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Genetics. Aug 2012; 191(4): 1367–1380.
PMCID: PMC3416014

A Network of Genes Antagonistic to the LIN-35 Retinoblastoma Protein of Caenorhabditis elegans

Abstract

The Caenorhabditis elegans pRb ortholog, LIN-35, functions in a wide range of cellular and developmental processes. This includes a role of LIN-35 in nutrient utilization by the intestine, which it carries out redundantly with SLR-2, a zinc-finger protein. This and other redundant functions of LIN-35 were identified in genetic screens for mutations that display synthetic phenotypes in conjunction with loss of lin-35. To explore the intestinal role of LIN-35, we conducted a genome-wide RNA-interference-feeding screen for suppressors of lin-35; slr-2 early larval arrest. Of the 26 suppressors identified, 17 fall into three functional classes: (1) ribosome biogenesis genes, (2) mitochondrial prohibitins, and (3) chromatin regulators. Further characterization indicates that different categories of suppressors act through distinct molecular mechanisms. We also tested lin-35; slr-2 suppressors, as well as suppressors of the synthetic multivulval phenotype, to determine the spectrum of lin-35-synthetic phenotypes that could be suppressed following inhibition of these genes. We identified 19 genes, most of which are evolutionarily conserved, that can suppress multiple unrelated lin-35-synthetic phenotypes. Our study reveals a network of genes broadly antagonistic to LIN-35 as well as genes specific to the role of LIN-35 in intestinal and vulval development. Suppressors of multiple lin-35 phenotypes may be candidate targets for anticancer therapies. Moreover, screening for suppressors of phenotypically distinct synthetic interactions, which share a common altered gene, may prove to be a novel and effective approach for identifying genes whose activities are most directly relevant to the core functions of the shared gene.

Keywords: Caenorhabditis elegans, lin-35, retinoblastoma, suppressors, slr-2

THE retinoblastoma gene (Rb) was originally implicated as a tumor suppressor whose inactivation led to a rare form of pediatric eye cancer (Knudson 1971). Subsequently, loss of Rb function has been shown to play a causative role in the majority of known human cancers (Sherr 1996; Sherr and McCormick 2002; Viatour and Sage 2011). This observation has led to intensive studies of the pRb/p105 protein along with its family members, p107 and p130, in an effort to define the molecular and cellular functions of the pRb “pocket protein” family. Currently, the best-characterized role of pRb is as an inhibitor of cell-cycle progression. In quiescent mammalian cells, pRb family members bind to E2F-family transcription factors, leading to the repression of genes required for entry into and progression through S-phase. Transcriptional repression by pRb family members is mediated both by directly blocking the transactivation domain of activator-type E2F proteins and by the recruitment of corepressors to genomic sites bound by inhibitory-type E2Fs, leading to heterochromatin formation and maintenance (Sherr and McCormick 2002; Knudsen and Knudsen 2006; van den Heuvel and Dyson 2008; Poznic 2009; Chinnam and Goodrich 2011). In addition, many studies have focused on elucidating the network of extracellular signals and intracellular factors that regulate the phosphorylation state and activity of pRb members (Sherr and McCormick 2002; van den Heuvel and Dyson 2008; Paternot et al. 2010; Witkiewicz et al. 2011).

A growing number of studies indicate that pRb family members also regulate non-cell-cycle processes during development. pRb physically interacts with >100 putative binding partners, of which E2F family members make up only a minority (Ji and Studzinski 2004; Balciunaite et al. 2005; Batsche et al. 2005; Morris and Dyson 2001; Thomas et al. 2001; Chen et al. 2007). These include physical and functional interactions with lineage-specific transcription factors that play well-characterized roles in development, including MyoD (muscle development), Id2 (erythrocyte production), Pdx1 (pancreas development), and C/EBP (adipocyte and monocyte differentiation) (Thomas et al. 2001; Rekhtman et al. 2003; Gery et al. 2004; Ji and Studzinski 2004; Lasorella et al. 2005; De Falco et al. 2006; Hua and Sarvetnick 2007; Kim et al. 2011). Although the majority of these proposed interactions still lack strong in vivo support, several have been verified in mouse models (Lasorella et al. 2000; Takahashi et al. 2004; Lasorella et al. 2005; Wikenheiser-Brokamp 2006; Walkley et al. 2008; Calo et al. 2010; Kim et al. 2011). Furthermore, in conjunction with E2F family proteins, pRb regulates many genes that have no known links to cell-cycle functions and may therefore influence a diverse range of cellular processes (Muller et al. 2001; Markey et al. 2002; Black et al. 2003). Finally, studies in non-mammalian systems, such as Caenorhabditis elegans, have identified a number of non-cell-cycle and developmental functions for the E2F-pRb network, some of which have correlates with proposed cancer-related pRb functions in mammals (van den Heuvel and Dyson 2008; Kirienko et al. 2010).

