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Loss of spr-5 bypasses the requirement for the C.elegans presenilin sel-12 by derepressing hop-1

2002; Springer Nature; Volume: 21; Issue: 21 Linguagem: Inglês

10.1093/emboj/cdf561

1460-2075

Stefan Eimer,

Genomics, phytochemicals, and oxidative stress

Article1 November 2002free access Loss of spr-5 bypasses the requirement for the C.elegans presenilin sel-12 by derepressing hop-1 S. Eimer S. Eimer ABI, Department of Biochemistry, Laboratory of Molecular Neurogenetics, Ludwig-Maximilians-Universität Munich, Schillerstraße 44, D-80336 Munich, Germany Search for more papers by this author B. Lakowski B. Lakowski Present address: Department of Neuroscience, Pasteur Institute, Paris, France Search for more papers by this author R. Donhauser R. Donhauser ABI, Department of Biochemistry, Laboratory of Molecular Neurogenetics, Ludwig-Maximilians-Universität Munich, Schillerstraße 44, D-80336 Munich, Germany Search for more papers by this author R. Baumeister Corresponding Author R. Baumeister ABI, Department of Biochemistry, Laboratory of Molecular Neurogenetics, Ludwig-Maximilians-Universität Munich, Schillerstraße 44, D-80336 Munich, Germany Search for more papers by this author S. Eimer S. Eimer ABI, Department of Biochemistry, Laboratory of Molecular Neurogenetics, Ludwig-Maximilians-Universität Munich, Schillerstraße 44, D-80336 Munich, Germany Search for more papers by this author B. Lakowski B. Lakowski Present address: Department of Neuroscience, Pasteur Institute, Paris, France Search for more papers by this author R. Donhauser R. Donhauser ABI, Department of Biochemistry, Laboratory of Molecular Neurogenetics, Ludwig-Maximilians-Universität Munich, Schillerstraße 44, D-80336 Munich, Germany Search for more papers by this author R. Baumeister Corresponding Author R. Baumeister ABI, Department of Biochemistry, Laboratory of Molecular Neurogenetics, Ludwig-Maximilians-Universität Munich, Schillerstraße 44, D-80336 Munich, Germany Search for more papers by this author Author Information S. Eimer1, B. Lakowski2, R. Donhauser1 and R. Baumeister 1 1ABI, Department of Biochemistry, Laboratory of Molecular Neurogenetics, Ludwig-Maximilians-Universität Munich, Schillerstraße 44, D-80336 Munich, Germany 2Present address: Department of Neuroscience, Pasteur Institute, Paris, France *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:5787-5796https://doi.org/10.1093/emboj/cdf561 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Presenilins are part of a protease complex that is responsible for the intramembraneous cleavage of the amyloid precursor protein involved in Alzheimer's disease and of Notch receptors. In Caenorhabditis elegans, mutations in the presenilin sel-12 result in a highly penetrant egg-laying defect. spr-5 was identified as an extragenic suppressor of the sel-12 mutant phenotype. The SPR-5 protein has similarity to the human polyamine oxidase-like protein encoded by KIAA0601 that is part of the HDAC–CoREST co-repressor complex. Suppression of sel-12 by spr-5 requires the activity of HOP-1, the second somatic presenilin in C.elegans. spr-5 mutants derepress hop-1 expression 20- to 30-fold in the early larval stages when hop-1 normally is almost undetectable. SPR-1, a C.elegans homologue of CoREST, physically interacts with SPR-5. Moreover, down-regulation of SPR-1 by mutation or RNA interference also bypasses the need for sel-12. These data strongly suggest that SPR-5 and SPR-1 are part of a CoREST-like co-repressor complex in C.elegans. This complex might be recruited to the hop-1 locus controlling its expression during development. Introduction Mutations in the presenilins PS1 and PS2 account for the majority of early-onset familial Alzheimer's disease (Selkoe, 2001). These mutations lead to the aberrant processing of the amyloid precursor protein (APP) and the generation of increasing amounts of the highly amyloidogenic form of Aβ, the amyloid β-peptide. Aβ is the predominant component of the plaques found in the brains of Alzheimer's patients. Presenilins are polytopic transmembrane proteins that are part of the proteolytic γ-secretase complex that