–3 of Strigamia maritima Su(H), synthesized by GeneWiz, Inc, and codon-optimized for expression in E. coli
Recombinant DNA reagentHIS-SmarSu(H)ex2-3 mut pRSET-C cloneThis paperHIS-tagged expression construct containing exons 2–3 of Strigamia maritima Su(H) with a PVDLS > AAAAA coding mutation, synthesized by GeneWiz, Inc, and codon-optimized for expression in E. coli
Recombinant DNA reagentpGEX-5X-3Sigma (formerly Amersham; discontinued)CAT#28-9545-53
Recombinant DNA reagentpRSET-CInvitrogenCAT#V35120
Commercial assay or kitChem Illumination ReagentsPierce ECL Western Blotting SubstrateCAT#32209
Resource, sequence databaseNCBINCBIRRID:SCR_006472
Software, algorithmNCBI BLASTNCBIRRID:SCR_004870
Software, algorithmGenePaletteSmith et al., 2017;Rebeiz and Posakony, 2004;http://www.genepalette.org
Software, algorithmDNA StriderMarck, 1988; Douglas, 1995
Software, algorithmBlastStation-Local64TM Software, Inc

Sequence searches, analysis, and annotation

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Genome and transcriptome sequences encoding Hairless, Suppressor of Hairless, S-CAP, and MTA proteins from a wide variety of species were recovered via BLAST searches, using either the online version at the NCBI website (Boratyn et al., 2013) or the version implemented by the BlastStation-Local64 desktop application (TM Software, Inc). Sequences were analyzed and annotated using the GenePalette (Rebeiz and Posakony, 2004; Smith et al., 2017) and DNA Strider (Marck, 1988; Douglas, 1995) desktop software tools. Analysis of predicted disordered regions in Hairless was conducted using DISOPRED3 on the PSIPRED server (Buchan et al., 2013; Jones and Cozzetto, 2015).

Generation of constructs for GST pulldown experiments

Strigamia maritima Su(H) protein constructs to test CtBP binding

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A codon-optimized fragment corresponding to exons 2 and 3 from S. maritima Su(H) mRNA was synthesized by Genewiz, Inc, and cloned into pRSET-C using Acc65I and BamHI restriction sites. The CtBP-motif mutant was subsequently generated by overlap extension PCR using the primers HISsmarSUH-f (CGCTGGATCCGCGGCCAGTATGAC), HISsmarSUH-r (CCATGGTACCAGTTATGCGTGGTG), HISsmarSUHctbpm-f (AACCACgCCGcaGcTGcGgCTAACAGCCATCGCGGTGAAGGCGGCCAC), HISsmarSUHctbpm-r (GCTGTTAGcCgCAgCtgCGGcGTGGTTGTCGGCGAAGTGAGGGGTCAG). After sequence confirmation, this fragment was also cloned into pRSET-C using the same enzymes. Binding of these constructs to Drosophila melanogaster CtBP was assayed using GST alone and a GST-CtBP fusion protein (Nibu et al., 1998).

Constructs to test potential Gro-binding motif in Strigamia maritima Su(H)