pRb is highly conserved across a wide range of taxa from Caenorhabditis elegans to mammals (Sabelli and Larkins 2006; Cao et al. 2010). Moreover, the pRb network in C. elegans is relatively simple as compared with mammals. In worms, there is only a single pRb ortholog (LIN-35), three E2F members (EFL-1, EFL-2, and F49E12.6), and one E2F dimerization partner (DPL-1) (van den Heuvel and Dyson 2008). LIN-35 was originally identified for its role in the regulation of vulval development (Ferguson and Horvitz 1989; Lu and Horvitz 1998). Interestingly, lin-35 single mutants do not show defects in vulval development, nor is lin-35 an essential gene under standard growth conditions. However, the combined inactivation of lin-35 and one of several “class A” synthetic-multivulval (synMuv) genes leads to the hyper-induction of vulval tissue during hermaphrodite larval development. Control of vulval cell induction by LIN-35 is carried out in conjunction with a number of transcriptional coregulators, including the E2F ortholog, EFL-1; its dimerization partner, DPL-1; and multiple members of the conserved DREAM and NuRD complexes, whose activities mediate transcriptional repression and chromatin silencing (Lu and Horvitz 1998; Thomas and Horvitz 1999; Beitel et al. 2000; Ceol and Horvitz 2001; Thomas et al. 2003; Harrison et al. 2006; Andersen and Horvitz; 2007). Together, these transcriptional regulators compose the majority of “class B” synMuv genes, and animals containing mutations in both a class A and a class B synMuv gene display a characteristic Muv phenotype (Fay and Yochem 2007).

The discovery of lin-35 as a synMuv gene marked the first non-cell-cycle function attributed to a pRb ortholog in a non-mammalian species. Since then, LIN-35 has been implicated in a number of distinct developmental and cellular processes, which include the characterization of seven lin-35-synthetic-lethal mutations that were identified by our laboratory through a forward genetic screen (Fay et al. 2002). Functions for LIN-35 identified through these screens include roles in cell-cycle regulation, pharyngeal morphogenesis, somatic gonad development, and asymmetric cell division (Fay et al. 2002, 2003, 2004; Bender et al. 2004, 2007; Cui et al. 2004). In addition, LIN-35 coregulates the expression of many intestinal genes with SLR-2, a C2H2-type zinc (Zn)-finger protein (Kirienko and Fay 2007; Kirienko et al. 2008). Whereas lin-35 and slr-2 single mutants develop normally, lin-35; slr-2 double mutants arrest uniformly as L1 larvae because of starvation resulting from intestinal malfunction. Here we describe a genome-wide RNA interference (RNAi) screen to identify suppressors of lin-35; slr-2 larval arrest. Although a majority of the RNAi clones identified specifically suppressed the lin-35; slr-2 larval-arrest phenotype, a number of the clones also led to the suppression of mechanistically unrelated lin-35-associated phenotypes. Specifically, we identified several chromatin-modification complex members whose functions appear to antagonize many or all of the activities of LIN-35 in C. elegans. As these genes are highly conserved, they may represent targets that could be exploited to neutralize the deleterious effects of pRb loss in mammals.

Materials and Methods

Strains

Nematodes were handled according to standard protocols (Stiernagle 2006). Animals were maintained at 20° unless otherwise stated. The following strains were used in this study: wild-type N2 (Bristol), lin-35(n745), MH1620 [lin-35(n745); slr-2(ku297); kuEx119], WY617 [dpl-1 (n2994); slr-2; kuEx119], WY83 [lin-35; ubc-18(ku354); kuEx119], MH1621 [lin-35; fzr-1(ku298); kuEx119], WY251[lin-35; spr-1(ar205); kuEx119], WY119 [lin-35; pha-1(fd1); kuEx119], WY142 [xnp-1(fd2) lin-35; kuEx119], JD118 [inx-6(rr5)], AA790 [lin-15AB (n765)], WY758 [lin-35unc-13; npp-5; kuEx119], WY761 [lin-35unc-13; hcf-1; kuEx119], WY760 [lin-35unc-13; wdr-5.1; kuEx119], RB777 [hcf-1(ok559)], VC1494 [npp-5 (ok1966)], RB1304 [wdr-5.1 (ok1417)], and JM63 [elt-2::GFP]. kuEx119 is an extrachromosomal array containing wild-type copies of lin-35 along with the SUR-5::GFP reporter (Gu et al. 1998; Fay et al. 2002).