liberates Aβ. Apart from their role in APP processing, presenilins are also required for proteolytic processing of the Notch receptors in their transmembrane domain. Ligand-induced cleavage and release of the intracellular domain of the Notch receptor (NICD) are crucial for nuclear Notch signalling (Struhl and Adachi, 1998). In agreement with these results, in all organisms tested so far, the loss of presenilin activity leads to phenotypes that resemble that of Notch loss-of-function mutants (Fortini, 2001). Similarly to humans, Caenorhabditis elegans has two somatically expressed presenilin genes, hop-1 and sel-12 (Levitan and Greenwald, 1995; Li and Greenwald, 1997). hop-1 and sel-12, like PS1 and PS2, show redundant activities since only double mutants display phenotypes associated with a complete loss of Notch signalling in C.elegans (Li and Greenwald, 1997; Westlund et al., 1999). A sel-12 null mutant can be rescued by transgenic expression of hop-1 (as well as either of the human PS1 or PS2). sel-12 is expressed rather uniformly at all developmental stages, while hop-1 expression is very weak and could not be detected by reporter gene fusions (Westlund et al., 1999). Probably as a consequence of these different levels of expression, hop-1 mutants are viable with no obvious phenotype, whereas sel-12 mutants display, along with other more subtle defects, an egg-laying (Egl) defect. The sel-12 Egl phenotype is caused by reduced LIN-12/Notch signalling, resulting in the failure to form a proper vulva–uterine connection (Cinar et al., 2001) and in sex-muscle patterning defects (Eimer et al., 2002). One approach to identify new molecules involved in the regulation of presenilin activity or molecules that are able to bypass the need for presenilin activity is to perform genetic screens for suppressors of the Egl defect of sel-12 mutant animals. Two sel-12 suppressors have been described already. Mutations in the C.elegans gene sel-10 directly influence LIN-12/Notch signalling by enhancing the half-life of the intracellular signalling domain of LIN-12 (Wu et al., 1998). The F-box/WD40 repeat protein SEL-10 is part of a ubiquitin ligase complex that mediates the ubiquitylation and degradation of the activated nuclear NICD (Lai, 2002). In contrast, the suppressor mutant spr-2 does not influence LIN-12/Notch signalling directly but requires HOP-1 activity in a manner that has not been characterized (Wen et al., 2000) but obviously does not affect the level of its transcription. SPR-2 belongs to a family of nuclear assembly proteins (NAPs) which have been implicated in chromatin remodelling and cell cycle control. The different modes of action of sel-10 and spr-2 function suggest that there exist distinct mechanisms to suppress sel-12. The functional conservation of presenilins suggests that a third possible mechanism to suppress the sel-12 mutant phenotype could involve transcriptional up-regulation of the expression of the second presenilin, in this case hop-1. Therefore, second site suppressors of sel-12 could include mutations in transcriptional activators that render them hyperactive, or mutations in repressors or repressor complexes that eliminate the down-regulation of hop-1. It is becoming increasingly obvious that histone deacetylase (HDAC)-containing repressor complexes are required for both transient and persistent transcriptional silencing of selected genes. Notably, HDAC complexes play an important role in the Notch-mediated transcriptional silencing of downstream genes (reviewed in Mumm and Kopan, 2000). HDACs associate with several distinct complexes, including the Sin-associated protein (SAP) complex and the nucleosome remodelling and histone deacetylation (NURD) complex, which exhibits ATP-dependent chromatin-remodelling activity (Jepsen and Rosenfeld, 2002). Recently, yet another HDAC complex was identified containing the transcriptional co-repressor CoREST and protein KIAA0601, which is a putative flavin–adenine dinucleotide (FAD)-dependent enzyme of still unknown function. We report here the identification of two new suppressors of the Egl defect of sel-12 mutant C.elegans animals, spr-5 and spr-1. The analysis of