Null mutations in lin-35, the Caenorhabditis elegans ortholog of the mammalian Rb protein, cause no obvious morphological defects. Using a genetic approach to identify genes that may function redundantly with lin-35, we have isolated a mutation in the C. elegans psa-1 gene. lin-35; psa-1 double mutants display severe developmental defects leading to early larval arrest and adult sterility. The psa-1 gene has previously been shown to encode a C. elegans homolog of yeast SWI3, a critical component of the SWI/SNF complex, and has been shown to regulate asymmetric cell divisions during C. elegans development. We observed strong genetic interactions between psa-1 and lin-35 as well as a subset of the class B synMuv genes that include lin-37 and lin-9. Loss-of-function mutations in lin-35, lin-37, and lin-9 strongly enhanced the defects of asymmetric T cell division associated with a psa-1 mutation. Our results suggest that LIN-35/Rb and a certain class B synMuv proteins collaborate with the SWI/SNF protein complex to regulate the T cell division as well as other events essential for larval growth.

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MEMBERS of the retinoblastoma tumor suppressor family of proteins (pRb, p107, and p130) play a fundamental role in cell cycle control, apoptosis, and development (reviewed by MORRIS and DYSON 2001; CLASSON and HARLOW 2002). A major cellular target of pRb is the E2F family of transcription factors that regulate the expression of genes required for G₁ to S phase transition as well as many genes unconnected to cell cycle control (DYSON 1998; STEVAUX and DYSON 2002). The formation of Rb-E2F complexes at the promoters of cell cycle genes blocks transcriptional activation by E2F's. In addition, Rb-E2F complexes can actively repress transcription through the recruitment of histone deacetylase (HDAC; BREHM et al. 1998; LUO et al. 1998; MAGNAGHI-JAULIN et al. 1998), histone methylase (ROBERTSON et al. 2000; NIELSEN et al. 2001; PRADHAN and KIM 2002), and members of the nucleosome remodeling complex (SWI/SNF; DUNAIEF et al. 1994; STROBER et al. 1996; ZHANG et al. 2000). Transfection studies indicate that class I HDACs and SWI/SNF members are recruited by pRb and may coordinate the sequential expression of E2F target genes to regulate the progression through G₁ and S phases (ZHANG et al. 2000). A genetic screen for modifiers of an E2F overexpression phenotype in the Drosophila eye identified enhancer mutations in Osa, Brahma, and Moira genes, which encode homologs of SWI1, SWI2, and SWI3, respectively, suggesting that the SWI/SNF complex downregulates E2F activity (STAEHLING-HAMPTON et al. 1999). First identified in the yeast Saccharomyces cerevisiae, SWI/SNF is a 2-MD multisubunit assembly that is highly conserved in eukaryotes. The SWI/SNF complex contributes to the regulation of gene expression by locally altering the structure of chromatin. Depending on the context, it can be involved in either transcriptional activation or repression (reviewed by MARTENS and WINSTON 2003). Growing genetic and molecular evidence indicate that subunits of the SWI/SNF complex act as tumor suppressors in humans and mice (reviewed by KLOCHENDLER-YEIVIN et al. 2002). In Caenorhabditis elegans, PSA-1/SWI3 and PSA-4/SWI2 have been shown to play a role in the asymmetric division of the hypodermal T cell during larval development (SAWA et al. 2000).

In contrast to Rb knockout mutations in flies and mice (for a review of mammalian studies see LIPINSKI and JACKS 1999), strong loss-of-function (lf) mutations in lin-35/Rb cause relatively subtle effects on viability and development in C. elegans (LU and HORVITZ 1998; BOXEM and VAN DEN HEUVEL 2002; FAY et al. 2002). To uncover the functions of lin-35 during C. elegans development, we have carried out a genetic screen to isolate mutations that are synthetically lethal with lin-35(n745) (FAY et al. 2002, 2003). Briefly, the strain used for the screen was homozygous for a strong loss-of-function mutation in lin-35(n745), but carried an extrachromosomal array (kuEx119) containing copies of the wild-type lin-35 gene and a ubiquitously expressed green fluorescent protein (GFP) reporter. Typically, extrachromosomal arrays fail to be transmitted to a certain percentage of progeny from parents that carry the array (kuEx119 is transmitted to 70% of self-progeny). After mutagenesis, mutant animals that are synthetically lethal with lin-35 were identified by their complete dependence on kuEx119 for viability. Using this strategy, our laboratories have previously reported the identification of mutations in fzr-1, a regulatory subunit of the APC proteasome (FAY et al. 2002), and mutations in ubc-18, the C. elegans homolog of human UBCH7 (FAY et al. 2003). lin-35; fzr-1 double mutants display a hyperproliferation phenotype, while lin-35; ubc-18 double mutants are defective at an early stage of pharyngeal morphogenesis. Here we describe our analysis of another mutation (ku355) isolated by this screen that shows defects that differ from that of fzr-1 and ubc-18.