RNAi screening and quantification of lin-35; slr-2 suppression

RNAi feeding screens were performed using the Geneservice RNAi library (Kamath et al. 2003), following standard protocols (Timmons 2006). Four or five lin-35; slr-2; kuEx119 L4 larvae were transferred to individual RNAi feeding plates, incubated at 20° for 3 days, and then qualitatively assessed for escape of L1 arrest in kuEx119-minus (GFP–) F1 progeny. Putative suppressors were retested by placing 15–20 L4 larvae on RNAi feeding plates for 12 hr and then transferring adults to fresh RNAi plates for 12 additional hours, after which adults were removed and total embryos were counted. kuEx119-positive (GFP+) F1 progeny were then removed every 12 hr and counted. After 4 days, the number of larvae developing beyond the L1 arrest stage were counted and divided by the calculated total number of kuEx119-negative (GFP–) F1 progeny. For statistical analysis, the experiments were repeated five times and numbers were pooled. A Fisher’s exact test was performed for each suppressor clone to assign significance using a P-value cutoff of 0.001. Suppressor clones were also sequenced to determine their correct identity.

inx-6 starvation arrest assay

inx-6(rr5ts) mutants arrest because of foregut defects that result in starvation at the nonpermissive temperature of 25° (Li et al. 2003). Twenty-five to 30 L4 inx-6 mutants were initially placed on vector-control (pPD129.36) or suppressor RNAi plates at 15°. Adult worms were transferred to corresponding RNAi plates 12 hr later and allowed to lay embryos for 3 hr. Total embryos (TEs) were counted and then allowed to develop at 15° for either 16 or 24 hr, at which time they were shifted to 25°. Plates were then kept at 25° for 4–5 days, and adult worms (AWs) were then counted. The percentage of adult worms was calculated as (AWs/TEs) × 100.

Oil Red O staining

Oil Red O staining was conducted as described but without the freeze–thaw steps (O’Rourke et al. 2009; Soukas et al. 2009). lin-35; slr-2; kuEx119 L4 larvae were placed on vector-control or suppressor RNAi feeding plates and allowed to develop into adults. Adult worms were bleached, and their liberated embryos were washed, transferred to corresponding RNAi plates, and allowed to develop to roughly synchronized L1 larvae, as monitored by an L1 apoptotic event in their tails (T.ppp) (Sulston and Horvitz 1977). Animals were mounted and images of kuEx119-minus (GFP–) larvae were captured.

Cycloheximide and geneticin (G418) assays

Cycloheximide dilutions were combined with cultured OP50 bacteria and allowed to dry on NGM plates. For G418-containing plates, G418 dilutions were added to NGM medium prior to pouring. Fifteen to 20 L4 lin-35; slr-2; kuEx119 or dpl-1; slr-2; kuEx119 worms were placed on plates and allowed to develop into adults. Adults were then transferred to identically treated plates and allowed to lay embryos for 4–5 hr, after which adults were removed and TEs were counted. kuEx119-postive (GFP+) worms (GWs) were removed and counted every 12 hr. After 3–4 days, the number of kuEx119-negative (GFP–) worms (i.e., suppressed worms, or SWs) that developed past the L1 arrest stage were counted, and the percentage suppression (S%) was calculated using the formula S% = [SWs/(TEs − GWs)] × 100.

Intestinal and distal tip cell hyper-proliferation assays

Intestinal hyper-proliferation was assayed using strains carrying an elt-2::GFP reporter (Fukushige et al. 1998). fzr-1(ku298); elt-2::GFP L4 larvae were placed on RNAi feeding plates for lin-35, vector-control, or double RNAi combinations of lin-35 and one of the suppressors. All double-RNAi experiments were controlled for optical density such that a 1:1 ratio of each bacterial solution was used at the time of plate seeding. Intestinal nuclei were counted using standard fluorescence and DIC optics. Distal tip cell (DTC) hyper-proliferation was assessed on the basis of gonad arm bifurcations in kuEx119-minus (GFP–) young adult progeny of lin-35; fzr-1; kuEx119 mutants grown on either vector-control or suppressor RNAi feeding plates.