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A truncated version of HLHmβ (HLHmβ-WRPWtrunc) was amplified from a pRSET-HLHmβ-WT construct using the primers HISmbeta-f (cgatggatccgaATGGTTCTGGAAATGGAGATGTCCAAG) and HISmbetatrunc-r (ccatggtaccagTCACATGGGGCCagaggtggagctggcctcgctgggcgc); a version of HLHmβ with the WRPW motif replaced with the amino acids GSLTPPDKV (HLHmβ+Smar-motifWT) was amplified from the WT construct with HISmbeta-f and mbetaSmarSuH-r (ccatggtaccagTCACACTTTATCAGGTGGAGTGAGAGAACCCATGGGGCCagaggtggagctggcc); and a version of HLHmβ with the WRPW motif replaced with a stretch of 9 alanine residues (HLHmβ+Smar-motifMUT) was amplified using HISmbeta-f and mbetaSmarSuHmut-r (ccatggtaccagTCAggctgccgctgcggctgccgctgctgcCATGGGGCCagaggtggagctggcc). Each construct was then subsequently cloned into pRSET-C using the restriction enzymes BamHI and Acc65I and sequence verified. Binding of these constructs to Drosophila melanogaster Gro was assayed using GST alone and a GST-Gro fusion protein. The latter construct was made by cloning the full-length Gro coding sequence into the pGEX-KG expression vector at the EcoRI and SalI restriction sites: gtggcgaccatcctccaaaatcggatctggttccgcgtggatccccgggaatttccggtggtggtggtggaattctaATG...TAAATCCACAAAACCATGCAGTTTTTTCATTTTGTAATAAGCTCGTATAGTTTTTATTACAACATGTTCGAAATCATGCAcccgggctgcaggaattcgatatcaagcttatcgataccgtcgactcgagctcaagcttaattcatcgtgactgactgacgatctg (underlined = pGEX KG vector; uppercase = gro cDNA; bold = gro start and stop codons; italic = linker)

S-CAP/Hairless constructs for Su(H) interaction analysis

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Codon-optimized fragments from Drosophila melanogaster Hairless (residues 192–389), and Parasteatoda tepidariorum cS-CAP (residues 233–432) as well as 5-alanine mutant substitutions (Dmel: GGRLQFFKDGKFILELARSKDGDKSGW - > GGRAQAFKDGKFIAEAARSKDGDKSGA; Ptep: VGSLKFFLGGRLVLKLNAQQDGGSGNKCQW - > VGSAKAFLGGRLVAKANAQQDGGSGNKCQA) were synthesized by Genewiz, Inc. Inserts were subsequently cloned into pRSET-C using the restriction enzymes BamHI and Acc65I. Binding of these constructs to Drosophila melanogaster Su(H) was assayed using GST alone and a GST-Su(H) fusion protein (Bailey and Posakony, 1995).

GST pulldowns using each of the above constructs were performed as previously described (Fontana and Posakony, 2009).

Synthesized, codon-optimized sequences

>Smar Su(H)ex2-3 WT (116 aa)

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CGCTGGATCCGCGGCCAGTATGACTACCCGCCGCCGTTAGCCAGCACATACAGCCGCGAGGCCGACCTGTGGAACGTGAACCTGGCCACCTACAGCAGCGCACCGACCACATGCACCGGTGCAACCCCGGCACCTAGCGTTACCGGTTTCTACGCCCAGGCCACCGGCAGCAACAGCGTTAGCCCGAGTAGCGTGAGCCTGACCACCCTGACCCCTCACTTCGCCGACAACCACCCGGTGGACCTGAGCAACAGCCATCGCGGTGAAGGCGGCCACCTGGATCTGGTGCGCTTCCAGAGCGACCGCGTGGATGCCTACAAGCACGCCAACGGCCTGAGCGTGCATATCCCGGACCACCACGCATAACTGGTACCATGG

>Smar Su(H)ex2-3 mut

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CGCTGGATCCGCGGCCAGTATGACTACCCGCCGCCGTTAGCCAGCACATACAGCCGCGAGGCCGACCTGTGGAACGTGAACCTGGCCACCTACAGCAGCGCACCGACCACATGCACCGGTGCAACCCCGGCACCTAGCGTTACCGGTTTCTACGCCCAGGCCACCGGCAGCAACAGCGTTAGCCCGAGTAGCGTGAGCCTGACCACCCTGACCCCTCACTTCGCCGACAACCACgCCGcaGcTGcGgCTAACAGCCATCGCGGTGAAGGCGGCCACCTGGATCTGGTGCGCTTCCAGAGCGACCGCGTGGATGCCTACAAGCACGCCAACGGCCTGAGCGTGCATATCCCGGACCACCACGCATAACTGGTACCATGG