Strains:

Nematodes were cultured using standard techniques (SULSTON and WHITE 1980). Mutations used were as follows: LGI, lin-35(n745); LGII, lin-8(n111); LGIII, lin-36(n766) and lin-37(n758); LGV, fog-2(q71), psa-1(ku355), psa-1(os22), rol-9(sc148), and unc-51(e369); LGX, lin-15(n765ts), lin-15b(n744), and lin-15a(n767). The Hawaiian strain CB4856 was used in the single-nucleotide polymorphism (SNP) mapping. In addition, we used a strain containing an extrachromosomal array, kuEx119 [lin-35(+), sur-5::GFP; FAY et al. 2002]. GFP marker strains used were JK2868 [qIs56 (IV or V; lag-2::GFP; BLELLOCH et al. 1999)], JR767 [SCM::GFP (TERNS et al. 1997)], and JM63 [elt-2::GFP (FUKUSHIGE et al. 1998)].

Isolation and mapping of psa-1(ku355):

psa-1(ku355) was isolated in a genetic screen for identifying mutations that cause synthetic phenotypes with lin-35(n745) in C. elegans (FAY et al. 2002). psa-1(ku355) was then mapped to LGV. Three-factor crosses using marker mutations in fog-2 and rol-9 further defined the map position of psa-1(ku355) to the region between fog-2 and rol-9. Among the progeny of hermaphrodites of genotype fog-2(q71) rol-9(sc148)/psa-1(ku355), 4 of 12 Rol non-Fog recombinants segregated psa-1(ku355). The strain rol-9(sc148) psa-1(ku355) and the Hawaiian strain CB4856 were used in a standard SNP mapping procedure to place psa-1(ku355) in a 96-kb region between fog-2 and the polymorphisms [vl30h01.s1@572,t,38] on the cosmid ZC15.

RNAi analysis:

RNAi was performed essentially as described (KAMATH et al. 2003). Feeding vectors containing lin-35, lin-36, lin-53, hda-1, and lin-15A were previously described (FAY et al. 2002). Feeding vectors containing lin-37, lin-9, let-418, C01G8.9, F01G4.1/psa-4, ZK1128.5, R07E5.3, Y71H2AM.17, F26D10.3, act-1, act-2, act-3, act-4, and act-5 were obtained from an RNAi library from the United Kingdom Human Genome Mapping Project Resource Centre (HGMP-RC). For psa-1, a 1055-bp SalI-SacI fragment from the psa-1 cDNA clone yk257g1 was subcloned into vector pPD129.36. For C18E3.2, a 1-kb PCR fragment of genomic DNA corresponding to nucleotides 26,000–27,000 of C18E3 (part of C18E3.2 coding region) was subcloned into vector pPD129.36. For ZK616.4, a 1369-bp PCR fragment of genomic DNA corresponding to nucleotides 2903–4272 of ZK616.4 (part of ZK616.4 coding region) was subcloned into vector pPD129.36. In most experiments, we placed synchronized L1 stage worms (parents or P0) onto a plate seeded with a bacterial strain carrying specific RNAi plasmids. We started scoring the phenotypes in the P0 or the F₁ generation 3 days later.

Embryonic lethal and larval arrest phenotypes:

For embryonic lethality, individual L4 larvae were transferred to fresh plates every day for 3 consecutive days, and the eggs laid on the plates were scored for hatching. For the larval arrest phenotype, the percentage of arrest animals was determined 60 hr after hatching. The larval stage of the arrest was evaluated mainly by the size and shape of the gonad.