High-temperature arrest assay

lin-35(n745) L4 larvae were transferred to vector-control or suppressor RNAi plates and incubated at 26°. Adult worms were then transferred to corresponding RNAi plates and allowed to lay embryos for 5 hr at 26°. Adults were removed and embryos were counted. After 2–3 days, larvae that had developed beyond the L1 arrest stage were counted and divided by the total number of embryos to calculate the percentage suppression.

Results

A genome-wide RNAi screen for suppressors of lin-35; slr-2 larval arrest

lin-35; slr-2 double mutants exhibit a nearly uniform arrest as L1 larvae because of defects in nutrient utilization (Kirienko et al. 2008). Homozygous lin-35; slr-2 double mutants, however, can be maintained by the presence of an extrachromosomal array (kuEx119) that contains lin-35-rescuing sequences as well as a ubiquitously expressed SUR-5::GFP reporter (Fay et al. 2002). Because extrachromosomal arrays are meiotically unstable, array-positive hermaphrodites fail to segregate the array to all of their self-progeny (Mello et al. 1991). In the case of lin-35; slr-2; kuEx119, ~30% of self-progeny fail to inherit the array and can be easily identified as L1-arrested larvae that lack GFP expression (Kirienko et al. 2008).

To identify suppressors of lin-35; slr-2 larval arrest, we carried out a genome-wide RNAi feeding screen using the GeneService library (Timmons et al. 2001; Kamath et al. 2003). Positive RNAi clones were identified by an increase in the proportion of GFP– self-progeny that developed past the L1 stage. Of the 16,757 genes represented in the RNAi feeding library, 26 suppressors were identified (0.16% of total clones screened). RNAi clones were sequence verified, and suppression by each clone was tested in five independent trials and shown to be statistically significant (P < 0.001, Fisher’s exact test; Figure 1A). We also confirmed suppression using genetic mutants for three of the identified suppressors: npp-5 (ok1966), hcf-1 (ok559), and wdr-5.1(ok1417) (Figure 1, B–D). Confirmation by genetic mutations, as well as the relatively low frequency of positive clones, indicates that our RNAi screening approach yielded results that were both reproducible and specific.

Figure 1
Suppressors of lin-35; slr-2 and dpl-1; slr-2 L1 larval arrest. (A) Percentage suppression of larval arrest by 26 sequence-confirmed RNAi clones. Vector denotes a control RNAi. Suppressors have been organized into functional classes. Suppression of L1 ...

There was considerable variability among the RNAi clones with respect to the percentage of suppressed animals as well as the terminal developmental stage to which lin-35; slr-2 animals progressed (supporting information, Figure S1; Table S1). For example, lin-35; slr-2 mutants treated with RNAi for hcf-1, mes-4, mrg-1, sin-3, hecd-1, or raga-1 were able to develop into fertile adults, whereas other RNAi clones resulted in sterility or arrest prior to adulthood. In fact, several of the strongest suppressors (e.g., C16A3.4, Y48A6C.4, phb-1, and phb-2) lead to arrest as sterile adults. In a number of cases, the identified suppressor genes have previously been shown to be required for late larval development or fertility, thereby precluding suppression to viable or fertile adults (Table S1) (Hanazawa et al. 2004; Checchi and Kelly 2006; Voutev et al. 2006; Rodenas et al. 2009).

Previous studies found slr-2 to be synthetically lethal with dpl-1, which encodes the sole E2F dimerization partner homolog in C. elegans and regulates transcription through its interaction with E2F family members (Ceol and Horvitz 2001; Page et al. 2001; Kirienko et al. 2008). The dpl-1; slr-2 arrest phenotype is morphologically indistinguishable from that of lin-35; slr-2 mutants, although the penetrance of L1 arrest is somewhat weaker: 93.9% for dpl-1; slr-2 mutants vs. 98.2% for lin-35; slr-2 mutants. We found that all but four of the lin-35; slr-2 suppressors also significantly suppress the dpl-1; slr-2 arrest phenotype (Figure 1A). In most cases, the percentage of suppressed animals was greater than that observed for lin-35; slr-2, which correlates with the slightly less penetrant phenotype of dpl-1; slr-2 mutants. The ability of the RNAi clones to suppress both lin-35; slr-2 and dpl-1; slr-2 mutants suggests that the RNAi clones correct an underlying defect that is shared in both double mutants (Kirienko et al. 2008). It also argues that suppression by the RNAi clones is not informational or due to the specific nature of the lin-35(n745) (opal stop) or dpl-1(n2994) (splice acceptor site mutation) genetic lesions. Because the phenotypes of lin-35; slr-2 and dpl-1; slr-2 mutants are very similar, the majority of our experiments were carried out using lin-35; slr-2 mutants only.