>Dmel Hairless192-389 WT

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CGATGGATCCGAGCAGTGGTTGCAGCAGCAGCTGGCACTGCCAAAATTGGTAAAGGCAGCAACAGCGGTGGCAGTTTTGATATGGGCCGCACACCGATCAGCACCCACGGCAACAATAGTTGGGGTGGCTATGGCGGCCGTTTACAGTTCTTTAAAGATGGCAAGTTTATTTTAGAACTGGCCCGCAGCAAAGATGGCGATAAAAGCGGCTGGGTGAGTGTGACCCGCAAAACCTTTCGCCCGCCGAGTGCAGCAACCAGCGCAACCGTGACCCCTACCAGTGCCGTGACCACCGCCTACCCGAAGAATGAAAACAGCACCTCTTTAAGCTTCAGCGACGACAATAGCAGCATTCAGAGCAGCCCGTGGCAGCGTGATCAGCCGTGGAAACAGAGTCGTCCGCGCCGTGGCATCAGCAAAGAACTGTCTTTATTTTTCCACCGCCCGCGCAATAGTACACTGGGTCGTGCAGCCTTACGTACCGCAGCCCGCAAACGTCGTCGTCCGCATGAACCGCTGACCACCAGCGAAGATCAGCAGCCGATCTTTGCCACCGCAATCAAAGCCGAGAACGGTGATGATACTTTAAAAGCCGAAGCAGCCGAATAACTGGTACCATGG

>Dmel Hairless192-389 5Amut

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CGATGGATCCGAGCCGTTGTGGCAGCAGCAGCTGGCACTGCCAAAATCGGCAAAGGCAGCAATAGCGGTGGTAGCTTTGACATGGGCCGCACCCCGATTAGCACCCATGGCAACAACAGCTGGGGTGGTTATGGTGGTCGTGCCCAAGCTTTTAAAGACGGCAAGTTCATCGCCGAAGCCGCACGCAGCAAAGATGGCGACAAAAGCGGTGCCGTGAGCGTGACCCGCAAAACCTTTCGTCCGCCGAGTGCAGCAACCAGCGCAACCGTTACCCCGACCAGCGCAGTTACCACCGCCTACCCGAAAAACGAAAACAGCACCTCTTTAAGCTTTAGCGACGACAACAGCAGCATTCAGAGCAGCCCGTGGCAGCGCGATCAGCCGTGGAAACAGAGCCGTCCTCGTCGCGGCATCAGCAAAGAGCTGTCTTTATTCTTTCATCGCCCGCGCAATAGCACTTTAGGTCGTGCAGCACTGCGCACAGCAGCACGTAAACGTCGTCGCCCGCATGAACCGCTGACCACCAGCGAAGACCAGCAGCCGATTTTTGCCACCGCAATCAAAGCCGAGAACGGCGATGATACTTTAAAAGCAGAAGCAGCCGAATAACTGGTACCATGG

>Ptep s-CAP233-432 WT

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CGATGGATCCGAACCGTGAATACCGAAGATCCGCCGAAGGATAGCATCAACTTTCTGGACCACAGCCGCGTGACCGATCCGTGTAGTGCCGCAAGCGAAACCAGCCTGCCGCAGGATGTGCCGGCAACAAGCACCGTGGGCAGCCTGAAATTTTTTCTGGGCGGTCGCCTGGTGCTGAAATTAAACGCCCAGCAGGATGGCGGCAGCGGCAATAAATGCCAGTGGGTGCAGAGCAACGATCTGCCGAAACATAGCAACCATAACAAAAAAGATAAACATAAGAAAAAATTTGCACCGTATAGCTATAGCAGCAGCGGCACTCAGAAACCGCTGAAGAAAGGCGACGATACCAGTGCCGTGCCGGACTGTGATCCGAGCGGCATCAAAAAGCCGCGCCTGAAAGAGTACGAGACCAGCGAGAATAGCGCCCTGGGTCTGCTGCTGTGCAGCAGCAGTTGGACCCCGCCGGTTGCAGATGGTCAGGAGAGCATTGACGTGGACGATACCAGCAGCAAAACCAGCGAGGGCTATATTAGCCCGATCCTGAGCAACAATAGCCGCACCAGCAAAATCGACACCATCAAGCACGATTTTGCCAGCAACCCGAACACCTAACTGGTACCATGG