Cell lineage analysis, microscopy, and dye-filling assays of the phasmids:

Cell morphology and lineage in living animals were observed using Nomarski optics (HERMAN and HORVITZ 1994). Phasmid dye filling as an indicator for the normal T cell polarity was scored as previously described (HERMAN and HORVITZ 1994). Briefly, adult animals were soaked in a cold solution of M9 salts containing 0.4 mg/ml of fluorescein isothiocyanate (FITC) for 4 hr, washed three times with cold M9 salts, and then transferred to a seeded agar plate at room temperature. For quantification and microphotography, dye filling was evaluated using a Zeiss Standard microscope equipped with epifluorescence. Generally, the amphids and phasmids of N2 animals were filled with the dye. In contrast, in psa-1(ku355) animals, the amphids were filled with the dye, but the phasmids often were not. Occasionally, neither the amphids nor the phasmids in N2 animals were filled with the dye. For this reason, we counted as phasmid defective only those individuals that showed amphid dye filling but no phasmid filling.

psa-1(ku355) and lin-35(n745) cause a synthetic larval arrest phenotype :

We initiated our study of the psa-1 gene by isolating the psa-1(ku355) allele in a genetic screen in C. elegans for mutations that cause synthetic mutant phenotypes with lin-35 (FAY et al. 2002, 2003). At 20°, lin-35(n745); psa-1(ku355) animals that carried an extrachromosomal array containing the lin-35(+) gene were viable. In contrast, 98% of the lin-35; psa-1 animals that failed to inherit the array arrested at the first larval stage (L1; Table 1), and those lin-35; psa-1 animals that reached adulthood were small and sterile (n = 50; Figure 1; Table 1). In addition, 16% of lin-35; psa-1 double mutants failed to progress through embryogenesis (n = 840). The arrested L1 animals tended to be curled and inactive, although no obvious pharyngeal morphological defects were observed in these animals. It was previously reported that lin-35(n745) single mutants are viable but show a substantial reduction in brood sizes when compared with wild type (LU and HORVITZ 1998; Table 1). At 20°, psa-1(ku355) single mutants were also viable but grew slowly and had reduced brood sizes (Table 1). In addition to larval arrest and embryonic lethality, psa-1(ku355) animals also displayed other pleiotropic phenotypes including extra vulval cell inductions (Muv, 1%, n = 309; Figure 2), egg-laying defectiveness (Egl, <1%, n = 400), and protruding vulvae (Pvl, 100%, n > 300). The Egl and Pvl phenotypes, but not the Muv phenotype, were mentioned previously to be associated with another psa-1 allele (SAWA et al. 2000). Furthermore, closer examination using a lag-2::GFP marker revealed that one arm of the gonad failed to elongate properly in 37% of psa-1(ku355) animals (n = 200) and that gonad arms folded in abnormal forms in 52% of the animals (n = 200; Figure 2). All these phenotypes associated with psa-1(ku355) were temperature sensitive (Table 1 and data not shown). The larval arrest and sterile phenotypes associated with lin-35(n745); psa-1(ku355) double mutants were also temperature sensitive (Table 1).

View this table: In this window In a new window   TABLE 1 Viability of lin-35 and psa-1 mutants

 

View larger version (42K): In this window In a new window Download PPT slide   FIGURE 1.— Larval arrest caused by a synergistic effect of mutations in both lin-35 and psa-1. Differential interference contrast (A), corresponding GFP fluorescence (B), and merged (C) images of lin-35(n745); psa-1(ku355) animals with and without an extrachromosomal array containing wild-type copies of lin-35. The large adult with GFP fluorescence contains the array kuEx119. Arrowhead indicates the position of an arrested lin-35; psa-1 larva that lost the extrachromosomal array. Bar, 100 µm.

 

View larger version (138K): In this window In a new window Download PPT slide   FIGURE 2.— Phenotypes of psa-1(ku355) at 20°. (A) Adult animal with a distinct protruding vulva (Pvl). (B) Adult animal with a Multivulva phenotype (Muv). Arrowheads indicate vulval tissue. (C and D) Animals with an abnormal U-turn of the gonad. Anterior is to the left. Bars, 100 µm in A (for A and B) and 10 µm in C (for C and D).