Suppressors of lin-35; slr-2 fall into several functional categories

Seventeen of the 26 suppressors identified from the lin-35; slr-2 screen can be placed into three functional classes: (1) ribosome biogenesis genes, (2) mitochondrial prohibitins, and (3) synMuv suppressors (Figure 1A; Table 1). The remaining 9 genes vary broadly in function but include several of the strongest suppressors (Figure 1A). A more detailed description of these genes, along with insights into their means of suppression, is discussed below. To functionally examine the suppressor classes, we placed lin-35; slr-2; sup triple mutants (where “sup” refers to one of the isolated suppressors) on RNAi plates targeting suppressors in either the same or a different class. In the case of the three synMuv suppressor genes hcf-1, mes-4, and wdr-5.1, no increase in suppression was observed when these genes were combinatorially inactivated vs. single-gene inhibition (Figure 1, B–D). This lack of an additive effect is consistent with their gene products acting within a single methyltransferase complex (Table 1) (Dehe and Geli 2006; Wu et al. 2008). In addition, no enhancement of lin-35; slr-2 suppression was observed following double RNAi of the two prohibitin-binding partners phb-1 and phb-2 relative to RNAi of either clone individually (data not shown). Conversely, additive suppression was observed in lin-35; slr-2 triple mutants with npp-5, hcf-1, and wdr-5.1 following RNAi of phb-2 and in lin-35; slr-2; npp-5 triple mutants treated with mes-4(RNAi) (Figure 1, B–D). Taken together, these findings are consistent with different gene classes acting through distinct mechanisms to suppress lin-35; slr-2 larval arrest.

Table 1
lin-35; slr-2 suppressor identities

Ribosome biogenesis genes

Seven genes implicated in ribosome biogenesis were isolated from the genome-wide screen (Figure 1A; Table 1). For this class, which includes pro-2, suppressed lin-35; slr-2 mutants arrested as sterile adults (Figure S1). The roles of these genes in ribosome biogenesis are inferred both from sequence homology to well-characterized orthologs in yeast and from studies in C. elegans showing that mutations in a subset of these genes lead to defects in rRNA processing (Bousquet-Antonelli et al. 2000; Vanrobays et al. 2001; Galani et al. 2004; Voutev et al. 2006). In yeast, Gar1/Y66H1A.4 and Ipi1/Y48A6C.4 are required for 18s and 35s pre-rRNA processing, respectively (Krogan et al. 2004), whereas orthologs of nst-1, pro-2, and W07E6.2 have demonstrated roles in the transport of pre-ribosomal subunits (Bassler et al. 2001; Milkereit et al. 2001; de la Cruz et al. 2005). Notably, worms with mutations in five of these genes (nst-1, pro-2, W07E6.2, Y56A3A.18, and Y48A6C.4) form proximal germline tumors resulting from defects in the developing somatic gonad (Killian and Hubbard 2004; Voutev et al. 2006).

The strongest suppressor identified by our screen, C16A3.4, may also act through the ribosome biogenesis pathway. In yeast, Rei1/C16A3.4 has defined roles in 60S ribosomal subunit maturation and subsequent recycling of subunit shuttle members (Albanese et al. 2010; Lo et al. 2010). Similar to other ribosome biogenesis suppressors, RNAi of C16A3.4 caused sterility in both N2 and suppressed lin-35; slr-2 backgrounds. Unlike other ribosome biogenesis genes, however, RNAi of C16A3.4 did not induce proximal germline tumors (data not shown), and C16A3.4 contains multiple C2H2 Zn-finger domains, suggesting a possible role in transcriptional regulation. Interestingly, the human ortholog of C16A3.4, ZNF622, has been proposed to be a transcriptional regulator of intestinal genes and is overexpressed in colorectal tumors (Kleivi et al. 2007). Several additional suppressors may also act through translational regulation mechanisms. ZK856.10 encodes a putative ortholog of Rpc25, a subunit of RNA polymerase III, which is required for the transcription of small RNAs including tRNAs and the 5S rRNA (Zaros and Thuriaux 2005). In addition, iff-1 encodes a protein with homology to EIF5A, a conserved protein with suggested roles in protein translation (Zanelli and Valentini 2007).

