Cytosolic Interaction Between Deltex and Notch Ankyrin Repeats Implicates Deltex in the Notch Signaling Pathway

Development 120, 473-481 (1994) 473 Printed in Great Britain © The Company of Biologists Limited 1994

Cytosolic interaction between deltex and Notch ankyrin repeats implicates deltex in the Notch signaling pathway

Robert J. Diederich, Kenji Matsuno, Huey Hing and Spyros Artavanis-Tsakonas Howard Hughes Medical Institute and Department of Cell Biology, Boyer Center for Molecular Medicine, Yale University, 295 Congress Avenue, New Haven, Connecticut 06536-0812, USA

SUMMARY

Genetic data from Drosophila have suggested a functional members. In addition, we present a new Notch allele, Nsu42c, relationship between the novel cytoplasmic protein that is associated with a missense mutation within the ﬁfth encoded by the deltex locus and the transmembrane ankyrin repeat. In addition to representing a new class of receptor encoded by Notch. We have demonstrated a direct viable Notch allele, this mutation behaves similarly to interaction between these proteins from expression studies mutations of deltex and further implicates the ankyrin conducted in cultured cells, in yeast, and in the imaginal repeats in Notch function. wing disc. deltex binds speciﬁcally to the Notch ankyrin repeats, a region that is crucial for Notch signaling and that Key words: ankyrin repeats, deltex, Drosophila, Notch, signal constitutes the most conserved domain among Notch family transduction

INTRODUCTION et al., 1986). It is a large transmembrane protein with an extracellular domain containing 36 tandem EGF-like repeats and 3 Cell-cell interactions are an important feature of the cell-fate Notch/lin12 repeats. The intracellular domain bears several decision processes that occur during the development of mul- common structural motifs including 6 cdc10/SWI6/ankyrin ticellular organisms. In Drosophila, numerous genetic and repeats (ANK repeats; Breeden and Nasmyth, 1987; Lux et al., molecular studies of the Notch locus have shown that it plays 1990; Bennett, 1992; Blank et al., 1992; Michaely and Bennett, a central role in regulative events controlling cell fate in a 1992) a polyglutamine stretch known as ‘opa’, and a PEST variety of tissue types (Artavanis-Tsakonas and Simpson, motif (Stifani et al., 1992). The remarkable degree to which 1991; Greenwald and Rubin, 1992). The manner by which these motifs have been conserved in homologs isolated from Notch is thought to influence determinative events is indirect, mice (Kopan and Weintraub, 1993), rats (Weinmaster et al., that is, not through the direct specification of cellular fates. 1991, 1992; Franco del Amo et al., 1993), humans (Ellisen et Instead, recent experimental studies (Coffman et al., 1993; al., 1991; Stifani et al., 1992), and Xenopus (Coffman et al., Fortini et al., 1993) indicate that Notch activity delays differ- 1990) implies that they will have a common biochemical mode entiation, and in this manner renders precursor cells competent of action. In particular, ANK repeats, which constitute the most to receive and/or interpret any number of specific develop- conserved region (approx. 70% amino acid identity) between mental cues (e.g., Cagan and Ready, 1989). In loss of function Notch and its vertebrate counterparts (Stifani et al., 1992), are mutants, this inhibition is lost and cells assume default thought to mediate protein-protein interactions among diverse pathways of differentiation. For example, during the develop- groups of proteins, including those involved in signal trans- ment of the Drosophila nervous system, cells that normally duction processes and cytoskeletal interactions (Breeden and would become epidermis instead adopt a neural fate in the Nasmyth, 1987; Lux et al., 1990; Bennett, 1992; Blank et al., absence of Notch function. However, a salient feature of Notch 1992; Michaely and Bennett, 1992). Recent experimental activity is its pleiotropy. Notch is required for the proper spec- studies have demonstrated that the ANK repeats are crucial for ification of many other cell types, including those of the Notch-mediated signaling events (Rebay et al., 1993; see also compound eye, ovary, and mesoderm (Cagan and Ready, 1989; Roehl and Kimble, 1993). Ruohola et al., 1991; Xu et al., 1992; Corbin et al., 1991). Our genetic attempts (Xu et al., 1990) to identify interact- Similarly, the widespread expression patterns exhibited by ver- ing partners of Notch have uncovered a small number of genes tebrate Notch cognates suggest also a broad-based functional collectively called the ‘Notch group’ (Artavanis-Tsakonas and role in these species (Coffman et al., 1990, 1993; Ellisen et al., Simpson, 1991). To date, this set comprises the genes Delta, 1991; Weinmaster et al., 1991, 1992; Franco del Amo et al., Serrate, Enhancer of split [E(spl)], mastermind, strawberry 1992; Stifani et al., 1992; Kopan and Weintraub, 1993). notch, and deltex (Vässin et al., 1985; Fleming et al., 1990; The structure of the Notch protein is consistent with its Coyle-Thompson and Banerjee, 1993; Xu et al., 1990; Gorman inferred role as a signaling receptor (Wharton et al., 1985; Kidd and Girton, 1992). The products of Delta and Serrate are EGF-