 
To further investigate the cause of the synthetic larval arrest of lin-35; psa-1 double mutants, we assayed psa-1 and lin-35; psa-1 mutants for proliferation defects. We failed to detect any hyperproliferation defects in both the single and the double mutants. We also failed to observe any proliferation defects in seam and intestinal cell lineages as well as gonadal distal-tip cells (DTCs) in both the single mutants and the double mutants, using a seam-cell-specific GFP marker (SCM::GFP; gift of J. Rothman, UCSB), a gut-cell-specific marker (elt-2::GFP; FUKUSHIGE et al. 1998), and a DTC-expressing lag-2::GFP marker (BLELLOCH et al. 1999). Therefore, there is no evidence that the synthetic larval arrest in double mutants is caused by proliferation defects.

ku355 is an allele of psa-1 encoding a homolog of the yeast SWI3:

We mapped psa-1(ku355) to a 96-kb region between fog-2 and the cosmid ZC15 using the genetic markers and the SNPs (see MATERIALS AND METHODS). This region contains 16 predicted genes including psa-1(Y113G7B.23). Complementation tests indicated that psa-1(ku355) failed to complement psa-1(os22). Sequencing of the psa-1 coding region of the genomic DNA from the psa-1(ku355) identified a missense mutation that leads to the replacement of leucine with proline at the amino acid position 86. This proline residue is completely conserved among homologs from different organisms.

The predicted PSA-1 protein is similar to the yeast SWI3 and its homologs in other organisms (SAWA et al. 2000). In yeast, Drosophila, and humans, the SWI3 family of proteins is known to be a component of the SWI/SNF complexes that are involved in remodeling chromatin structure by destabilizing the histone-DNA interaction (KINGSTON and NARLIKAR 1999). Rb interacts with certain SWI/SNF complexes to coordinate the sequential expression of E2F target genes to regulate progression through G₁ and S phases (ZHANG et al. 2000). Previously, it has been shown that psa-1 and other components of the SWI/SNF complex are required for asymmetric T cell divisions in C. elegans (SAWA et al. 2000).

To further investigate the synthetic phenotype of complete loss of both lin-35 and psa-1 genes, we inactivated the psa-1 gene in both wild-type and lin-35(n745) animals using the RNAi feeding method (see MATERIALS AND METHODS). When synchronized L1 wild-type animals were fed with psa-1 dsRNA containing bacteria, a sterile phenotype associated with these P0 animals was observed (Table 2). However, when lin-35(n745) animals were treated the same way, a larval arrest phenotype was observed (Table 2). These animals arrested at the third (L3) or the fourth (L4) larval stage as determined by the size and shape of the gonad (n = 80).

View this table: In this window In a new window   TABLE 2 Identification of C. elegans homologs of subunits of the SWI/SNF complex and their RNAi phenotype in N2 and lin-35(n745)

 

lin-35 interacts with other components of the SWI/SNF complex:

To further confirm that the SWI/SNF complex and lin-35/Rb collaboratively regulate larval development in C. elegans, we examined the possibility that loss of function of other components of the SWI/SNF complex may lead to synthetic phenotypes with lin-35(n745). A BLAST search of the C. elegans genome identified the homologs for a number of subunits of the SWI/SNF complex in Drosophila and yeast (Table 2). In the case of Swp73/BAF60, two C. elegans proteins, C18E3.2 and ZK1128.5, which are 45 and 49% identical to the Drosophila homologs, were identified. There are also five actin proteins in C. elegans that display high homology to Drosophila ACT1/2. In addition, the predicted nematode protein C01G8.9 displays weak homology to the Drosophila ELD/OSA protein.

We performed RNAi analysis with the genes coding for potential SWI/SNF subunits in both wild-type and lin-35(n745) animals to test for synthetic mutational effects. psa-1(RNAi) in wild-type animals led to a sterile (Ste) and Pvl phenotype in the P0 worms. RNAi of four other genes, C18E3.2, ZK1128.5, ZK616.4, and Y71H2AM.17, led to Pvl and sterile phenotypes in the F₁ generation in the wild-type background. However, no larval arrest phenotype was observed when RNAi was applied to wild-type worms for any of these genes. In contrast, RNAi of all five of these genes produced the synthetic larval arrest phenotype in the lin-35(n745) background (Table 2). When RNAi was applied to lin-35(n745) animals (Table 2), 100% of larval arrest of P0 worms (for psa-1) or of the F₁ generation (for the other four genes) was observed. These results indicate that lin-35/Rb cooperates with the SWI/SNF complex to control C. elegans larval development.