spr-5 revealed that it acts by de-repession of hop-1 expression in developmental stages where hop-1 normally is not expressed. Therefore, sel-12 presenilin activity is replaced by hop-1 activity. SPR-5 exhibits homology to the human polyamine oxidase (PAO)-like protein KIAA0601, which is an integral component of the CoREST co-repressor complex. Our screens also identified by133, an spr-1 allele that, like RNA interference (RNAi) inhibition of a C.elegans homologue of CoREST, phenocopies the spr-5 suppressor function. Furthermore, we show that D1014.8/SPR-1 forms a complex with SPR-5. We suggest that a CoREST-like co-repressor complex also exists in C.elegans and participates in the transcriptional repression of the presenilin hop-1 during development. Results spr-5 suppresses the sel-12 Egl phenotype in a non-allele-specific way In a screen using the mutator strain sel-12(ar171) unc-1(e538); mut-7(pk204), we recovered a mutation on chromosome I, by101, that suppressed the Egl defect of sel-12(ar171) hermaphrodites. Genetic mapping of the mutant loci placed the suppressor in the interval between unc-101 and unc-59 close to unc-59 (Figure 1; for details see Materials and methods). As it complements the previously mapped but not yet cloned presenilin suppressor spr-4(ar208) on LGI (Wen et al., 2000), the mutant locus we found corresponds to a new gene, which we will refer to as spr-5. Subsequently, we identified five additional alleles of spr-5 in similar screens using chemical mutagens (details of the experimental procedures will be published elsewhere; B.Lakowski and R.Baumeister, unpublished data). Figure 1.Physical map of the spr-5 region on LGI. The location and the extension of the fosmids H14o4, H37o19 and H18N7 relative to the Y40B1B locus, as well as their rescuing activity, are indicated. The scale bar below represents base pairs. Download figure Download PowerPoint All spr-5 mutants result in a very strong suppression of all aspects of the sel-12 Egl defect. Eighty-five to 95% of all spr-5; sel-12 animals are non-Pvl and non-Egl (Table I). Furthermore, the brood size of these animals is much larger than that of sel-12 and is in the range of that of the wild type (Table I). spr-5 mutations suppress all sel-12 alleles tested (Table I): ar171, a truncation after the fifth transmembrane domain (W225stop); ar131, a C60S missense mutation (Levitan and Greenwald, 1995); and lg1401, a deletion of sel-12 and part of the promoter that is a clear null allele (Eimer et al., 2002; Table I). This indicates that the mechanism of suppression is not allele specific and does not depend on the presence of SEL-12 protein. Table 1. spr-5 bypasses the need for sel-12 in a non-allele-specific way Genotype Pvl (%) Egl (%) Brood size n Wild-type N2 0 0 316 ± 8 20 sel-12(ar171) 80 100 62 ± 7 25 sel-12(ar171); spr-5(by113) 0 5 188 ± 15 20 sel-12(ar171); spr-5(by128) 10 15 230 ± 20 20 sel-12(ar171); spr-5(by134) 10 10 219 ± 12 20 sel-12(ar171); spr-5(by101) 5 10 208 ± 15 40 sel-12(lg1401); spr-5(by101) 0 5 214 ± 12 19 sel-12(ar131); spr-5(by101) 0 5 231 ± 17 20 spr-5(by101) 0 0 276 ± 12 27 In addition, as opposed to sel-12 animals, spr-5; sel-12 hermaphrodites also lay eggs in response to the drugs serotonin and imipramine, suggesting that they have a functional egg-laying system that can be stimulated pharmacologically (Trent et al., 1983). spr-5 mutations also restore male mating that is defective in sel-12 males (Eimer et al., 2002). Therefore, spr-5 rescues not only the structural defects of the egg-laying system of sel-12 animals, but also the functional defects. When genetically separated from sel-12 alleles, spr-5 mutants alone display no obvious phenotype, suggesting that spr-5 is a specific suppressor of sel-12. Neither egg-laying, egg motility nor other behaviours were different from those of wild-type animals under the conditions tested (data not shown). In addition, the brood size of all spr-5 alleles is in the range of wild type (Table I). spr-5 mutants do not enhance LIN-12/Notch signalling directly In order to determine the mechanism of suppression, we first tested whether lin-12 is the prime target of spr-5 genetic interactions. Since