474 R. J. Diederich and others homologous transmembrane proteins that have been shown to monoclonal antibody (line C458.2H; R. Fehon and S. Artavanis- interact with Notch as presumed ligands (Fehon et al., 1990; Tsakonas, unpublished data), which recognizes the Notch extracel- Heitzler and Simpson, 1991; Rebay et al., 1991). E(spl) and lular domain, and fixed in PLP fixative (Tomlinson and Ready, mastermind encode nuclear proteins, but no direct biochemi- 1987). Cells were then permeabilized and incubated with rat anti- cal links to Notch have been established (Smoller et al., 1990; deltex polyclonal or monoclonal (line C645.17A) antibodies Delidakis et al., 1991). No interacting partners in the form of (Busseau et al., 1994) followed by incubation with FITC- and Texas Red-conjugated goat anti-mouse and anti-rat antibodies (Jackson prospective ligands or cytoplasmic components have been ImmunoResearch Laboratories, Inc.) according to Fehon et al. uncovered for other Notch family members. (1990). Third instar larvae from transgenic line 124AD (Busseau et The relationship of deltex to Notch was first discovered (Xu al., 1994) were heat shocked and allowed to recover as outlined and Artavanis-Tsakonas, 1990; Xu et al., 1990) based upon the above to induce deltex expression. Dissected wing discs were fixed ability of loss-of-function deltex mutations to suppress, in a in PLP fixative and incubated simultaneously with anti-Notch and dominant manner, the lethality associated with certain het- anti-deltex antibodies. Confocal images were obtained as described eroallelic combinations of the Abruptex class of Notch alleles (Xu et al., 1992); cropping and pseudo-coloring were performed (so-called ‘negatively complementing’ alleles; Foster, 1975; using Adobe Photoshop (Adobe Systems, Inc.) computer program. Portin, 1975). The Abruptex mutations represent gain-of- Standard procedures (Harlow and Lane, 1988) were used for the function Notch alleles resulting from missense mutations in preparation of cell lysates, PAGE-electrophoresis (3-15% gel) and immunoblot analysis. The anti-Delta monoclonal antibody C584.9B specific EGF-like repeats (Hartley et al., 1987; Kelly et al., (Fig. 2C; R. Fehon and S. Artavanis-Tsakonas, unpublished data) 1987). That deltex is integrated within the Notch signaling was used in conjunction with goat anti-mouse peroxidase-conjugated pathway is suggested also by the gene dosage-dependent inter- secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.) actions it exhibits with Notch, Delta, and mastermind alleles and the LumiGLOTM (Kirkegaard & Perry Laboratories, Inc.) chemi- (Xu et al., 1990). Dosage-dependent genetic interactions are a luminescent detection kit according to manufacturer’s instructions. hallmark of a number of signal transduction pathways in Drosophila and have been exploited successfully to uncover S2 cell expression constructs additional interacting components (see Hoffman, 1991; Rubin, The deltex expression construct pCaSpeR-hsdx was based on the 1991). pCaSpeR-hs vector and has been described by Busseau et al. (1994). The deltex gene has recently been cloned and sequenced, All other constructs were based on the expression vector pRmHa-3 (Bunch et al., 1988), which uses the inducible Drosophila metalloth- and its temporal and spatial patterns of expression have been ionein promoter to drive expression. Construction of Notch deletion examined: it encodes a novel cytoplasmic protein with an plasmids (Fig. 2A) has been described (Rebay et al., 1993). Encoded apparently ubiquitous tissue distribution throughout develop- Notch ANK repeats were isolated as a PCR-derived DNA fragment ment (Busseau et al., 1994). This makes deltex the only cyto- containing artificial NdeI and BspEI sites. The following synthetic plasmic component