The results are more complicated for several other genes, the five actin genes, C01G8.9, R07E5.3, and F26D10.3. RNAi of these genes produced a complete sterile phenotype or complete larval arrest in the P0 animals in both the wild-type and the lin-35(n745) background (Table 2). One exception was that RNAi of psa-4, encoding a homolog of yeast Swi2/Snf2 and Drosophila brm, failed to produce any obvious phenotype either in the wild-type or in the lin-35 mutant background. In the C. elegans genome, there are >20 proteins with the Snf2 motif. It is possible that some of these proteins have redundant functions with PSA-4.

psa-1 interacts with a subset of synMuv B genes:

lin-35 is a member of a large synthetic Multivulva (synMuv) gene family. synMuv genes are commonly assigned to one of two classes, A or B. In general, animals carrying mutations in genes from either class undergo normal vulval development, as do most double mutants containing two mutations in the same class. However, animals containing mutations in both classes (A and B) display a Muv phenotype, the result of extra vulval cell inductions (FERGUSON and HORVITZ 1989). To determine the spectrum of psa-1 genetic interactions, we inactivated synMuv genes in both wild-type and psa-1 mutants by the RNAi feeding method and assayed for the larval arrest phenotype (Table 3). RNAi of the two class B genes, lin-9 and lin-37 (BEITEL et al. 2000; BOXEM and VAN DEN HEUVEL 2002), in psa-1(ku355) animals produced a highly penetrant larval arrest phenotype similar to that observed in lin-35; psa-1 double mutant. In the control experiment, no larval arrest was observed in the wild-type background. RNAi of the RbAp46/48 homolog, lin-53 (LU and HORVITZ 1998), histone deactylase, hda-1 (LU and HORVITZ 1998; SOLARI and AHRINGER 2000; DUFOURCQ et al. 2002), and let-418 (VON ZELEWSKY et al. 2000; SOLARI and AHRINGER 2000) rendered all P0's of wild-type animals sterile (for lin-53 and hda-1) or complete larval arrest in the F₁ generation (for let-418), thus preventing the test of the synthetic interaction between these three genes and psa-1. RNAi treatment of another class B gene, lin-36 (THOMAS and HORVITZ 1999), and a class A gene, lin-15a, failed to produce the larval arrest phenotype in either the psa-1 mutant or wild-type animals (Table 3). To further verify the RNAi results, we also constructed double mutants containing psa-1(ku355) and a mutation in lin-37, lin-36, or lin-15a. At 20°, psa-1; lin-37 double-mutant animals displayed 100% larval arrest, while double-mutant animals of psa-1 with lin-36 or lin-15a were indistinguishable from psa-1 single mutants with respect to larval development (Table 3). Concurrently, we also constructed a double mutant between psa-1(ku355) and lin-8(n111) and found no synthetic larval arrest phenotype associated with it (Table 3).

View this table: In this window In a new window   TABLE 3 Genetic interactions between psa-1 and synMuv genes

 
Several synMuv genes, including lin-35, lin-36, and lin-9, are involved in the repression of cell proliferation and regulation of the G₁ to S phase transition (BOXEM and VAN DEN HEUVEL 2002). In contrast, lin-37, let-418, lin-53, hda-1, and synMuv A genes (including lin-15a, lin-8, and lin-38) do not appear to function in cell cycle control (BOXEM and VAN DEN HEUVEL 2002). We have shown here that lin-36, lin-8, and lin-15a mutations do not cause the synthetic larval arrest phenotype with the psa-1 mutation, as do lin-35, lin-37, and lin-9. Taken together, these results are consistent with an idea that the synthetic larval arrest phenotype may not result from perturbation in cell cycle control per se, although these results firmly rule out this possibility.

