spr-2, a suppressor of the egg-laying defect caused by loss of sel-12 presenilin in Caenorhabditis elegans, is a member of the SET protein subfamily

Abstract

Presenilin plays critical roles in the genesis of Alzheimer's disease and in LIN-12/Notch signaling during development. Here, we describe a screen for genes that influence presenilin level or activity in Caenorhabditis elegans. We identified four spr (suppressor of presenilin) genes by reverting the egg-laying defective phenotype caused by a null allele of the sel-12 presenilin gene. We analyzed the spr-2 gene in some detail. We show that loss of spr-2 activity suppresses the egg-laying defective phenotype of different sel-12 alleles and requires activity of the hop-1 presenilin gene, suggesting that suppression is accomplished by elevating presenilin activity rather than by bypassing the need for presenilin activity. We also show that SPR-2 is a nuclear protein and is a member of a protein subfamily that includes human SET, which has been identified in numerous different biochemical assays and at translocation breakpoints associated with a subtype of acute myeloid leukemia.

A key factor in the development of Alzheimer's disease is the deposition of plaques formed from Aβ peptides. These peptides are released when the single-pass transmembrane protein β-amyloid precursor protein (β-APP) is cleaved at the β site in the extracellular domain and at a γ site in the transmembrane domain. Studies of familial Alzheimer's disease led to the identification of the presenilin 1 and presenilin 2 genes (reviewed in ref. 1). Presenilin is required for γ-secretase cleavage of β-APP (2, 3), and the two presenilins seem to be functionally interchangeable (4–6). Studies with putative aspartyl protease-active site inhibitors indicate that presenilin may itself be the long elusive γ-secretase (7, 8).

Presenilin is being intensively pursued as a key therapeutic target for the amelioration or prevention of Alzheimer's disease. The identification of factors that influence presenilin activity, synthesis, and stability will be important for maximizing the efficacy of drugs that are targeted against γ-secretase and perhaps for identifying new therapeutic targets. Genetic analysis of presenilin in Caenorhabditis elegans offers one approach to identifying such factors.

In C. elegans, there are two presenilins, sel-12 and hop-1, that can be studied by virtue of their involvement in LIN-12/Notch signal transduction (9, 10). LIN-12/Notch proteins are transmembrane proteins that act as receptors for intercellular signals that specify cell fates. Ligand binding to LIN-12/Notch receptors leads to proteolytic cleavage within the transmembrane domain, which releases the intracellular domain so that it may translocate to the nucleus and activate transcription of target genes (see 11, 12). The transmembrane-cleavage event is analogous to the γ-secretase-processing event that generates Aβ from β-amyloid precursor protein, and presenilin is essential also for the proteolytic cleavage that releases the intracellular domain of LIN-12/Notch proteins (11, 12). Indeed, in C. elegans, concomitant depletion of both sel-12 and hop-1 activity causes the phenotypes associated with the loss of all LIN-12/Notch signaling (10), and this observation, along with similar findings from genetic studies in Drosophila (12, 13) and mice (3, 6, 14, 15), has suggested that the key essential function of presenilin during animal development may be in LIN-12/Notch signaling.

We describe here one genetic approach to identifying factors that influence presenilin activity, synthesis, and stability: the identification of suppressors of the egg-laying defective (Egl) phenotype caused by loss-of-function mutations in the C. elegans presenilin sel-12. The suppressor approach mitigates potential difficulties arising from functional redundancy of members of gene families, functional redundancy of different regulatory mechanisms, or pleiotropy. We analyze one such suppressor of presenilin, spr-2, in some detail. We show that spr-2 seems to suppress the Egl phenotype resulting from the absence of sel-12 activity by elevating the level or activity of another C. elegans presenilin, hop-1. We also show that SPR-2 is a nuclear protein that is related to mammalian SET, a protein that has been identified in numerous different contexts.

Materials and Methods

Genetic Materials and Methods.