sel-12 mutations reduce LIN-12 signalling, spr-5 mutations might act by increasing LIN-12 expression or activity. From other experiments not reported here, we had concluded that mRNA levels expressed from both C.elegans Notch genes, lin-12 and glp-1, did not differ between wild type and the spr mutants we had recovered in our screens (B.Lakowski and R.Baumeister, unpublished data). Therefore, it is unlikely that spr-5 mutants act by transcriptional up-regulation of Notch expression. The lin-12(n676n930) reduction of function allele displays a temperature-sensitive Egl defect (Sundaram and Greenwald, 1993), which is similar to the sel-12 Egl phenotype (Eimer et al., 2002). Therefore, we wondered whether spr-5 might suppress the Egl defect of lin-12(n676n930) at 25°C. However, spr-5 did not rescue any aspect of the lin-12(n676n930) Egl defect (Table II). We also tested whether spr-5 is able to enhance another well-characterized process that is defective in lin-12 mutants, the AC/VU decision (Greenwald et al., 1983). In the AC/VU decision, two initially equipotent cells of the somatic gonad adopt different cell fates through a LIN- 12-dependent process called lateral inhibition. The lin-12(n302) gain-of-function allele is vulva-less and therefore Egl, because it develops two VU cells at the expense of an AC (Greenwald et al., 1983). As the lin-12(n302) mutation is semi-dominant, 50% of the hetero zygote animals are Egl. spr-5 mutants show no increase in the 0 AC phenotype in heterozygotes, indicating that the LIN-12-dependent lateral inhibition is also not affected in spr-5 mutants (Table II). From these results, we conclude that spr-5 does not directly influence any of the characterized modes of LIN-12 signalling. Therefore, we consider it unlikely that spr-5 suppresses sel-12 by directly up-regulating LIN-12 signalling. Table 2. Genetic interactions of spr-5 with lin-12 and hop-1 sel-12 suppression by spr-5 is dependent on hop-1 Genotype No. of sterile/total (%) sel-12(ar171); hop-1(lg1501)a 36/36 (100%) sel-12(ar171); hop-1(lg1501) spr-5(by101)a 43/43 (100%) No. of Egl/total (%) sel-12(ar171); hop-1(lg1501)b 50/50 (100%) sel-12(ar171); hop-1(lg1501) spr-5(by101)b 48/49 (98%)c No effect on the 0 AC defect caused by elevating lin-12 activity Genotype No. of Egl/total (%) lin-12(n302)/ unc-32(e189) 40/79 (51%) lin-12(n302)/ unc-32(e189); spr-5(by101) 77/141 (55%) No enhancement of a lin-12 hypomorphic mutation Relevant genotype No. of Egl/total (%) at 25°C lin-12(n676n930)d 50/50 (100%) lin-12(n676n930)d; spr-5(by101) 50/50 (100%) a These animals segregated from sel-12(ar171); hop-1(lg1501)/unc-73(e936) or sel-12(ar171); hop-1(lg1501) spr-5(by101)/unc-73(e936) spr-5(by101) mothers. The genotype is therefore: sel-12 m z; hop-1 m z. b These animals segregated from sel-12(ar171) unc-1(e538)/++; hop-1(lg1501) or sel-12(ar171) unc-1(e538)/++; hop-1(lg1501) spr-5(by101) mothers. The genotype is therefore: sel-12 m z; hop-1 m z. c One animal was sterile. d This strain also carries an unc-32(e189) mutation that is linked to lin-12. The Egl phenotype was scored with animals raised at 25°C. The suppressor activity of spr-5 requires functional HOP-1 presenilin To determine whether spr-5 bypasses the need for functional presenilins in LIN-12 signalling or may act through modulating the activity of the second presenilin hop-1, we constructed hop-1 spr-5; sel-12 triple mutants. Both sel-12 and hop-1 have partial maternal effects, so the phenotype of hop-1; sel-12 double mutants depends on how they are constructed. Double mutant animals that have maternally supplied hop-1 are sterile, whereas, if sel-12 is provided maternally, hop-1; sel-12 mutants become Egl, with embryos that do not hatch (Westlund et al., 1999). hop-1 spr-5; sel-12 triple mutants, with maternally supplied sel-12, are also Egl (Table II) and only produce dead embryos. Furthermore, triple mutants with maternally supplied hop-1 are sterile like the similarly constructed sel-12; hop-1 double mutants (Table II). We therefore conclude that spr-5 mutants require hop-1 activity for sel-12 suppression. Molecular cloning of spr-5 Initially, spr-5 was mapped near unc-59 on the right