Mutations in a subset of synMuvB genes enhanced the T cell lineage defects of psa-1(ku355):

In wild-type males and hermaphrodites, the bilaterally symmetric T cells divide during the early L1 stage to produce a hypodermal cell (T.aa) that joins the large hyp7 syncytium, a hypodermal blast cell that divides later in a sex-specific manner (T.ap), and a group of neural cells (derived from T.p). Cells T.paa and T.pap are the phasmid socket cells PHso1 and PHso2, which function to provide channels for the phasmid neurons PHA and PHB to communicate with the environment (SULSTON and WHITE 1980). When animals are soaked in the fluorescent dyes FITC or DiO, the two neurons of each of the two phasmids, as well as six of the eight amphidial neurons in the head, take up these dyes, an effect that can be visualized in living animals (HERMAN and HORVITZ 1994). Abnormalities in the T cell lineage will cause defects in phasmids and there will often be no dye filling in the phasmids position. Therefore dye-filling assays of phasmids are well-established methods used to detect abnormalities in the T cell lineage (HERMAN and HORVITZ 1994; ZHAO et al. 2002).

Previously psa-1 was shown to be required for asymmetric T cell division in C. elegans; at 18°, 16% of psa-1(os22) animals had T cell lineage defects (SAWA et al. 2000). Since inactivation of lin-35, lin-37, and lin-9 in the psa-1(ku355) background produced a dramatic larval arrest phenotype, we investigated the possibility of these genes interacting with psa-1 in regulation of asymmetric T cell division. As RNAi of these synMuv genes in the psa-1(ku355) animals produced nearly 100% larval arrest at 20°, we performed the dye-filling assay on the animals beyond the fourth larval stages at 18°. A total of 50% of the phasmids in psa-1(ku355) animals were found to be filled with FITC (Table 4). In contrast, only 11% of the phasmids in the lin-35(n745); psa-1(ku355) animals at the same stage were filled with FITC (Table 4). After inactivating lin-35, lin-37, and lin-9 in psa-1(ku355) animals by RNAi, a very low percentage of the phasmids were found to be filled with FITC (Table 4). We also determined the T cell lineage in the psa-1(ku355) single and lin-35; psa-1 double mutants at 18°. Two of the seven psa-1 mutant animals examined displayed abnormal T cell lineage, while four of the six lin-35; psa-1 mutant animals displayed abnormal T cell lineage (Figure 3). These results are consistent with the idea that these class B synMuv genes play a role in the regulation of T cell divisions.

View this table: In this window In a new window   TABLE 4 Genetic interactions between psa-1 and synMuvB genes in regulating T cell division

 

View larger version (12K): In this window In a new window Download PPT slide   FIGURE 3.— Abnormal T cell lineages in psa-1(ku355) and lin-35(n745); psa-1(ku355) mutants during the L1 stage. All animals were grown at 18°. The numbers of animals that showed the corresponding lineages are indicated below the diagrams. The fate of cells was determined by nuclear morphology (HERMAN and HORVITZ 1994). hyp, hypodermal.

 

psa-1 does not function as a class A or class B synMuv gene, but its mutation enhances the Muv phenotype of lin-15(n765):

Only 1% of the psa-1(ku355) single mutants displayed a Muv phenotype (n = 309). To determine if psa-1 acts as a class A or class B synMuv gene, we constructed double mutants containing psa-1(ku355) and either a class A or a class B synMuv mutation. For double mutants with class A mutations, lin-15a(n767) and lin-8(n111) that display no Muv phenotype on their own were used. Double mutants containing either of these class A synMuv mutations exhibited no synthetic Muv phenotype (data not shown), indicating that psa-1 is not a class B synMuv gene. For double mutants with class B mutations, lin-35(n745), lin-37(n758), lin-36 (n766), and lin-15b(n744) were used. No synthetic Muv phenotype was observed in any of these double-mutant strains, further indicating that psa-1 is not a class A synMuv gene. However, psa-1(ku355) displayed a significant interaction with a hypomorphic mutation in the lin-15(n765) (FERGUSON and HORVITZ 1989). The lin-15 locus is an operon containing a class A synMuv gene, lin-15a, and a class B synMuv gene, lin-15b (CLARK et al. 1994; HUANG et al. 1994). lin-15(n765) is a temperature-sensitive allele that causes a strong Muv phenotype (100%, n = 300) at 20°, but displays only 27% of Muv phenotype (n = 324) at 15°. The severity of the Muv phenotype of this allele at different temperatures reflects the relative level of lin-15 gene activity (HSU and MEYER 1994). At 15°, 78% of psa-1(ku355); lin-15(n765) double mutants were Muv (n = 400), suggesting that psa-1 may play an important role in lin-15 gene expression or the lin-15-mediated repression of vulval differentiation.