Standard methods were used for handling, maintenance, ethyl-methanesulfonate mutagenesis, and genetic analysis. The wild-type parent for most strains used in this study is C. elegans var. Bristol strain N2 (16). Experiments were conducted at 20°C unless otherwise indicated. The following single-nucleotide polymorphism (SNP) from LG IV were used for mapping as described below; these SNPs were identified in C. elegans var. Hawaii strain CB4856 by the Genome Sequencing Consortium (http://genome.wustl.edu/gsc/CEpolymorph/snp.shtml): vm23 g02.s1, vc86f02.s1, vd48b08.s1, v125f08.s1, and vr89 g03.s1. sel-12 alleles are described in ref. 9. The markers used for mapping or for facilitating genetic analysis mentioned in the text are described at http://biosci.umn.edu/CGC/CGChomepage.htm.

Mapping of spr Mutations.

The spr mutations were initially mapped to autosomal linkage groups with visible dpy markers (data not shown) and then to LG V, LG IV, LG X, and LG I intervals.

LG V.

spr-1(ar200) maps between unc-68(e540) and rol-3(e754): unc-68 (6/10) spr(ar200) (4/10) rol-3. Because ar201 [unc-68 (5/11) spr(ar201) (6/11) rol-3] and ar205 [unc-68 (14/18) spr(ar205) (4/18) rol-3] map to the same genetic interval, they may also be spr-1 alleles. Preliminary results suggest that spr(ar212) maps much closer to rol-3, and, hence, may not be an allele of spr-1 (data not shown). Because spr(ar212) has not been mapped to a different interval, however, we have not given it an independent spr gene designation.

LG IV.

spr-2(ar199), spr-2(ar211), and spr-2(ar214) IV were all mapped between unc-8(e49) and dpy-20(e1282). ar199: unc-8 (7/17) ar214 (10/17) dpy-20. ar214: unc-8 (7/12) ar214 (5/12) dpy-20. ar211: unc-8 (2/12) ar211 (10/12) dpy-20. The ar216 mutation was not mapped further but was shown to be an allele of spr-2 by DNA sequence analysis. spr-2(ar211) was mapped between the single-nucleotide polymorphisms (SNPs) markers vd48b08.s1 and vl25f08.s1, and spr-2(ar199) was mapped between the SNPs markers vr89 g03.s1 and vm23 g02.s1, essentially as described (17).

LG X.

spr-3(ar209) was mapped between dpy-3(e27) and unc-2(e55): dpy-3 (9/13) spr-4(ar209) (4/13) unc-2. ar198 also maps in this interval: dpy-3 (20/23) spr(ar198) (3/23) unc-2. ar217 was found to be X-linked but was not further mapped.

LG I.

Three spr mutations, ar197, ar204, and ar208, mapped to LG I, and we have tentatively assigned ar208 as the canonical allele of spr-4. In our initial experiments, we were unable to map any of these mutations unambiguously to a single interval; further work will be necessary to determine why.

Identification of hop-1(0) Alleles.

We used the protocol of R. Barstead (http://pcmc41.ouhsc.edu/Knockout/) to screen for an internal deletion within the hop-1 gene. We used the primers TMP-F₂ (5′-CACAGTAACCTTCAAAACCACAC) and TMP-R2 (5′-GTTAGACGATCTCCACCATC), which give a wild-type PCR product of 2,509 bp and recovered two deletion alleles, hop-1(ar179) and hop-1(ar180) (data not shown). hop-1(ar179) is predicted to be a protein null and was used for the genetic studies described in this paper. hop-1(ar179) is a 716-bp deletion with breakpoints in exon 2 (at codon Ser-57) and exon 4 (at codon Tyr-218). This deletion shifts the reading frame after the first transmembrane domain, resulting in a stop codon immediately after residue Asp-56 and a newly introduced residue Thr.

We note that ar179 affects only sequences internal to the hop-1 locus. In contrast, the coordinates reported for other hop-1-deletion alleles suggest that neighboring genes are affected. hop-1(nr2003), a deletion from 23,744 (or 23,743) to 22,549 (or 22,548) of cosmid C18E3 (18), should remove part of C18E3.2; hop-1(lg1501), a deletion of 20,359–22,238 of cosmid C18E3 (19), should remove part of C18E3.3.

Transgenic Lines.

DNA was injected into the germline of C. elegans hermaphrodites (20). PMH86[dpy-20(+)] (21) was used as a cotransformation marker so recipient strains would contain the chromosomal marker dpy-20(e1282).

Rescue experiments.