identified among the Notch group proteins. oligonucleotide primers (HHMI facility, Yale University) were used: Because this subcellular localization raised the possibility of a 5′ GCG CAT CAG GAT CAT ATG AAG CAC GAT GTG GAT direct interaction with the intracellular domain of Notch, we GCA 3′ and 5′ GGC CAC ATC GTC CGG AAA TCG ATC CAT sought to investigate this possibility. In this paper, we present GTG ATC 3′. To generate pMTDl/NANK, the following three-piece a series of experiments in which we demonstrate that deltex ligation was performed: a 2.6-kb EcoRI-NdeI DNA fragment from interacts specifically with the Notch ANK repeats. In addition, pMTDl1 (Fehon et al., 1990) and the NdeI/BspEI-digested PCR fragment were inserted into the EcoRI and XmaI sites of pRmHa-3. we describe a new class of viable Notch allele that is associ- The resulting construct encoded Notch protein sequences, beginning ated with a missense mutation within the ANK repeat region four residues before the first ANK repeat and ending nine residues and that, like mutations of deltex, behaves as a dominant (intra- after the sixth ANK repeat. For pMTDl/CANK, the same three-piece genic) suppressor of the Abruptex ‘negative complementation’. ligation was performed except a 0.7-kb AseI-BspEI DNA fragment from the cactus cDNA pcact5B (generously provided by R. Geisler and C. Nüsslein-Volhard; Geisler et al., 1992) replaced the fragment encoding the Notch ANK repeats. The resulting construct encoded MATERIALS AND METHODS eight residues of cactus before the first ANK repeat and five residues of cactus after the sixth ANK repeat. For both constructs, a TAG stop Cell culture and immunological methods codon was provided by a XbaI site located within the vector polylinker Cell culture conditions and media were according to Fehon et al. sequence. (1990). Cell transfections were performed as follows. Cells were passaged in fresh medium to 6-well plates (Falcon) and allowed to Yeast expression constructs attach to plastic for 1-3 hours (cell density approx. 3/4 confluent). In The ‘interaction trap’ method is described in Zervos et al. (1993). R. polystyrene tubes, 10 µg total plasmid DNA in 0.5 ml serum-free Finley and R. Brent generously provided yeast strain EGY40 and medium (SFM) was mixed with 0.5 ml SFM containing 50 µl Lipo- plasmids LEX202+PL (to make LexA fusions), pJG4-5 (to make fectACETM (Life Technologies, Inc.). After sitting for 15-30 acidic activation domain fusions), pSH18-34 (LexAop-lacZ reporter minutes, this mixture was added to cells previously rinsed twice with gene) and pRFHMI (LexA-bicoid fusion). The entire groucho coding SFM. After approx. 6 hours incubation, the transfection mixture was region (Delidakis et al., 1991) was isolated by PCR and inserted into replaced with fresh medium plus serum. Expression from the metal- the EcoRI restriction sites of LEX202+PL and pJG4-5 to create LexA- lothionein promoter was induced approx. 24 hours later by CuSO4 groucho and pJG4-5-groucho. A PCR-derived DNA fragment con- addition to 0.7 mM, followed 12-18 hours later with induction of the taining artificial EcoRI and SalI sites and encoding amino acids 1827- Hsp70 promoter consisting of two 30-minute incubations at 37¡C 2258 of Notch (Wharton et al., 1985) was inserted into LEX202+PL with an intervening 30 minutes at 25¡C. A final recovery period of to create LexA-Notch ICN1. The entire coding region of deltex was at least 4 hours significantly improved the resolution of the co-local- recovered by PCR and inserted into the XhoI site of pJG4-5 to create ization by allowing excess deltex protein accumulation to abate. pJG4-5-deltex. Yeast transformations were performed according to Cells were then incubated for 30 minutes with mouse anti-Notch Gietz et al. (1992). deltex/Notch ANK repeat interaction 475