>Ptep s-CAP233-432 5Amut

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CGATGGATCCGAACCGTGAACACCGAAGACCCGCCGAAAGATAGCATCAACTTTTTAGACCATAGCCGCGTGACAGACCCGTGCAGTGCCGCAAGTGAAACCTCTTTACCGCAAGATGTGCCGGCAACCAGCACCGTGGGTAGCGCCAAAGCCTTTCTGGGCGGTCGTCTGGTGGCCAAAGCCAATGCCCAGCAAGATGGTGGTAGTGGTAACAAATGCCAAGCTGTGCAGAGCAACGATCTGCCGAAACACAGCAATCACAATAAGAAAGACAAACACAAGAAAAAATTTGCCCCGTATAGCTATAGCAGCAGCGGCACCCAGAAACCGCTGAAAAAAGGCGATGACACCAGCGCAGTGCCGGATTGCGATCCGAGCGGCATTAAGAAACCGCGTTTAAAGGAGTACGAGACCAGCGAAAACAGTGCTTTAGGTTTACTGCTGTGCAGCAGCAGTTGGACACCGCCGGTGGCCGATGGTCAAGAAAGTATCGATGTGGACGACACCAGCAGCAAAACCAGCGAAGGCTACATCAGCCCGATTCTGAGCAACAATAGCCGCACCAGCAAAATTGATACCATTAAACATGATTTTGCAAGCAATCCGAATACCTAACTGGTACCATGG

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Decision letter

  1. Patricia J Wittkopp
    Senior and Reviewing Editor; University of Michigan, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Hairless as a novel component of the Notch signaling pathway" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Patricia Wittkopp as the Senior and Reviewing Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

This paper examines the evolution of the Hairless gene and how it became part of the Notch signaling pathway. Solid experimental and computational analyses support the conclusions. It is a revealing case study for understanding how new protein functions evolve.

That said, there was extensive discussion post-review about the major contributions of this work, with concerns raised that the authors were inaccurately claiming to be the first to have discovered Hairless as part of the Notch signaling pathway. My own reading is that this is mostly an issue with word choices in the title and elsewhere in the paper that gave some reviewers this impression that the authors were claiming to have discovered Hairless as a new (novel) component of Notch signaling rather than describing the evolutionary novelty that lead to Hairless being a part of the Notch pathway in some species. For example, I suggest changing the title from "Hairless as a novel component of the Notch signaling pathway" to something like "Evolutionary recruitment of Hairless to the Notch signaling pathway" (although I don't really love "recruitment", as it sounds too active.).

Summary:

Suppressor of Hairless (SuH) is the major transcriptional effector of Notch signals. This study explores the evolutionary origins of Hairless and its mode of interaction with SuH and the two co-repressors Groucho (Gro)and the C-terminal Binding Protein (CtBP). The work persuasively argues that Hairless mediated recruitment of the co-repressors Gro and CtBP to SuH was evolutionarily preceded by direct interactions between SuH and co-repressors. Thus, ancestral direct recruitment of these co repressors, through specific sequences in each protein, evolved into a Hairless mediated (i.e. indirect) recruitment. Hairless proteins seem confined to the Pancrustacea while evidence is presented showing that Myriapods and Chelicerates do harbor a protein with Hairless like "qualities" but not Hairless.