Cosmid DNA spanning the spr-2 region was injected at 5 μg/ml each in pools together with pMH86 (10 μg/ml) and pBluescript (80 μg/ml) into recipient strain spr-2(ar211) dpy-20(e1282); sel-12(ar171) unc-1(e538). Transgenic lines were established and their egg-laying ability was checked for antisuppression, which is indicative of spr-2(+) activity. When rescue was seen with a pool at the F₂ stage, individual cosmid DNAs from that pool were injected into the same strain at a higher concentration (50 μg/ml) together with pMH86 (10 μg/ml) and pBluescript (40 μg/ml). Only one cosmid clone, F18C7, showed rescuing activity in 6 of 12 transgenic lines at the F₂ stage. Five predicted genes are completely contained in F18C7, and two others are partially contained. Five PCR fragments, each of which contains one or two of these predicted genes, were injected into the recipient strain at 50 μg/ml. Four of these PCR fragments generated three to eight transgenic lines each, and none showed antisuppression. The fifth PCR fragment contained C27B1.1 and C27B2.2; lines could not be obtained at 50 μg/ml, but a single line was obtained at 5 μg/ml, and antisuppression was seen in the F₂ generation. In all cases where antisuppression was seen at the F₂ generation, the antisuppression activity subsequently disappeared, although the dpy-20(+) marker was still functional. We did not investigate this behavior further.

SPR-2∷GFP expression.

p21XXGFP encodes a SPR-2∷GFP protein and contains all genomic sequences from C27B7.1 described above, with green fluorescent protein (GFP) inserted in frame after the last codon of SPR-2. p21XXGFP is a derivative of p21XX, which contains a 2.1-kb XbaI/XhoI genomic fragment containing 0.7 kb of 5′ flanking region and 0.35 kb of 3′ flanking region. The coding region of p21XX was sequenced and confirmed. Additional details are available on request.

We generated transgenic lines in a dpy-20(e1282) background. p21XXGFP was injected at 50 μg/ml into dpy-20(e1282) hermaphrodites with pMH86 [dpy-20(+)] as a cotransformation marker. Six independent extrachromosomal arrays were generated, and (GFP) fluorescence was observed in all six lines; however, the expression pattern from the extrachromosomal arrays was highly mosaic. We used a standard method (20) to generate the integrated transgene arIs57, used for analyzing the SPR-2∷GFP expression pattern. dpy-20(e1282); arIs57 showed a reproducible expression pattern from animal to animal. In addition, the SPR-2∷GFP fusion protein expressed from this transgene is functional, because it displays antisuppression activity: 40% of hermaphrodites of genotype spr-2(ar199) dpy-20; sel-12(ar131) unc-1; arIs57 [spr-2∷gfp] are Egl.

RNA-Mediated Interference (RNAi).

RNAi was performed as described (22). cDNA clone yk81B12 was used for spr-2 double-stranded (ds)RNA; genomic DNA was used to generate D2096.8 dsRNA. dsRNA was microinjected into the pseudocoelomic space of L4 hermaphrodites. Injected hermaphrodites were cultured individually overnight and then transferred to fresh plates. Progeny of injected hermaphrodites were scored for their ability to lay eggs.

Results

Identification of Extragenic Suppressors of sel-12(ar171).

Hermaphrodites that are homozygous for the putative null allele sel-12(ar171) are Egl. The Egl phenotype of sel-12(−) resembles that of a lin-12 partial loss-of-function mutant (9). The cellular basis for the Egl phenotype in either case is not completely understood, although it is likely to reflect cell fate abnormalities involving the π cells of the ventral uterus (ref. 23 and A. Newman, personal communication).

We screened for suppressors of sel-12(ar171) by mutagenizing sel-12(ar171) unc-1(e538) hermaphrodites with ethyl methanesulfonate (16) and screening for normal egg-laying (Egl⁺) revertants in the F₁, F₂, and F₃ generations. Approximately 13,800 F₁ hermaphrodites (representing 27,600 mutagenized haploid genomes) and their progeny were examined for suppression, and Egl⁺ hermaphrodites or eggs were picked to establish potentially suppressed strains. We kept only strains displaying relatively high penetrance, a minimum of approximately 80% Egl⁺. Then 14 independent, highly penetrant Egl⁺ revertants were obtained, for a frequency of approximately 5 × 10⁻⁴ suppressor mutations/mutagenized haploid genome. Because these suppressor mutations seem to define four distinct loci, this frequency is comparable to the average ethyl methanesulfonate mutagenesis-induced forward mutation rate of 1.3 × 10⁻⁴ per gene (16, 24), suggesting that at least some spr mutations recovered in this screen are likely to be hypomorphic or null alleles.