DNA sequencing with a set of Notch deletion constructs (Fig. 2A) in the S2 cell Genomic DNA was obtained by PCR and subcloned into Bluescript culture assay. The phenotypic consequences of expressing KS- (Stratagene). Several clones of at least two different PCR these same deletions of Notch were also recently examined in reactions were sequenced (both strands) by the dideoxy chain termi- transgenic flies (Rebay et al., 1993). The constructs ∆ B-S and nation method (Sanger et al., 1977) and covered nucleotides 2850- ∆ S-S, which delete sequences C-terminal of the ANK repeats, 8880 (numbering based on Wharton et al., 1985). Eleven base-pair did not visibly impede deltex association (Fig. 1E,F). Overex- changes were discovered, three of which predicted amino acid sub- pression of these deletions within the fly produced only mild stitutions. Of these, one at position 3584 corresponded to the previ- phenotypes similar to those resulting from overexpression of ously described Ax9B2 mutation (Hartley et al., 1987; Kelly et al., 1987) and another at 7510 coincided with a formerly described strain an intact version of Notch (Rebay et al., 1993). This stands in polymorphism (Kidd et al., 1986). A third base-pair change at position contrast to the results obtained with deletions that removed the 6920 was taken to be the su42c mutation and was confirmed by direct ANK repeats (ECN or ∆ cdc10). In the S2 cell assay, we sequencing of the PCR product. detected no association of deltex with these versions of Notch (Fig. 1G,H). In the fly, overexpression of both the ECN and ∆ cdc10 constructs resulted in severe dominant-negative pheno- RESULTS types, demonstrating an essential role for the ANK repeats in Notch signaling activity (Rebay et al., 1993). Combined, these deltex interacts with Notch but not with Delta or results not only implicated the ANK repeats in mediating Serrate Notch/deltex interaction, but also suggested a role for deltex in We explored the possibility of protein-protein interactions intracellular signaling events (see Discussion). between deltex and Notch by examining the relative subcellu- To determine whether the ANK repeats alone were sufficient lar localization of the two proteins after co-expressing them in to promote deltex binding, we produced an expression Drosophila Schneider 2 (S2) cultured cells (Schneider, 1972). construct, pMTDl/NANK, that affixed the Notch ANK repeats S2 cells were co-transfected with plasmid expression con- to the cytoplasmic domain of Delta (Fig. 2B). Because Delta structs that placed Notch and deltex under the inducible control shows no physical association with deltex (Fig. 1C), co-local- of the Drosophila metallothionein and Hsp70 promoters, ization of the two proteins at the cell surface would be a con- respectively. S2 cells do not express endogenous Notch (Fehon sequence of the Notch ANK repeats. Fig. 1I and J show this to et al., 1990) and express deltex at levels too low for immuno- be the case. Indeed, the deltex/Notch-ANK-repeat interaction fluorescent detection (R. J. Diederich and S. Artavanis- is emphasized in Fig. 1J, which is an electronic merging of the Tsakonas, unpublished data; see also Busseau et al., 1994). side-by-side image displayed in Fig. 1I. Unlike the staining Notch expression was induced first to ensure proper cell patterns obtained from other transfection experiments, this surface localization, followed by a brief heat-shock to induce shows that the co-localization is offset, i.e., not coincident, deltex expression. These cells were then aggregated with cells reflecting the labeling of Notch in one cell and that of deltex expressing Delta, a presumptive membrane-bound ligand of (through hybrid Delta/Notch-ANK-repeat protein) in an Notch (Fehon et al., 1990; Heitzler and Simpson, 1991), to adjacent cell. Thus, the Notch ANK repeats are both necessary produce a ‘mutual capping’ (Singer, 1992) of Notch and Delta and sufficient for deltex binding to occur. at the point of cellular contact (Fehon et al., 1990). Fig. 1A Because ANK repeats, in general, are a conserved feature of shows co-localization between deltex and the ‘capped’ Notch, many proteins, we sought to address the specificity of deltex indicating molecular interaction between the two proteins. binding for those repeats of Notch. We replaced the ANK Moreover, this co-localization was evident even in the absence repeats of pMTDl/NANK with those of the Drosophila gene, of capping with Delta, although in this case Notch and deltex cactus, to produce the expression plasmid, pMTDl/CANK were mis-localized within peri-nuclear regions of the cell (Fig. (Fig. 2B). cactus encodes an I-κ B cognate that has been shown 1B). This latter result suggests that there is no ligand depen- to bind, via ANK repeats, a Drosophila NF-κ B cognate, dorsal dency of deltex association with Notch. (Geisler et al., 1992; Kidd, 1992). In the S2 cell expression When the aggregation experiment was performed such that assay, no association of deltex for the ANK repeats of cactus deltex was expressed in the same cell along with either Delta was observed (Fig. 1K), although immunoblot analysis or Serrate (another presumptive ligand of Notch; Rebay et al., indicated a hybrid Delta/cactus protein of proper size was 1991), no co-localization was observed between deltex and expressed in these cells (Fig. 2C). This result indicates a these transmembrane proteins (Fig. 1C,D). This indicates that specific association of deltex for the ANK repeats of Notch and the interaction is specific for Notch and that overexpression serves as a control for the concern that overexpression of ANK itself is not causing deltex to concentrate in the regions of repeats, per se, promotes deltex association. cellular contact where Notch and Delta accumulate. Moreover, the results suggest that the genetic interaction previously noted Notch and deltex co-localization in vivo between deltex and Delta (Xu and Artavanis-Tsakonas, 1990) The above demonstration of Notch/deltex interaction in trans- is likely an indirect reflection of a direct Notch/deltex interac- fected cells prompted us to explore whether such interaction tion. was detectable in vivo. We examined the imaginal wing disc, a tissue that requires both proteins for its proper development Notch ANK repeats are both necessary and (Xu and Artavanis-Tsakonas, 1990). Because the endogenous sufficient for deltex association level of deltex protein accumulation is below the threshold The 938-amino-acid intracellular domain of Notch contains necessary for confident immunofluorescent detection (Busseau several structural motifs (Stifani et al., 1992). To identify those et al., 1994), we used a transformant fly line carrying an regions involved in deltex binding, we expressed deltex along inducible deltex construct to elevate transiently the in vivo pool 476 R. J. Diederich and others