More specifically, outside of the Pancrustacea Su(H) is able to directly recruit transcriptional repressors, which is argued to represent the ancestral phenotype. To support this hypothesis, the authors surveyed many arthropod Su(H) genes to identify repressor recruitment domains, and in so doing identified a novel Gro recruitment motif. In the Pancrustacea, a loss of Su(H)'s ability to directly recruit repressors is linked to the emergence of hairless. The main conclusion of the paper is that hairless evolved from S-CAP proteins found in myriapods and chelicerates. S-CAPs are similar to hairless in that they have a functional Su(H) binding and Groucho and CtBP recruitment motifs. The similarity to Hairless argues that the S-CAP proteins represent an intermediate step in Hairless evolution. This claim is supported by the fact that S-CAPs contain a functional Su(H) binding domain, which is encoded on different exons with exactly the same splice sites as the SBD found in hairless. The authors also identify chelicerate S-CAPs that retain conserved domains found in MTA proteins, and both hairless and S-CAP proteins maintain microsynteny with MTA in a large number of clades. These observations suggest that S-CAP proteins evolved by following tandem duplication of the MTA protein. Their data suggests a mechanism by which a critical component of animal biology (the ability to turn off Notch targets) could adapt a completely novel mechanism (a transition from direct to indirect recruitment of repressors to Su(H)) by tandem duplication and neofunctionalization of one paralog.

Essential revisions:

1) Revise to make it more clear throughout that there is a body of literature already establishing Hairless as part of the Notch signaling pathway, at least in Drosophila. Examples of reviewer comments related to this point: "I find the title of the paper somewhat misleading or disingenuous as there is a fairly deep literature establishing Hairless as a component of the Notch signaling pathway." and "The claim in the title, in the impact statement and in the text that Hairless is a novel component of the Notch signaling pathway is incorrect"

2) As one reviewer wrote "The discussion as to why this evolution has occurred and what possible benefits such a transition may have led to (e.g. simultaneous CtBP and Gro binding) is also interesting but still only speculation." and anther wrote "The speculative discussion in Figure 7 is interesting, but it was not clear how likely the putative sources are to being the actual source. Are these the only obvious choices or are there many more? As it stands there is no evidence to argue that these are the sources and it isn't clear that those data should be included without over emphasis on the speculative nature of this figure." Please moderate the Discussion to provide some more context for the likelihood of the particular possibilities described.

3) Please make it more clear that Maier (who has numerous papers related to Hairless evolution) previously reported the existence of a protein with properties like those described in this work.

https://doi.org/10.7554/eLife.48115.020

Author response

[…] There was extensive discussion post-review about the major contributions of this work, with concerns raised that the authors were inaccurately claiming to be the first to have discovered Hairless as part of the Notch signaling pathway. My own reading is that this is mostly an issue with word choices in the title and elsewhere in the paper that gave some reviewers this impression that the authors were claiming to have discovered Hairless as a new (novel) component of Notch signaling rather than describing the evolutionary novelty that lead to Hairless being a part of the Notch pathway in some species. For example, I suggest changing the title from "Hairless as a novel component of the Notch signaling pathway" to something like "Evolutionary recruitment of Hairless to the Notch signaling pathway" (although I don't really love "recruitment", as it sounds to active.).

We wish to make clear that we most certainly were NOT claiming in this paper to be reporting Hairless’s role in the Notch pathway for the first time. We find it very difficult to understand how this interpretation could have gained any traction, in view of the following obvious facts:

• Our lab has a long published history – extending back to 1991 – of investigating the nature and function of the Hairless gene and protein as a regulator of cell fate in the fly PNS. And we have made very substantial contributions to defining its role in the Notch pathway as an adaptor protein that mediates the indirect recruitment of two co-repressors to Suppressor of Hairless. As a result, we are of course fully aware of Hairless’s known membership in the pathway. We are utterly mystified how anyone could infer that we were attempting to mislead our colleagues in the field with an obviously false claim.