Our initial assessment suggested that most of the spr mutations are somewhat semidominant (data not shown); thus, we relied principally on genetic-map position rather than complementation tests to assess the number of spr genes. Initial linkage experiments identified potential loci on LG I (three alleles), IV (four alleles), V (four alleles), and X (three alleles); two alleles were lost. We have tentatively designated four spr genes: spr-1 V, spr-2 1V, spr-3 X, and spr-4 I. The map data for most of the spr mutations are consistent with their being alleles of one of these four loci, with possible exceptions described in Materials and Methods.

We note that the LG V spr mutations map to a distinct position from sel-10, a partial suppressor of the Egl phenotype of sel-12(ar171) that had originally been identified based on genetic interactions with lin-12 (see ref. 25). We did not expect to recover sel-10 here, because loss of sel-10 activity results in only 20% suppression of sel-12(ar171) (25), less than the 80% threshold of this screen.

The genomic location of spr-2 helped make it especially tractable to molecular analysis, and the remainder of this report is concerned with this gene.

spr-2 Suppression of the Egl Phenotype of sel-12(−) Is Not Allele-Specific and Requires the Activity of hop-1.

There are two C. elegans presenilin genes, sel-12 and hop-1 (9, 10). sel-12 is expressed in many different cell types (4); hop-1 expression seems to be too low to detect in cells by using conventional GFP and lacZ reporter-gene approaches (X.L. and I.G., unpublished observations). Expression of a hop-1 cDNA under the control of sel-12 regulatory sequences rescues the sel-12(ar171) Egl phenotype (10).

In principle, mutations that suppress the Egl phenotype of sel-12(ar171) might have one of the following effects: (i) corrective interaction: the spr mutations might function as informational suppressors or somehow enable the truncated SEL-12(ar171) product to function; (ii) bypass mechanism: the spr mutations might bypass the need for presenilin activity altogether; or (iii) augment hop-1 activity: the spr mutations might augment the activity, level, or stability of HOP-1 protein, either directly or indirectly.

These three possibilities may be distinguished by two genetic tests: allele-specificity and hop-1 dependence. A corrective interaction or informational suppression would be allele specific and would not depend on hop-1 activity. A bypass mechanism would be allele nonspecific and would not depend on hop-1 activity. A mechanism that augments hop-1 activity would be allele-nonspecific and would depend on hop-1 activity.

When we performed these two genetic tests, we found a lack of allele specificity and dependence on hop-1. First, spr-2 mutations efficiently suppress sel-12(ar131), a C60S missense change in the first transmembrane domain, as well as sel-12(ar171), a W225STOP change in the fifth transmembrane domain (ref. 9; Table 1). Second, the presence of the hop-1(ar179) null allele (see Materials and Methods) prevented spr-2 mutations from suppressing the Egl phenotype of sel-12(ar171) (Table 2A). These results taken together indicate that spr-2 mutations augment hop-1 activity.

Table 1.

Allele-nonspecific suppression

Relevant genotype	Egl+/total, %
sel-12(ar171)	0/30 (0%)
sel-12(ar131)	0/33 (0%)
spr-2(ar199); sel-12(ar171)	28/30 (93%)
spr-2(ar199); sel-12(ar131)	31/31 (100%)
spr-2(ar211); sel-12(ar171)	30/30 (100%)
spr-2(ar211); sel-12(ar131)	32/33 (97%)

All strains also contained unc-1(e538). 

Table 2.