of deltex protein. Previously, we have shown that over- and/or dence of deltex and Notch proteins at the apical surface of the ectopic-expression from this transposon rescues deltex mutant wing disc epithelium, in agreement with the co-localization defects and has no obvious phenotypic consequences in data of the S2 cell culture assay. Although we cannot rule out otherwise wild-type animals (Busseau et al., 1994). In the wing the possibility that deltex is apically localized irrespective of disc epithelium, Notch displays a polarized distribution to the Notch, the results nonetheless suggest that Notch/deltex inter- apical surface (Fehon et al., 1991). Fig. 1L shows a coinci- actions normally occur in vivo. deltex/Notch ANK repeat interaction 477

Fig. 1. deltex interacts with Notch ANK repeats. Confocal (C,D,I-K) to induce cell surface ‘capping’. Immunoﬂuorescent microscope images of Drosophila S2 cells (A-K) and distal portion labeling of cells was performed (see Materials and methods) using of imaginal wing disc (L) are presented as split images (except J) mouse anti-Notch monoclonal antibody speciﬁc for the extracellular with Notch expression shown in green and deltex in red. For S2 domain and rat anti-deltex polyclonal or monoclonal antibodies. cells, each panel represents a co-transfection experiment involving Delta and Serrate expression (C,D) were viewed indirectly through the deltex expression plasmid pCaSpeR-hsdx (Busseau et al., 1994) their ‘mutual capping’ with Notch. Extraneous cytoplasmic Notch and each of the expression constructs depicted in Fig. 2 or described expression was minimized by reacting cells with anti-Notch elsewhere: (A,B) pMtNcDNA (full-length Notch; Rebay et al., antibody prior to cell permeabilization and subsequent incubation 1993); (C) pMTDl1 (Delta; Fehon et al., 1990); (D) Ser-mtn with anti-deltex antibodies. Excess deltex expression was (Serrate; Rebay et al., 1991); (E) pMT∆B-S; (F) pMT∆S-S; minimized by allowing at least a 4-hour recovery period after the (G) pMTECN; (H) pMT∆cdc10; (I,J) pMTDl/NANK; last heat shock induction of deltex. In some instances, aggregated (K) pMTDl/CANK. Cells expressing these constructs were mixed cell partners were torn apart during manipulation. The diameter of (except in B) with cells expressing either Delta (A,E-H) or Notch S2 cells is 7-10 µm.

A C

Fig. 2. Plasmid expression constructs used in S2 cell transfections. (A) Notch intracellular domain (top; drawn to scale); extracellular domains are not shown but are intact for all constructs (TM, transmembrane region). Structural motifs and restriction sites used to generate deletion constructs (black bars) are indicated. Deleted amino acids are presented in parentheses (numbering from Wharton et al., 1985). (B) Intracellular domains of Delta-based expression constructs (same scale as in A). NdeI site adjacent to translational stop codon was used as insertion site for Notch- (pMTDl/NANK) and cactus- (pMTDl/CANK) ANK repeat coding sequences. Results of deltex binding are summarized. (C) Immunoblot analysis of S2 cell lysates after transfection and expression of plasmid constructs depicted in B (NT, non-transfected cells). Integrity of other expression constructs (A) was also conﬁrmed by immunoblot analysis (not shown; I. Rebay and S. Artavanis-Tsakonas, unpublished data).

Fig. 3. Direct Notch/deltex interaction in yeast. Each column lists Drosophila genes that were fused to sequences encoding a LexA DNA-binding domain or an acidic transcription activation domain. These were co-transformed into yeast containing a LexA operator-lacZ reporter gene plasmid (pSH18-34; S. Hanes and R. Brent, unpublished data) and were plated onto glucose- and galactose-Ura− His− Trp− X- gal-indicator plates. Three independent yeast isolates are shown for each experiment. Only transformations involving LexA-Notch ICN1 and deltex-ACT resulted in detectable β-galactosidase activity.

Direct Notch/deltex interaction indicated by yeast called ‘interaction trap’ technique (Zervos et al., 1993). Two expression studies expression plasmids were constructed. One (LexA-Notch The above demonstration of a speciﬁc association between ICN1) encoded a LexA DNA-binding domain fused to a deltex and Notch ANK repeats leaves open the question of portion of the Notch intracellular domain (amino acids 1827- whether the interaction is mediated directly or through other 2258; Wharton et al., 1985) that included the ANK repeats. cellular components. To address this question, we have The other plasmid (pJG4-5-deltex) encoded the entire deltex conducted similar expression studies in yeast using the so- protein fused to an acidic transcription activation domain 478 R. J. Diederich and others