Using a genetic screen to identify mutations that are synthetically lethal with lin-35/Rb, we have isolated a mutation in psa-1, a gene encoding a central component of the SWI/SNF complex. Strong loss-of-function mutations in lin-35 cause only subtle effects on viability and larval development (LU and HORVITZ 1998; FAY et al. 2002). A reduction-of-function mutation in psa-1 causes the low-penetrance larval arrest. However, lin-35; psa-1 double-mutant animals are almost completely arrested in the L1 larval stage. Inactivation of several other components of the SWI/SNF complex in the lin-35(n745) background by RNAi also causes a synthetic larval arrest phenotype. These results revealed a previously unknown function of lin-35/Rb in larval development and indicated that lin-35/Rb functions concertedly with the SWI/SNF complex. The expression patterns of both lin-35 and psa-1 genes are also consistent with their roles in larval development. LIN-35 has been determined to be expressed throughout development in most or all cell types by antibody staining (LU and HORVITZ 1998). Through the use of a functional psa-1::GFP construct, psa-1 has been shown to be expressed ubiquitously in the nuclei of all somatic cells (SAWA et al. 2000).

Genetic studies in Drosophila showed that the SWI/SNF complex downregulates E2F activity (STAEHLING-HAMPTON et al. 1999). These studies imply that, like Rb family members, SWI/SNF proteins function in the regulation of E2F activity. Transfection studies indicate that class I HDACs and SWI/SNF are recruited by Rb and may coordinate the sequential expression of E2F target genes to regulate progression through G₁ and S phases (ZHANG et al. 2000). Our study presented here shows for the first time that Rb functions in a redundant manner with the SWI/SNF complex to regulate animal development in vivo. As we have obtained no evidence for cellular hyperproliferation in lin-35; psa-1 double mutants, the synthetic larval arrest does not appear to be caused by overexpression of E2F cell cycle targets. Interestingly, however, E2F's have been reported to regulate a number of non-cell cycle genes (ISHIDA et al. 2001; MULLER et al. 2001) and has been shown in C. elegans to play a role in the establishment of early asymmetries in the embryo (PAGE et al. 2001).

SAWA et al. (2000) have already reported that the SWI/SNF complex is involved in regulating T cell division. egl-27, which encodes a protein homologous to MTA1 (a component of NURD complex), has also been found to be required for T cell division (HERMAN et al. 1999). The observation that the loss-of-function mutation in lin-35/Rb strongly enhanced the T cell division defects in a psa-1 mutant allele suggests that lin-35/Rb may play a role in T cell division. Since lin-35(lf) alone has no defects in T cell division, we propose that lin-35 and the SWI/SNF complex may function redundantly to affect the expression of a common set of targets. It has been shown that T cell division is regulated by Wnt signaling (HERMAN et al. 1995; SAWA et al. 1996; ROCHELEAU et al. 1999; ZHAO et al. 2002). It is also known that Wnt signaling and synMuv pathways are both involved in vulval induction in C. elegans (GLEASON et al. 2002). The elucidation of the interaction between lin-35 and Wnt signaling in T cell division could be crucial in understanding how lin-35 acts in asymmetric cell divisions.

The exact cause of the synthetic larval arrest of lin-35; psa-1 double mutant is presently unknown. While a lin-35 mutation can enhance the defects in the T cell asymmetric division of a psa-1 mutant, these defects alone cannot be responsible for the larval arrest. On the basis of the pleiotropic phenotypes of these mutant animals, it is hard to assign the cause of the synthetic larval arrest to one specific developmental event. The discovery of common targets of lin-35/Rb and the SWI/SNF complex will be a key step toward understanding the mechanism of the synthetic larval arrest.

We thank E. B. Kim for assistance; D. Eastburn, Y. Suzuki, and E.B. Kim for a critical reading of this manuscript; members of our and W. Wood's laboratory for discussion; and the Caenorhabditis Genetics Center (supported by the National Institutes of Health National Center for Research Resources) for providing some of the strains used in this work. This work was supported by National Institutes of Health grant GM-47869 to M.H. and a grant from the American Cancer Society to D.S.F. M.C. is a research associate and M.H. is an associate investigator of the Howard Hughes Medical Institute.

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