• The very first figure in our manuscript includes diagrams summarizing for the reader — especially one unfamiliar with the prior studies — Hairless’s function in the Notch pathway. The legend to Figure 1B began as follows in the original version: “Summary of Hairless’s mode of action as an adaptor protein that recruits the global co-repressors C-terminal Binding Protein (CtBP) and Groucho (Gro) to Suppressor of Hairless [Su(H)], the transducing transcription factor for the Notch (N) cell-cell signaling pathway.” Again, it is extremely puzzling how this could be viewed as in any way consistent with an attempt to claim that we were reporting on a new discovery. A summary figure like that would appear last, not first, in a paper describing Hairless’s function for the first time. (Indeed, Figure 1B is based on Figure 6 – the last one – in our 2002 paper in Genes and Development.)

• Our use of the word “novel” in the paper’s title (which seems to have evoked this concern) is fully consistent with standard nomenclature in the field of evolutionary biology. We of course meant to convey that Hairless is an evolutionary novelty. Moreover, even a superficial reading of the paper’s Abstract (never mind the paper as a whole) makes it very clear that it is about the evolution of Hairless, and not about its current function.

Essential revisions:

1) Revise to make it more clear throughout that there is a body of literature already establishing Hairless as part of the Notch signaling pathway, at least in Drosophila. Examples of reviewer comments related to this point: "I find the title of the paper somewhat misleading or disingenuous as there is a fairly deep literature establishing Hairless as a component of the Notch signaling pathway." and "The claim in the title, in the impact statement and in the text that Hairless is a novel component of the Notch signaling pathway is incorrect"

To avoid any possible further confusion, we have made several changes to the manuscript to solidify the reader’s understanding of the topic and intent of our paper.

a) Along the line suggested, we have changed the paper’s title to: “Evolutionary emergence of Hairless as a novel component of the Notch signaling pathway”.

b) We have altered the Impact Statement to read: “Hairless emerged as an evolutionarily novel regulatory protein that replaced an ancestral paradigm of direct co-repressor recruitment by Suppressor of Hairless, its partner transcription factor in the Notch signaling pathway.”

c) We have edited the legend to Figure 1B to start with the phrase “Summary of Hairless’s known mode of action.….”, and have added references to two review articles on Hairless function, including one by Maier.

With regard to other possible wording changes, we have searched the entire manuscript for the words “novel” and “novelty”. Excluding the title and Impact Statement (see above), “novel” appears five times — three times referring to the Gro recruitment motif we found in protostome Su(H)’s, and twice in sentences containing the word “evolutionary” (thus making our intent clear). “Novelty” appears only three times, in each case immediately preceded by the word “evolutionary”. Thus, we see no need for any additional edits (beyond those above) to address this first concern.

2) As one reviewer wrote "The discussion as to why this evolution has occurred and what possible benefits such a transition may have led to (e.g. simultaneous CtBP and Gro binding) is also interesting but still only speculation."

We fully agree that this suggestion is speculative, but we feel that we had already made that quite clear. Please note the following in the relevant part of the original Discussion:

- includes the phrase “…what might have led”….

- includes the phrase “…What capability might it have conferred….”

- begins “One appealing possibility….”

- begins “We suggest that this might be compatible….”

Nevertheless, to be fully explicit about the speculative nature of our suggestion, we have revised the opening sentence of the paragraph referred to as follows:

“One appealing (but of course speculative) possibility is that Hairless may have permitted Su(H) for the first time to recruit both CtBP and Gro simultaneously to the same target genes.”

And another wrote "The speculative discussion in Figure 7 is interesting, but it was not clear how likely the putative sources are to being the actual source. Are these the only obvious choices or are there many more? As it stands there is no evidence to argue that these are the sources and it isn't clear that those data should be included without over emphasis on the speculative nature of this figure." Please moderate the Discussion to provide some more context for the likelihood of the particular possibilities described.

We’re unclear as to the basis for this concern, since the original version of the manuscript contained multiple explicit indicators that we were offering only illustrative possibilities:

- Discussion: “The extensive reconfiguration of this paralog also included the eventual acquisition of the SBD motif and the addition of one or more CtBP recruitment motifs (see Figure 7 for some possible sources of these components).”

- The title of the Figure 7 legend reads as follows: “Speculative possible sources for key elements of the Hairless and S-CAP proteins.”