arm of chromosome I (Figure 1). A Tc3 transposon was found to co-segregate with the suppression phenotype in spr-5(by101) (for details see Materials and methods). Sequencing of the genomic DNA flanking the transposon revealed that the Tc3 had inserted into the seventh exon in codon 606 of the predicted reading frame Y40B1B.6 (Figure 1). When injected into sel-12; spr-5 animals, fosmids H14o4 (four out of four transgenic lines) and H37o19 (five out of six transgenic lines) that contain the entire coding region of Y40B1B.6 rescued the spr-5 mutant phenotype, and restored a sel-12 phenotype (Egl). In contrast, fosmid H18N7 that terminates after the sixth exon of Y40B1B.6 was not able to rescue the suppression by spr-5 (none out of three transgenic lines), suggesting that Y40B1B.6 indeed corresponds to spr-5 (Figure 1). Further analysis of the genomic organization of the open reading frames (ORFs) on Y40B1B indicated that the genes Y40B1B.8, Y40B1B.5 and Y40B1B.6/spr-5 belong to an operon consisting of three genes (Figure 2). In operons, the downstream genes are trans-spliced to a 22 nucleotide splice leader SL2 (Spieth et al., 1993), whereas the most upstream gene is trans-spliced to an SL1 splice leader. We used RT–PCR with SL1 and SL2 primers to determine the 5′ ends of all three putative ORFs. We found that Y40B1B.8 is spliced exclusively to SL1, Y40B1B.5 is trans-spliced to a mixture of SL1 and SL2, and Y40B1B.6/spr-5 is spliced primarily to SL2 (Figure 2). The existence of a three-gene operon containing spr-5 is supported further by DNA array data (Blumenthal et al., 2002). Based both on their orientation and their encoded amino acid sequence, these three genes are highly conserved in the related nematode Caenorhabditis briggsae, consistent with being in an operon. However, the exon–intron structure of the C.elegans and C.briggsae genes differs to some extent (Figure 2). Although the genes are highly conserved on a protein level, Y40B1B.8 contains seven exons in C.elegans compared with nine in C.briggsae, and the fourth exon of Y40B1B.6/spr-5 is represented by two exons in C.briggsae (Figure 2). Figure 2.Exon–intron structure of the spr-5 operon on LGI. The specific splice leader and the locations of the mutations in the different spr-5 alleles are shown. In addition, the organization of the homologous operon in C.briggsae is included, along with the identity and similarity scores of the C.elegans and C.briggsae proteins. Download figure Download PowerPoint We examined in all spr-5 alleles the expression of the two downstream reading frames of the operon. On a mixed stage northern blot, most spr-5 alleles show clear alterations in either the size and/or the intensity of bands detected with an Y40B1B.6 probe, and no obvious differences in the transcript of Y40B1B.5 (Figure 3). To support this notion further, we performed RNAi experiments against each of the three genes in this operon by bacterial feeding of double-stranded RNA (dsRNA) (Timmons and Fire, 1998; Timmons et al., 2001). Although it has been reported that RNAi may also target the pre-mRNA (Bosher et al., 1999), only the dsRNAi against spr-5/Y40B1B.6 led to suppression of the Egl defect of sel-12 animals that is typical for the different spr-5 alleles (Table III). dsRNAi against any of the upstream genes was not able to suppress the Egl defect of sel-12 (Table III). Therefore, we conclude that spr-5 is Y40B1B.6. Subsequently, we confirmed the genomic structure predicted in Wormbase by sequencing. Y40B1B.6/spr-5 encodes a protein of 770 amino acids (Figure 4A). The positions of all mutations and alterations found in the various spr-5 alleles are shown in Figures 2 and 4A. Figure 3.Northern blot of mixed staged total RNA from the different alleles probed with spr-5, Y40B1B.5, and ama-1 as a loading control. Download figure Download PowerPoint Figure 4.