Genetic interactions with hop-1 and with lin-12^*

hop-1dependence
Relevant genotype	No. of Egl+/total, %
hop-1(ar179); sel-12(ar171)	0/28
hop-1(ar179); spr-2(ar199); sel-12(ar171)	0/30
hop-1(ar179); spr-2(ar211); sel-12(ar171)	0/40
No effect on the 0 AC defect caused by elevating lin-12 activity
Complete genotype	No. of 0 AC/total, %†
lin-12(n302)/unc-32; dpy-20/+	27/50 (54%)
lin-12(n302)/unc-32; spr-2(ar211) dpy-20	33/60 (55%)
lin-12(n302)/unc-32; spr-2(ar211)  dpy-20/spr-2(+) dpy-20	88/168 (52%)
No effect on the 2 AC defect caused by reducing lin-12 activity
Complete genotype	No. of 2 AC/total, %§
lin-12(ar170)‡ (25°C)	39/50 (78%)
lin-12(ar170); spr-2(ar211) (25°C)	25/39 (64%)
lin-12(ar170)‡ (20°C)	10/52 (19%)
lin-12(ar170); sel-12(ar171) unc-1 (20°C)	39/43 (91%)
lin-12(ar170); spr-2(ar211); sel-12(ar171)  unc-1 (20°C)	40/49 (82%)

^*Hermaphrodites of the relevant genotype shown segregated from parents that also carried the free duplication mnDp68 [sel-12(+)]. Maternal sel-12(+) activity provided by the duplication enables hop-1(−); sel-12(−) progeny to survive to adulthood and to produce progeny, which arrest as embryos (see ref. 18). spr-2 mutations do not suppress the maternal-effect lethality of embryos produced by hop-1(−); sel-12(−) mothers (data not shown). All strains also contained unc-1(e538). 

^† The 0 AC defect was scored by determining egg-laying ability; for lin-12(n302), the ability to lay eggs correlates absolutely with the presence of an AC (38). AC, anchor cell. 

^‡ lin-12(ar170) behaves as a partial loss-of-function allele at all temperatures but is most hypomorphic at 25°C (39). 

^§The number of ACs was scored directly using Nomarski microscopy. 

These observations raise the question of whether the effect of spr-2 is specific to hop-1, or whether spr-2 might be able also to augment sel-12 presenilin activity. In principle, this question could be addressed genetically by examining whether spr-2 can suppress the effects of removing hop-1 activity, and whether such suppression depends on sel-12. Because there is no phenotype caused by removing hop-1 activity in a sel-12(+) or lin-12 mutant background (refs. 10 and 18; X.L. and I.G., unpublished observations), this question cannot be answered at this time.

Genetic Interactions Between spr-2 and lin-12 Have Not Been Detected.

We considered whether spr-2 might seem to augment hop-1 activity because lin-12 activity, rather than hop-1 presenilin activity per se, has been elevated. If spr-2 affected lin-12 directly, then we might expect to see genetic interactions between spr-2 and lin-12. However, we have not detected any such interactions. First, the Egl phenotype of the partial loss-of-function allele lin-12(n676n930) is not suppressed by spr-2(ar211) and spr-2(ar199) (data not shown). Second, we did not see any evidence for spr-2 involvement in a well characterized lin-12-mediated process, the decision of two gonadal cells between the anchor cell (AC) and ventral uterine precursor cell (VU) fates (reviewed in ref. 26): spr-2(ar211) and spr-2(ar199) do not influence the penetrance of AC/VU defects caused by elevating lin-12 activity or by partially reducing lin-12 activity (Table 2). However, spr-2 may not function in the AC/VU pair, because spr-2 mutations do not suppress the increased penetrance of the two-AC defect caused by combining a partial loss-of-function allele of lin-12 with sel-12(ar171) (Table 2).

Molecular Cloning of spr-2, Mutations, and RNAi.

spr-2 was mapped between single-nucleotide polymorphisms on cosmids D2096 and F49C12, and cosmids and derivative PCR fragments were assayed for antisuppression, the ability to cause an Egl phenotype in a recipient strain of genotype spr-2(ar211) dpy-20(e1282); sel-12(ar171) unc-1(e538) (see Materials and Methods). Ultimately, a PCR fragment containing C27B7.1 and C27B7.2 showed antisuppression, suggesting that spr-2 might correspond to one of these two genes. We therefore sequenced the coding regions of C27B7.1 and C27B7.2 from all four spr alleles that mapped to LG IV. In three alleles, we found single nucleotide changes in the coding region of C27B7.1 (see Fig. 1) but no change in the coding region of C27B7.2. The fourth allele, ar199, was found to contain a 134-bp deletion in the predicted 5′ flanking region of C27B7.1, 64 bp upstream of the predicted start codon.

Figure 1.