(ACT-deltex). These were co-transformed into yeast carrying tion trap’ assay, to demonstrate a specific, and presumably a reporter gene (LexA operator-lacZ) plasmid. Notch/deltex direct, interaction between deltex and the ANK repeats of the interactions would be expected to mediate the formation of a Notch intracellular domain. This identifies deltex as the first complex between the LexA-Notch ICN1 and ACT-deltex cytoplasmic protein known to interact with Notch and confirms proteins resulting in the restoration of transcriptional activity. earlier genetic inferences about deltex function. As discussed This would be detected as a blue yeast colony due to induced below, the implication drawn from this data is that deltex con- β-galactosidase synthesis. As presented in Fig. 3, a specific stitutes a component of the Notch signaling pathway that also interaction was indeed detected between Notch and deltex includes Delta and Serrate as presumed ligands. within the yeast cell. This result suggests that the Notch/deltex interaction observed within Drosophila cells is the conse- deltex as a regulator of Notch activity quence of a direct protein-protein interaction. The functional relationship of deltex to Notch was uncovered by the phenotypic interactions noted with the Abruptex alleles, Intragenic suppressor mutation maps within ANK which represent single amino acid substitutions clustering repeat within adjacent EGF-like repeats (Hartley et al., 1987; Kelly In the same genetic screen (Xu and Artavanis-Tsakonas, 1990; et al., 1987). These gain-of-function alleles are thought to Xu et al., 1990) that identified deltex as a potentially interact- reflect ‘activated’ forms of Notch (Foster, 1975; Palka et al., ing partner of Notch, an unusual allele of Notch was also 1990; Heitzler and Simpson, 1993), and their phenotype is recovered. This allele, denoted Nsu42c, is homozygous viable, mimicked by truncated forms of Notch lacking the extracellu- and like mutations of deltex, Delta or mastermind, suppresses lar domain (Rebay et al., 1993; Struhl et al., 1993). The under- completely the pupal lethality associated with the AxE2/Ax9B2 lying basis for the dominant ‘activated’ phenotype produced ‘negative complementation’. by the Abruptex mutations is not known. On the basis of Intragenic recombination analyses (W. Welshons, unpub- genetic mosaic studies, Heitzler and Simpson (1993) have lished data) indicated that the Nsu42c lesion was positioned cen- suggested that Abruptex molecules may have an increased tromere-proximal, and thus 3′, to the Ax9B2 mutation located affinity for Delta, but do not represent constitutively active within the genomic region encoding the 24th EGF-like repeat. forms of the receptor, as evidenced by their dependency upon We have sequenced Ax9B2 su42c genomic DNA encompassing the Delta ligand. the 17th EGF-like repeat to the C-terminal end of the protein and find a missense A mutation that results in an alanine to valine substitution within the fifth ANK repeat (Fig. 4). This alanine is conserved among all Notch homologs isolated to date from mice, rats, humans, and Xenopus (Coffman et al., 1990; Ellisen et al., 1991; Weinmaster et al., 1991, 1992; Franco del Amo et al., 1993; B Stifani et al., 1992; Kopan and Weintraub, 1993), but does not fall within a conserved position in the consensus sequence compiled for ANK repeats in general (Lux et al., 1990; Blank et al., 1992; Michaely and Bennett, 1992; Robbins et al., 1992). The su42c mutation is the first mutation known to affect the ANK region of Notch. Interestingly, the Ax9B2 su42c combination also confers upon adult flies a subset of deltex-like mutant phenotypes (not shown). These include outstretched wings and variably-fused ocelli (Xu and Artavanis- Tsakonas, 1990; Gorman and Girton, 1992), which are not displayed by the parental C Ax9B2 mutant. Thus, both the genetic behavior of this mutation as well as its position within the ANK domain implicate this region in Notch/deltex interactions. Fig. 4. Intragenic suppressor mutation maps within fifth Notch ANK repeat. (A) Diagram 9B2 DISCUSSION of Notch protein. The su42c mutation was induced in flies containing the Ax mutation. (B) DNA sequencing gels showing C to T transition at position 6920 in Ax9B2 su42c genomic DNA. This residue is unaffected (i.e., wild-type) in the parental Ax9B2 DNA. We present three lines of evidence: (1) the (C) Amino acid sequence (single letter code) of fifth ANK repeat showing alanine to S2 cell expression assay, (2) in vivo co- valine substitution (residue number 2060) resulting from the su42c mutation. All localization data, and (3) the yeast ‘interac- numbering is based on Wharton et al. (1985). deltex/Notch ANK repeat interaction 479