- The legend to Figure 7B included the following: “A pre-existing gene encoding a protein that utilizes the same PLNLS recruitment motif is a possible source of this exon. Example shown is a portion of the senseless gene….”

We believe the quoted wording had already made it quite clear that we were not claiming to have identified the actual sources of the protein segments referred to.

Still, to make this point even more explicit, we have added the following sentence after the title of the Figure 7 legend:

“Note that these are intended to be only illustrative examples; other sources are of course possible.”

3) Please make it more clear that Maier (who has numerous papers related to Hairless evolution) previously reported the existence of a protein with properties like those described in this work.

We thought we had already done this in the original version of the manuscript. Please note that the Results section explicitly included the following sentence: “We note that the occurrence of this protein in the centipede Strigamia maritima has also recently been reported by Maier (Maier, 2019).” Then, in the legend to Figure 4A (the sequence alignment panel illustrating the S-CAP similarities to the Hairless SBD), we again cited Maier’s paper in connection with the centipede protein.

However, to clarify this point further, we have now added a sentence to the Figure 4A legend crediting Maier more explicitly:

“We note that Maier (Maier, 2019) has previously described the presence of the SBD-like element in the Strigamia maritima sequence.”

https://doi.org/10.7554/eLife.48115.021

Article and author information

Author details

  1. Steven W Miller

    Division of Biological Sciences, Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, United States
    Contribution
    Supervision, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7610-6336
  2. Artem Movsesyan

    Division of Biological Sciences, Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, United States
    Contribution
    Investigation, Writing—original draft, Writing—review and editing
    Competing interests
    No competing interests declared
  3. Sui Zhang

    Division of Biological Sciences, Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, United States
    Contribution
    Investigation, Writing—original draft, Writing—review and editing
    Competing interests
    No competing interests declared
  4. Rosa Fernández

    Bioinformatics and Genomics Unit, Center for Genomic Regulation, Barcelona, Spain
    Present address
    Department of Life Sciences, Barcelona Supercomputing Center, Barcelona, Spain
    Contribution
    Resources, Investigation, Writing—review and editing
    Competing interests
    No competing interests declared
  5. James W Posakony

    Division of Biological Sciences, Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, United States
    Contribution
    Conceptualization, Data curation, Supervision, Funding acquisition, Investigation, Methodology, Writing—original draft, Writing—review and editing
    For correspondence
    jposakony@ucsd.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6377-1732

Funding

National Institute of General Medical Sciences (R01GM046993)

  • James W Posakony

National Institute of General Medical Sciences (R01GM120377)

  • James W Posakony

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We are especially grateful to our many colleagues who made their genome and transcriptome sequence assemblies freely available; without their generosity, this study would not have been possible. We thank Scott Rifkin and Mark Rebeiz for helpful discussion and input during the preparation of the manuscript. We also thank the following artists for making available the illustrations shown in Figure 2A: crab, by Firkin (https://openclipart.org/detail/270221/crab-silhouette); monarch butterfly, by carolemagnet (https://openclipart.org/detail/263384/monarch-butterfly); centipede, by Firkin (https://openclipart.org/detail/261126/centipede-3); spider, by liftarn (https://openclipart.org/detail/179190/spider); horseshoe crab, by Gosc (https://openclipart.org/detail/174556/horseshoe-crab); tick, by Juhele (https://openclipart.org/detail/279073/simple-tick-ixodes-ricinus-silhouette). This work was supported by NIH Grants R01GM046993 and R01GM120377 (to JWP).

Senior and Reviewing Editor

  1. Patricia J Wittkopp, University of Michigan, United States

Publication history

  1. Received: May 3, 2019
  2. Accepted: September 23, 2019
  3. Accepted Manuscript published: September 23, 2019 (version 1)
  4. Version of Record published: October 4, 2019 (version 2)
  5. Version of Record updated: October 8, 2019 (version 3)

Copyright

© 2019, Miller et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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