Previous work in our laboratory demonstrated that lin-35 and slr-2 coregulate a large number of intestinal genes and that genes upregulated in single mutants are in many cases synergistically misregulated in lin-35; slr-2 and dpl-1; slr-2 double mutants (Kirienko et al. 2008). We therefore postulated that suppression of larval arrest by inhibition of ribosome biogenesis might reduce the translation of intestinal mRNAs that are deleterious when overexpressed. To test this model, we asked whether two well-characterized inhibitors of protein translation, cycloheximide and G418, could suppress lin-35; slr-2 and dpl-1; slr-2 larval arrest. Although toxicity was evident with these drugs, lin-35; slr-2 and dpl-1; slr-2 strains showed significant suppression of L1 arrest when grown on sublethal concentrations of either compound (Figure 2). Thus, a net reduction in protein translation may reduce the levels of misexpressed intestinal proteins in lin-35; slr-2 and dpl-2 slr-2 mutants, such that their negative impact on intestinal functions is diminished. At present we have no direct evidence to indicate whether translational inhibition of one or multiple targets of LIN-35 and SLR-2 is required to achieve suppression. However, the absence of intestinal targets of LIN-35 and SLR-2 from the suppressor screen suggests that a reduction in the expression of individual targets may not be sufficient to achieve suppression.

Figure 2
Chemical inhibition of protein translation suppresses lin-35; slr-2 and dpl-1; slr-2 double mutants. (A) Cycloheximide and (B) G418 treatments significantly suppress L1 larval arrest of lin-35; slr-2 and dpl-1; slr-2 strains as compared with the no-treatment ...

Mitochondrial prohibitins

Two genes identified by our screen, phb-1 and phb-2, encode orthologs of the mitochondrial prohibitins (Figure 1A; Table 1). PHB1 and PHB2 are structurally related proteins that are highly conserved from yeast to humans. PHB1 and PHB2 form large megadalton complexes within the inner mitochondrial membrane where they are thought to regulate mitochondrial morphology and maintain membrane potential (Artal-Sanz and Tavernarakis 2009a,b; Osman et al. 2009; Van Aken et al. 2010). Work in several organisms further indicates that prohibitins also reside in the nucleus where they may control transcription through a number of distinct mechanisms (Mishra et al. 2006, 2010). We identified an RNAi clone corresponding to phb-2 as a strong suppressor of lin-35; slr-2 arrest (32.2%; Figure 1). lin-35; slr-2; phb-2(RNAi) mutants arrested as sterile adults, an outcome consistent with phb-2(RNAi) inducing sterility in wild type (Kamath et al. 2003; Rual et al. 2004). We also generated a phb-1(RNAi) feeding construct and observed strong suppression of lin-35; slr-2 (41.9%). As noted above, double RNAi of phb-1 and phb-2 conferred no additive suppression, consistent with both proteins acting as partners within a complex. Interestingly, mammalian PHB1 and PHB2 physically interact with all three mammalian pRb family proteins as well as E2F (Wang et al. 1999a,b, 2002; Fusaro et al. 2002). However, given that the mammalian prohibitins are thought to repress E2F target-gene expression in a pRb-dependent manner, their loss would not be expected to compensate for pRb inactivation. This suggests that suppression of lin-35; slr-2 and dpl-1; slr-2 by phb-1 and phb-2 occurs through a mechanism that does not involve direct interactions with C. elegans pRb or E2F family members. However, it is also possible that LIN-35 does bind to C. elegans prohibitin members and that their regulatory relationship is different from that described in mammals.

In aging wild-type adult worms, RNAi of phb-1 and phb-2 leads to a strong reduction in intestinal fat stores (Artal-Sanz and Tavernarakis 2009b, 2010). Given that lin-35; slr-2 mutants arrest because of a defect in nutrient utilization, we tested whether prohibitin knockdown could also alter fat storage at the L1 stage of arrest. Notably, lin-35; slr-2 treated with RNAi of phb-1 or phb-2 showed a dramatic decrease in L1 fat stores based on staining with Oil Red O (Figure 3A). This decrease in fat stores at the L1 stage suggests a mechanism for suppression by the prohibitins. Namely, utilization of fat reserves in nutrient-deprived lin-35; slr-2 mutants may allow these animals to bypass the L1 diapause developmental arrest that is normally initiated under conditions of starvation (Fukuyama et al. 2006; Kirienko et al. 2008). Thus, L1 arrest in lin-35; slr-2 mutants is likely not due to a complete failure of the intestine to process nutrients but may result from a reduction in metabolic capacity that falls below a set threshold, thereby activating a nutrient-sensing developmental checkpoint.

Figure 3
Analysis of suppression by mitochondrial prohibitins. (A) Oil Red O staining, which detects fat, is markedly reduced in lin-35; slr-2 L1 larvae treated with phb-1/2(RNAi) vs. control RNAi. Bar, 10 µm. (B) Suppression of larval arrest in inx-6 ...