The mechanism by which certain heteroallelic combinations Gertler et al., 1993), an outcome that resembles our current of Abruptex alleles ‘negatively complement’ to result in pupal knowledge of deltex. It is only when these mutations are lethality is not understood. This phenomenon is thought to combined with those of second-site loci that dramatic gene- reflect homotypic interactions between Notch molecules dosage-sensitive phenotypes are revealed, which is very likely (Foster, 1975; Kelly et al., 1987). Such interactions are often an indication of an evolved regulatory redundancy (Hoffman, ligand dependent and have been documented for a number of 1991; Gertler et al., 1993). Similarly, deltex mutants display other receptor proteins (Ullrich and Schlessinger, 1990). dominant, and usually lethal, interactions when combined with However, the deleterious effects of negative complementation heterozygous mutations of Notch, Delta, or mastermind (Xu do not appear to result from dominant-negative interactions and Artavanis-Tsakonas, 1990). (Foster, 1975). Rather, this particular combination of Abruptex The strongest evidence to suggest a direct role for deltex in molecules appears to manifest an even greater gain-of-function the Notch signaling pathway comes from recent studies impli- activity than that observed with Abruptex homozygotes. This cating the ANK repeats in this process. In transgenic flies made is suggested by the severe loss of sensory bristles exhibited by to express various Notch deletion constructs, it is only those these animals (so-called ‘anti-neurogenic’ phenotype; Palka et that delete the ANK repeats that produce severe dominant- al., 1990) when compared with the normally viable Abruptex negative phenotypes (Rebay et al., 1993). This outcome, homozygotes (Foster, 1975; R. J. Diederich and S. Artavanis- combined with the ‘activated’ phenotypes that result from Tsakonas, unpublished data). overexpressing just the Notch intracellular domain (Rebay et In light of our demonstration of a physical Notch/deltex al., 1993; Fortini et al., 1993; Struhl et al., 1993), points to an interaction, the finding that lowering the gene dosage of deltex essential role for the ANK motif in Notch signaling. This view attenuates this apparent hyperactivity implies that deltex is also supported by the finding that a missense mutation within normally functions to positively ‘regulate’ Notch activity. This the ANK domain (Nsu42c; Fig. 4) suppresses the apparent inference is supported by other genetic evidence: in double hyperactivity associated with negatively complementing mutants deltex suppresses the dominant wing-vein-gap Abruptex alleles. Recent experiments involving glp-1, a Notch phenotype of Abruptex alleles (Xu and Artavanis-Tsakonas, family member from the nematode Caenorhabditis elegans 1990), and lethal interactions result when deltex mutants (reviewed by Greenwald and Rubin, 1992), also strengthen and contain the hypomorphic Notch allele notchoid1 (nd1) or when extend this conclusion. Roehl and Kimble (1993) have shown deltex mutants bear only one wild-type copy of Notch (Xu and that overexpression of the ANK repeats from glp-1 results in Artavanis-Tsakonas, 1990). Thus, in genetic terms, deltex can an anchor cell-independent multi-vulva phenotype typically be considered a ‘positive regulator’ of Notch activity. The rela- observed with gain-of-function glp-1 alleles. Thus, the ANK tionship of deltex to truncated forms of Notch defining repeat domain alone is sufficient to regulate cell fate. We think dominant-activated and dominant-negative versions of the it reasonable, therefore, to suggest that by association with the receptor (Rebay et al., 1993; Fortini et al., 1993) is currently ANK repeats, deltex may constitute a component of the Notch under investigation. signaling mechanism. It is possible that deltex represents one of several proteins Possible roles of deltex in the Notch signaling that bind to Notch ANK repeats (or to remaining parts of an pathway otherwise large intracellular domain) in order to modulate What functional role does deltex perform within the Notch Notch activity in a number of distinct cell types. This may signaling pathway? As a likely ‘positive regulator’, deltex may explain, in part, why extant deltex alleles reveal mild pheno- serve several indirect roles, perhaps in organizing the types and why overexpression of deltex does not produce phe- cytoskeleton around Notch or in controlling the trafficking of notypes like those associated with ‘activated’ Notch, although the Notch receptor. The data from S2 cells (Fig. 1B) and from in the latter case other elements of the pathway most likely are yeast (Fig. 3) indicating that deltex associates with Notch in a limiting. Additional genetic and molecular analyses will ligand independent manner is consistent with these possibili- address these questions. Mosaic studies involving deltex and ties. other Notch group members should address whether deltex However, several genetic observations combined with the influences the reception and/or transmission of Notch-mediated data presented here suggest that deltex may play a more direct signals. Moreover, we expect that genetic screens seeking to role in the Notch signaling pathway. Phenotypic analysis of identify second-site phenotypic suppressors and/or enhancers deltex mutants by themselves has not been particularly infor- will identify additional