Alignment of SPR-2 and human SET. Identical amino acids are shaded. The SPR-2 sequence is based on our cDNA sequence analysis (see text). The positions of spr-2(ar211) Y206N, spr-2(ar214) E270stop, and spr-2(ar216) P203S are indicated by asterisks (*). The region used for construction of the tree shown in Fig. 2 ranges from K76 to V275 of SPR-2 (each marked with X).

We sequenced three cDNA clones corresponding to C27B7.1: yk81b12, yk312a7, and yk274d1, kindly provided by Yuji Kohara (http://www.ddbj.nig.ac.jp/htmls/c-elegans/html/CE_INDEX.html). All intron/exon junctions predicted by GENEFINDER were confirmed with one exception: the 3′ acceptor site of the first intron appears to be three nucleotides downstream of the predicted acceptor. Our analysis suggests that the predicted SPR-2 protein has 312 amino acids, shown in Fig. 1.

The nature of the three point mutations, spr-2(ar211) Y206N, spr-2(ar214) E270stop, and spr-2(ar216) P203S, does not allow us to conclude that any are likely molecular-null alleles. However, loss of spr-2 function can result in suppression, because spr-2(RNAi) has suppressor activity: many Egl⁺ progeny were produced after injection of sel-12(ar131) hermaphrodites or sel-12(ar171) unc-1(e538) hermaphrodites (summarized in Table 3).

Table 3.

Summary of RNAi experiments

dsRNA injected	Wild type	sel-12(ar171)*	sel-12(ar131)
none†	+ (many)	Egl (4/4)	Egl (5/5)
spr-2	ND	Egl+ (6/15)	Egl+ (10/10)
D2096.8	ND	Egl (13/13)	Egl (10/10)
spr-2 + D2096.8‡	+ (10/10)	Egl+ (9/15)	Egl+ (10/10)

See text for details. +, no novel phenotypes, such as overt lethality, were seen. Egl, egg-laying defective. Egl⁺, egg-laying ability was restored, and no novel phenotypes were seen. The numbers in parentheses indicate the number of broods displaying the phenotype indicated/total injected hermaphrodites. 

^*Also carried unc-1(e538). 

^†Distilled water lacking dsRNA was injected. 

^‡ The extent of suppression of the Egl phenotype sel-12(ar171) and sel-12(ar131) was comparable to the extent of suppression seen with spr-2(RNAi) alone (data not shown). 

SPR-2 Is a Member of the SET/NAP Protein Family.

blast searches of the GenBank database and CLUSTALW analysis have revealed that SPR-2 is a member of a large family of proteins that includes human SET and yeast Nap1 (Fig. 1). SPR-2 falls within a distinct subfamily of SET/Nap proteins, referred to here as the SET subfamily. The SET subfamily includes mammalian SET and TSPY proteins, as well as proteins from eukaryotes as diverse as Arabidopsis and Plasmodium. (SET does not contain a “SET domain,” a motif found in certain DNA binding proteins.)

SET has been identified in many different assays and experimental systems, but its function in vivo is not clear. SET was identified first as the product of a gene located at a translocation breakpoint associated with a subtype of acute myeloid leukemia (27). SET was identified also in HeLa cell extracts as template-activating factor in a biochemical assay for proteins that enable replication of the adenovirus genome complexed with viral core proteins (28), as an inhibitor of PP2A (29), in a yeast two-hybrid screen for proteins that bind to the human chromatin protein HRX (30), and in Xenopus extracts as a protein that binds to cyclin B (31).

Another group of proteins related in sequence to SET and identified in BLAST searches with SPR-2 are the Nap proteins. SET and Nap1 share many biochemical activities, but the implication of the biochemical activities of Nap1 for its in vivo function is not clear. Nap1 was first identified as a nucleosome assembly protein (32), and this activity has been the focus of biochemical studies of Nap1 and closely related Naps. In yeast, Nap1p has nucleosome-assembly activity (33), but, in addition, Nap1p was found to bind cyclin B and to be involved in the specific functions of cyclin B/p34cdc2 kinase complexes (31, 34). BLAST searches and CLUSTALW analyses reveal that there are multiple genes in mammals that are more related to Nap1 than to SET, i.e., these Nap-related genes seem to be distinct from SPR-2 and the SET subfamily (Fig. 2). (We note that another protein, Nck Associated Protein, has been called “Nap1” also, but is unrelated to SET/Nap proteins.) BLAST searches performed using SPR-2 and mammalian Nap proteins also reveal another C. elegans-predicted protein, D2096.8, which is related in sequence to SET and Nap1-like proteins. D2096.8 is not clearly orthologous to any available Nap sequences and is quite divergent also from SPR-2 and its SET homologs (Fig. 2).