To test our model, we asked whether prohibitin knockdown was capable of suppressing starvation-induced L1 arrest in an independent genetic background. INX-6 is required for pharyngeal gap junction formation and muscle contractions that are necessary for feeding (Li et al. 2003). Temperature-sensitive inx-6(rr5ts) mutants are viable at 15° but arrest uniformly as L1 larvae at 25° because of starvation (Li et al. 2003). A series of temperature-shift experiments was carried out in which inx-6(rr5ts) embryos were incubated at 15° for either 16 or 24 hr, at which point animals were late-stage embryos or L1 larvae. Plates were then transferred to 25° for the remainder of development. inx-6 mutants grown on vector RNAi plates rarely developed to adulthood after temperature up-shift at either 16 or 24 hr (Figure 3B). In contrast, inx-6 mutants grown on phb-1, phb-2, or phb-1/2 RNAi plates showed a >10-fold increase in the percentage of worms that reach adulthood (Figure 3B). In addition, inx-6 mutants treated with phb-1 or phb-2 RNAi showed reduced levels of L1 fat stores on the basis of Oil Red O staining, consistent with effects observed for lin-35; slr-2 larvae (data not shown). The ability of phb-1 and phb-2 to suppress the mechanistically disparate phenotypes of inx-6 and lin-35; slr-2 mutants indicates that the loss of prohibitins leads to suppression of starvation-induced L1 arrest through the utilization of normally unavailable fat stores. In addition, we tested the other 24 lin-35; slr-2 suppressors identified from our screen for fat storage defects and inx-6 suppression, including raga-1, a GTPase target of the nutrient-responsive TOR pathway (Figure 3B and data not shown) (Schreiber et al. 2010). These tests failed to identify other similarly acting suppressors, indicating that the prohibitins act through a unique mechanism of bypass suppression.

synMuv suppressors

Among the 26 lin-35; slr-2 suppressor genes, 8 were previously shown to suppress the synMuv phenotype of lin-15AB(n765) mutants (Table 1) (Andersen et al. 2006; Cui et al. 2006). This class of genes consists primarily of transcriptional regulators, including a number of chromatin-modifying factors. Homologs of proteins encoded by mes-4, hcf-1, wdr-5.1, and dpy-30 are components of the set1/COMPASS histone H3K4 methylation complex, suggesting that they may act within a similar complex in C. elegans (Xu and Strome 2001; Wysocka et al. 2003; Bender et al. 2006; Simonet et al. 2007; Halbach et al. 2009; Murton et al. 2010; Li and Kelly 2011). However, although homologous to the Set1 component of the Set1/COMPASS complex, studies in C. elegans suggest that MES-4 acts primarily as an autosome-specific H3K36 methyltransferase (Furuhashi et al. 2010; Rechtsteiner et al. 2010). MRG-1 is a member of the NuA4 complex, which promotes acetylation of histone H4 and subsequent exchange with the Htz histone variant (Eisen et al. 2001; Takasaki et al. 2007). The remaining three synMuv suppressor genes and their orthologs in other systems are more poorly characterized at a functional level; proteins encoded by hecd-1, sin-3, and zfp-1 are homolgous to the yeast Ufd4 ubiquitin ligase (Xie and Varshavsky 2000), the Sin3 histone deacetylase (Bernstein et al. 2000; Silverstein and Ekwall 2005), and the Nto1 histone acetyltransferase complex member, respectively (Shi et al. 2007; Hang and Smith 2011).

Given that about one-third of the identified lin-35; slr-2 suppressors were previously shown to suppress the lin-15ab synMuv phenotype, we tested the remaining lin-35; slr-2 suppressors by RNAi to see if they, too, could suppress synMuv. None of the remaining suppressors were capable of suppressing the lin-15ab synMuv phenotype (Figure S2), consistent with their absence from the published genome-wide RNAi screen (Cui et al. 2006). Furthermore, we verified that all eight of the synMuv suppressors identified by our screen suppressed the synMuv phenotype (data not shown), indicating that our assay for synMuv suppression was robust. Finally, we tested 24 additional synMuv suppressors isolated by Cui and colleagues to see if these could suppress the lin-35; slr-2 phenotype (Table S2). None of these RNAi clones suppressed lin-35; slr-2 (data not shown). Thus, although there is clear overlap between the genes identified as lin-35; slr-2 and synMuv suppressors, these results suggest that the majority of suppressors within each class act through mechanisms that are specific to the individual phenotypes.