interaction partners that will enlarge the mative in ascribing a role for deltex: extant alleles do not Notch group, which we do not consider to be complete. display a ‘neurogenic’ phenotype typically observed with loss- of-function Notch alleles (Xu and Artavanis-Tsakonas, 1990), Other considerations of the Notch/deltex protein although null mutations of deltex are not yet known and there interaction is a significant maternal contribution of deltex mRNA Models analogous to those that have emerged from studies of deposited into the oocyte, which would mask embryonic Iκ B and NF-κ B have been proposed recently (LaMarco et al., requirements for deltex (see Busseau et al., 1994). However, 1991; Kidd, 1992; Kodoyianni et al., 1992) to explain the identical phenotypes are not necessarily an expected outcome activity of Notch family members: Notch may represent a for mutations affecting all components of a signal transduction novel Iκ B-like protein that, in response to an external pathway and, in this regard, the genetics of deltex is very stimulus, causes the dissociation of an ANK repeat-bound tran- similar to that of other signaling pathways. For example, loss- scriptional regulator, which is then transported into the of-function mutations of the Drosophila Abelson (abl) protein- nucleus. However, in S2 cells expressing either the ECN or ∆ tyrosine kinase result in mild phenotypes (Hoffman, 1991; cdc10 deletion constructs (Fig. 1G,H) or in embryos and 480 R. J. Diederich and others imaginal tissues over-expressing the deltex protein (Busseau et Bennett, V. (1992). Ankyrins: Adaptors between diverse plasma membrane al., 1994; R. J. Diederich and S. Artavanis-Tsakonas, unpub- proteins and the cytoplasm. J. Biol. Chem. 267, 8703-8706. κ lished data), we see no nuclear accumulation of deltex at the Blank, V., Kourilsky, P. and Israel, A. (1992). NF- B and related proteins: Rel/dorsal homologies meet ankyrin-like repeats. Trends Biochem. Sci. 17, level of resolution provided by immunofluorescent 135-140. microscopy. Truncations of Notch that delete the extracellular Breeden, L. and Nasmyth, K. (1987). Similarity between cell-cycle genes of and transmembrane regions result in the production of an budding yeast and fission yeast and the Notch gene of Drosophila. Nature ‘activated’ intracellular domain (Nnucl; Fortini et al., 1993) that 329, 651-654. localizes within the nucleus (Fortini et al., 1993; Struhl et al., Bunch, T. A., Grinblat, Y. and Goldstein, L. S. B. (1988). Characterization and use of the Drosophila metallothionein promoter in cultured Drosophila 1993), although nuclear localization does not appear to be a melanogaster cells. Nucl. Acids Res. 16, 1043-1061. precondition for an ‘activated’ response (Fortini et al., 1993). Busseau, I., Diederich, R. J., Xu, T. and Artavanis-Tsakonas, S. (1994). A In S2 cells co-expressing deltex and Nnucl, deltex expression member of the Notch group of interacting loci, deltex encodes a cytoplasmic remained cytoplasmic (R. J. Diederich and S. Artavanis- basic protein. Genetics, in press. Cagan, R.L. and Ready, D. F. (1989). Notch is required for successive cell Tsakonas, unpublished data). The question of whether deltex decisions in the developing retina. Genes Dev. 3, 1099-1112. nucl is capable of preventing N from translocating into the Coffman, C., Harris, W. and Kintner, C. (1990). Xotch, the Xenopus nucleus is currently under investigation. homolog of Drosophila Notch. Science 249, 1438-1441. Although we have demonstrated that the ANK repeats are Coffman, C. R., Skoglund, P., Harris, W. A. and Kintner, C. R. (1993). both necessary and sufficient to promote deltex binding, other Expression of an extracellular deletion of Xotch diverts cell fate in Xenopus embryos. Cell 73, 659-671. regions of the Notch intracellular domain may influence the Corbin, V., Michelson, A. M., Abmayr, S. M., Neel, V., Alcamo, E., Notch/deltex protein complex. This possibility is raised by the Maniatis, T. and Young, M. W. (1991). A role for the Drosophila lethal interaction that results when deltex mutants also contain neurogenic genes in mesoderm differentiation. Cell 67, 311-323. the Notch mutation nd1, which is associated with missense Coyle-Thompson, C. A. and Banerjee, U. (1993). The strawberry notch gene functions with Notch in common developmental pathways. Development mutations near the C terminus (Xu et al., 1990). 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J. and Artavanis-Tsakonas, region among the various Notch homologs (Stifani et al., S. (1990). The gene Serrate encodes a putative EGF-like transmembrane 1992), the interaction between deltex and Notch ANK repeats protein essential for proper ectodermal development in Drosophila suggests that deltex cognates will exist in higher eukaryotes melanogaster. Genes Dev. 4, 2188-2201. and that these may function through similar biochemical modes Fortini, M. E., Rebay, I., Caron, L. A. and Artavanis-Tsakonas, S. (1993). An activated Notch receptor blocks cell-fate commitment in the developing of action. Drosophila eye. Nature 365, 555-557. Foster, G. G. (1975). Negative complementation at the Notch locus of We thank Robert Geisler and Christiane Nüsslein-Volhard for Drosophila melanogaster. Genetics 81, 99-120. providing the cactus cDNA; Ilarhino Rebay for plasmid expression Franco del Amo, F., Gendron-Maguire, M., Swiatek, P. J., Jenkins, N. 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