Figure 2.

The SET/Nap family. BLAST searches were done by using SPR-2, SET, and Nap1 as query sequences. To generate this tree, a region that seemed well conserved among all members of this family (as marked in Fig. 1) was used for CLUSTALW analysis. The two C. elegans SET/Nap proteins, SPR-2 and D2096.8, are encased within ovals, and the SET subfamily is boxed in gray. For clarity, we have in some cases renamed proteins, as various names are seen on the BLAST report because of the different synonyms for SET, and we have omitted sequences from other mammals, other species of Plasmodium, other plants, and relatively short expressed sequence tags. Accession numbers: SPR-2, AF321546; DmSET (Drosophila melanogaster), AE003708; MusSET (Mus musculus), AB015613; HsSET (Homo sapiens), Q01105; XenopusTAF-Ia (Xenopus laevis), AB022691; XenopusTFA-Ib, AB022692; TetraodonSET (Tetraodon fluviatilis), AF007219; MusTSPY, AF042180.1; Hs TSPY, U58096; HsTSPY-like, AAF03521.1; ArabidopsisSET2 (Arabidopsis thaliana), AC011765; ArabidopsisSET1, AC011809; PlasmodiumSET-like (Plasmodium falciparum), AJ238237; pombeNap-like2 (Schizosaccharomyces pombe), T40114; pombeNap-like, T41330; cerevisiaeNap1, NP 012974.1; CeD0296.8, T15896; AnisakisNap-like (Anisakis simplex), AJ237977; MusNap1–4, NP 032698.1; HsNap1–4 and MusNap1–1, NP 056596.1; HsNap1–1, NP 004528.1; HemicentrotusNap-like (Hemicentrotus pulcherrimus), D21877; MusNap1–2, NP 032697.1; HsNap1–2, BAA84706; HsNap1–3, NP 004529; and ArabidopsisNap-like, AAA50234.

The relationship between SET and Nap proteins led us to investigate whether D2096.8 is functionally related to spr-2, despite the considerable sequence divergence. We performed RNAi with double-stranded D2096.8 RNA (Table 3). We saw no evidence for suppression of sel-12(-): no Egl⁺ progeny were produced after injection of sel-12(ar131) hermaphrodites or sel-12(ar171) unc-1(e538) hermaphrodites, suggesting that D2096.8 activity is not a major influence on hop-1 or sel-12 activity (Table 3). Furthermore, we saw no synthetic lethality or other novel phenotypes when D2096.8 activity was concomitantly depleted along with spr-2 activity by RNAi in a wild-type (N2) background, suggesting that D2096.8 and spr-2 may not be functionally redundant.

A Functional SPR-2∷GFP Protein Is Localized to the Nucleus.

To determine the subcellular localization of SPR-2, we constructed the integrated array arIs57, which expresses a SPR-2∷GFP reporter protein. The SPR-2∷GFP protein seems to retain spr-2 antisuppression activity (see Materials and Methods). SPR-2∷GFP was localized to the nucleus in all cells and at all stages in which it could be visualized (Fig. 3; data not shown). In particular, we note that SPR-2∷GFP was visualized in the nuclei of π cells (Fig. 3), a likely cellular focus for the Egl phenotype of sel-12 and lin-12 mutants (ref. 23; A. Newman, personal communication). In mammals, SET is found predominantly in the cell nuclei (35), whereas yeast Nap1 has been found to localize to the cytoplasm (31), and Drosophila Nap1 has been found to change its subcellular localization from nucleus to cytoplasm in a cell cycle-dependent manner (33, 36). The finding of SPR-2∷GFP in the nucleus is consistent with our assignment of SPR-2 to the SET subfamily based on sequence analysis.

Figure 3.

Expression of SPR-2∷GFP. All L3 stage. (A) Nomarski photomicrograph. The π cells (23) are indicated with arrowheads. (B) GFP fluorescence in the nuclei of π cells of the same hermaphrodite shown in A. (C) GFP fluorescence in the nucleus of the anchor cell (marked with arrowhead) and other gonadal cells. (D) GFP fluorescence in intestinal nuclei.