(A) Alignment of SPR-5 with the human KIAA0601 (DDBJ/EMBL/GenBank accession No. BAA25527), Drosophila melanogaster CG17149 (accession No. AAF49051), C.elegans T08D10.2 and Zea mays PAO (accession No. CAA05249). Identical residues are shaded in black, whereas similar residues are shaded in grey. Asterisks indicate the residues contacting the FAD cofactor (Binda et al., 1999), and open circles the residues contacting the substrate (Binda et al., 2001) in the maize PAO. Underlined is the signature motif characteristic for the flavoprotein family (Dailey and Dailey, 1998). The positions of the spr-5 mutations are indicated by black triangles above the sequence. (B) ClustalW-aligned tree of the different protein families showing homologies to PAOs. SPR-5 defines a distinct subgroup including KIAA0601, DmCG17149 and T08D10.2. For details on nomenclature, see Supplementary data available at The EMBO Journal Online. Download figure Download PowerPoint Table 3. Summary of dsRNAi feeding experiments RNAi construct RNAi phenotype in strains Wild type sel-12(ar171) sel-12(ar131) – wt (15/15) Egl (10/10) Egl (10/10) spr-5 wt (10/10) Non-Egl (39/40)a Non-Egl (20/20) Y40B1B.5 wt (10/10) Egl (20/20) Egl (20/20) Y40B1B.8 wt (10/10)b Egl (20/20)b Egl (20/20)b T08D10.2 wt (11/11) Egl (20/20) Egl (20/20) D1014.8 wt (10/10) Non-Egl (30/30) ND Y74C9A.4 wt (10/10) Egl (30/30) ND a One animal was sterile. b There were sick worms on the plates at a low penetrance. spr-5 is homologous to amine oxidases found in transcriptional repressor complexes SPR-5 exhibits strong similarity to a large class of FAD-dependent amine oxidases. SPR-5 is most similar to the class of FAD-dependent PAOs (Figure 4). The PAOs are found in all organisms from bacteria to mammals and plants, and catalyse the oxidation of secondary amine groups of straight chain aminoalkanes (Sebela et al., 2001). Despite the differences in the site of action on the secondary amine group between plants and mammals, PAOs are monomeric soluble enzymes with non-covalently bound FAD cofactor (Seiler, 1995). All PAOs have a two-domain organization, with one domain binding the FAD cofactor while the other binds the substrate (Binda et al., 1999, 2001). Even though they only share 20–30% identity, the maize PAO has the same three-dimensional structure as the vertebrate monoamine oxidase, known to act on primary amines (Binda et al., 2002). SPR-5 and the maize PAO share the same FAD-binding signature motif (Dailey and Dailey, 1998), and the residues known to bind the FAD cofactor in maize PAO are conserved (Binda et al., 1999), while those residues that recognize the substrate are not conserved (Binda et al., 2001). Therefore, we conclude that it is likely that SPR-5 binds FAD as a cofactor while the substrate specificity may have diverged. SPR-5 defines a subfamily of the PAOs along with a second C.elegans protein T08D10.2, the Drosophila melanogaster protein encoded by expressed sequence tag (EST) CG17149 and the human protein encoded by EST KIAA0601 (Nagase et al., 1998). Members of this subfamily of proteins are more similar to each other than to the maize PAO (Figure 4A and B). SPR-5 has 27% amino acid identity (45% similarity) to hKIAA0601 and Dm CG17149, whereas it has 44% identity (62% similarity) to the C.elegans paralogue. The protein that corresponds to KIAA0601 was co-purified as an integral component of the human CoREST–HDAC complex (Tong et al., 1998; Humphrey et al., 2001; You et al., 2001). The CoREST complex was shown to be a functional co-repressor that is required for REST-mediated repression of neuronal genes in non-neuronal cells (Andres et al., 1999; Ballas et al., 2001; Griffith et al., 2001). Additional components of the human CoREST complex are HDACs 1 and 2 and the SANT domain protein CoREST (Humphrey et al., 2001; You et al., 2001), which binds to the zinc finger factor REST (Andres et al., 1999; Ballas et al., 2001). REST recruits the CoREST complex to specific DNA sites, suggesting that the complex may regulate individual genes. Loss of spr-5 leads to derepression of hop-1 expression Due to the sequence similarity of SPR-5 to a member of a complex involved in transcriptional repression, it is possible that SPR-5 may have a similar function in C.elegans. Thus, we searched for candidate target genes in the Notch signalling pathway that may be regulated by spr-5. The suppression of sel-12 by spr-5 was shown not to up-regulate LIN-12/Notch signalling but was dependent on hop-1 activity. Since hop-1 and sel-12 are functionally redundant, hop-1 was an attractive candidate for spr-5-mediated transcriptional regulation. Therefore, we analysed the stage-specific transcriptional regulation of hop-1 by northern blot analysis. sel-12 is ubiquitously expressed at high levels throughout all developmental stages (Baumeister et al., 1997). In contrast, hop-1 expression dramatically changes throughout development. hop-1 is almost undetectable in the L1 and L2 larvae and its expression gradually increases throughout further development and reaches a maximum at the adult stage (B.Lakowski and R.Baumeister, unpublished data). As a consequence of the high expression levels in the adult stage, probably a high level of hop-1 mRNA is supplied to the embryo maternally. In sel-12 mutant animals, hop-1 expression levels are indistinguishable from wild-type levels, suggesting that sel-12 does not control hop-1 expression at the transcriptional level. Next, we investigated hop-1 expression in spr-5; sel-12 suppressor strains. hop-1 expression was detectable already in the L1 stage, while it is almost absent in wild-type L1 animals (Figure 6). The up-regulation of hop-1 expression was between 20- and 30-fold depending on the spr-5 allele tested and independent of the presence of sel-12 (data not shown). spr-5 is expressed in all stages at nearly the same level, indicating that it may act as a general co-repressor (Figure 5). Figure 5.Stage-specific northern blot of wild-type total RNA probed with spr-5, and ama-1 as a loading control. Download figure Download PowerPoint Figure 6.Northern blot of L1-specific total RNA N2, sel-12(ar171) and different sel-12(a171); spr-5 double mutants probed with hop-1- and ama-1-specific probes. *Every spr-5 allele was in a sel-12(ar171) mutant background. Download figure Download PowerPoint These data suggest that spr-5 is required to repress hop-1 expression in the first larval stages and that loss of spr-5 leads to a derepression of hop-1 in these stages. Derepression of hop-1 expression is sufficient to replace the lack of sel-12 activity and, therefore, suppresses the Egl phenotype of the sel-12 mutants. A CoREST-like complex might be involved in transcriptional repression of hop-1 If SPR-5 functions in a HDAC–CoREST complex of transcriptional regulation, then one might assume that manipulating the expression of other members of this complex should result in a phenotype similar to that of spr-5 mutants. We identified two predicted open reading frames, D1014.8 and Y74C9A.4, encoding proteins with similarity to CoREST. dsRNAi experiments were performed for both genes in a sel-12 mutant background (Table III). RNAi against Y74C9A.4 did not reveal any novel phenotype in a sel-12 background. Strikingly, however, dsRNAi against D1014.8 suppressed the sel-12 Egl defect as strongly as spr-5 (Table III). D1014.8 maps to a genomic region on LGV where another sel-12 suppressor, spr-1, was mapped previously (Wen et al., 2000), suggesting that D1014.8 corresponds to spr-1. In our screens for sel-12 suppressors, we had identified a mutant with properties similar to spr-5 alleles. We had mapped this suppressor allele, by133, close to D1014.8, suggesting that it might encode an allele of spr-1. To determine if by133 is an allele of spr-1, we performed rescue experiments with the cosmid D1014 using an spr-1(by133); sel-12(ar171) strain. Seven of seven transgenic F2 lines clearly and profoundly rescued the spr-1(by133)-mediated suppression of sel-12. Subsequently, we sequenced the coding region of the by133 mRNA and found that it contains a T→A mutation at position +905, converting a TTA (leucine) to TAA (stop). This mutation truncates the protein at amino acid 301 after the first SANT domain, but before the second SANT domain. Thus, mutations in two members of the CoREST complex suppress the presenilin defect in C.elegans. RNAi against the three C.elegans class I HDACs hda-1, hda-2 and hda-3, alone or in combination, resulted in a pleiotropic phenotype that was difficult to interpret. This result was not unexpected since it had been shown before that these HDACs in C.elegans are involved in a variety of different fu