Discussion

We have found that loss of spr-2 activity can suppress the Egl phenotype caused by loss of sel-12 presenilin activity in C. elegans. Suppression is not sel-12 allele specific and depends on the activity of hop-1, another C. elegans presenilin: when hop-1 activity is removed also, spr-2(−) cannot suppress the Egl phenotype caused by sel-12(−). This latter result implies that spr-2(−) does not bypass the need for presenilin per se and instead has the effect of augmenting hop-1 presenilin activity. spr-2(−) might have a direct effect on the level of hop-1 gene expression or on HOP-1 protein stability or activity. Alternatively, the elevation of hop-1 activity might be indirect, reflecting an increase in the level of expression, stability, or activity of another component required for presenilin function in the development of the egg-laying system.

One of these other components might be, in principle, lin-12 or limiting components of lin-12 signaling. However, we have not been able to detect genetic interactions between alleles of spr-2 and alleles of lin-12. In this context, we note that there is no evidence that presenilin activity is normally rate limiting for LIN-12/Notch signaling in C. elegans, such that elevating presenilin activity beyond the wild-type level may not increase lin-12 activity, and might not be detectable via a genetic interaction with lin-12 alleles. If presenilin activity is not rate limiting, then the lack of genetic interaction between spr-2 and lin-12 would favor the possibility that spr-2 affects presenilin activity per se rather than lin-12 activity.

We have found that SPR-2 is a member of the SET/Nap family of proteins. Our analysis suggests that there is a clear subfamily of SET-related proteins in multicellular organisms, which includes SPR-2 and human SET. This subfamily seems to be distinct from the nucleosome assembly protein Nap1 and Nap1-related proteins. Furthermore, we used RNAi to investigate the role of the C. elegans protein D2096.8, which is more Nap1-like, and found no evidence that D2096.8 is a potential suppressor of sel-12(−) or functionally redundant with spr-2.

Biochemically, SET has been identified in different systems based on different properties. One group of properties pertains to chromatin structure: there is evidence that the template-activating factor activity of SET involves remodeling the chromatin structure of the adenovirus core (37), and SET is found in a protein complex with HRX, a chromatin-remodeling protein (30). Biochemical studies of Nap1 and Nap1-related proteins have also pointed to a role in chromatin structure (32, 33). However, SET has been identified also as an inhibitor of PP2A enzymatic activity (29), and both SET and Nap1 have been identified as factors that bind to cyclin B in Xenopus extracts (31). Thus, whether there is a single biochemical mechanism of SET function in vivo remains unclear.

We have found that the major site of accumulation of a functional SPR-2∷GFP protein is in the nucleus. This observation suggests that SPR-2 functions in the nucleus to facilitate hop-1 activity. In the context of biochemical data suggesting a role in chromatin structure, a simple hypothesis is that loss of spr-2 activity alters chromatin structure, which derepresses expression of hop-1. We have not detected an alteration in the level of hop-1 mRNA by Northern analysis (S. Jarriault, C.W., and I.G., unpublished observations); however, this method would not detect an alteration restricted to specific cells, a possibility we have not been able to explore further because we have been unable to detect expression of a hop-1∷gfp transgene (X.L. and I.G., unpublished observations). It is possible also that loss of spr-2 activity depresses expression of a gene that facilitates presenilin activity. Alternatively, another of the diverse biochemical properties of SET, or other properties that remain to be discovered, may underlie the mechanism of sel-12(−) suppression by spr-2(−).

Our finding that loss of spr-2 activity suppresses the Egl phenotype of sel-12(−) offers a system for the investigation of SET structure and function in vivo. spr-2 is one of an apparently small group of genes that can be identified as strong suppressors of the Egl phenotype of sel-12(−). Other spr genes identified in this screen may prove, therefore, to be conserved factors that cooperate with SPR-2/SET in regulating hop-1 activity. If so, they may provide insight as to the mechanism of SET function or additional tools for the biochemical analysis of SET function in other systems.

Abbreviations

Egl

egg-laying defective

Egl⁺

normal egg-laying

GFP

green fluorescent protein

RNAi

RNA-mediated interference