Suppression of multiple lin-35-associated phenotypes by a subset of lin-35; slr-2 suppressors

We next decided to test all 26 lin-35; slr-2 suppressors for suppression of six independent lin-35-synthetic genotypes previously characterized in our laboratory. These include (1) lin-35; fzr-1, which shows defects in cell-cycle regulation (Fay et al. 2002); (2) lin-35; ubc-18 and lin-35; pha-1, which are defective at pharyngeal morphogenesis (Fay et al. 2003, 2004; Qiu and Fay 2006; Mani and Fay 2009); (3) lin-35; spr-1 and lin-35; xnp-1, which are defective at somatic gonad and germline development (Bender et al. 2004, 2007); and (5) lin-35; psa-1, which arrests as larvae and is defective at asymmetric cell division (Cui et al. 2004). Notably, of the 26 lin-35; slr-2 suppressors, 9 suppressed multiple lin-35-synthetic phenotypes (Figure 4; Figure S3, Figure S4, Figure S5, Figure S6, Figure S7, and Figure S8). These multi-phenotypic lin-35 suppressors included five synMuv suppressors as well as four lin-35; slr-2 suppressors that did not suppress the synMuv phenotype. We also tested the 24 synMuv suppressors that failed to suppress lin-35; slr-2 and found that 8 of them were capable of suppressing multiple lin-35-synthetic phenotypes (Figure 4; Figure S3, Figure S4, Figure S5, Figure S6, Figure S7, and Figure S8; Table S2). In total, we identified 19 RNAi clones that suppress multiple lin-35-synthetic phenotypes.

Figure 4
Suppressors of multiple lin-35-synthetic phenotypes. Suppression by RNAi clones (left-most column) of the indicated genotypes (top row) was scored as negative (red), moderate (yellow: 10–30% suppression), and strong (green: 30–100% suppression). ...

Notably strong suppressors were hcf-1, mes-4, and mrg-1, which suppressed nearly all lin-35-synthetic phenotypes. These findings suggest that these conserved chromatin modification factors regulate, directly or indirectly, the same core targets as LIN-35 and that their normal function is antagonistic to that of LIN-35. Also among the strongest suppressors were isw-1, htz-1, ZK1127, and F54D11.2, none of which significantly suppressed lin-35; slr-2 mutants. Given that hcf-1 and mes-4 encode homologs of Set1/COMPASS H3K4 methyltransferase complex members, we tested nine additional putative complex members for their ability to suppress lin-35-synthetic phenotypes. Notably, the H3K4 methyltransferase, set-2, and the yeast Cps40 homolog, F52B11.1, suppressed multiple phenotypes. (Figure 4, Figure S3, Figure S4, Figure S5, Figure S6, Figure S7, and Figure S8). Consistent with our genome-wide screen, however, RNAi of these genes did not suppress lin-35; slr-2. Interestingly, several of these multi-phenotypic lin-35 suppressors are not known to regulate chromatin, including raga-1, a target of TOR signaling (Schreiber et al. 2010), and osm-11, an osmosensation factor with suggested roles in Notch signaling (Wheeler and Thomas 2006; Komatsu et al. 2008).

Of particular interest to us were eight suppressors of lin-35; fzr-1 embryonic lethality (Figure 4; Figure S3). To examine these suppressors in more detail, we assayed proliferation of two cell types known to undergo excess divisions in lin-35; fzr-1 mutant larvae (Fay et al. 2002). We found that, among the lin-35; fzr-1 suppressors, only hcf-1 RNAi and iff-1 RNAi inhibited extra divisions of the DTCs (the Shv phenotype) in lin-35; fzr-1 mutants (Figure 5A). The presence of at least one DTC per gonad arm in lin-35; fzr-1 mutants treated with hcf-1 or iff-1 RNAi indicates that inhibition of these genes does not prevent DTC differentiation per se. Furthermore, our previous studies indicate that the extra DTCs present in lin-35; fzr-1 gonads are derived from a single precursor DTC in each arm and are not due to mis-specification of the DTC fate by other cell types (Fay et al. 2002). We also observed that RNAi of hcf-1 or mes-4 suppressed ectopic intestinal cell divisions (Figure 5B). The inability of other suppressors of lin-35; fzr-1 embryonic arrest to significantly reduce excess cell divisions during postembryonic development may reflect a lack of sensitivity in these particular assays or possibly a mechanism of embryonic arrest suppression by these genes that is not related to the cell cycle. This latter possibility is consistent with our previous findings that LIN-35 does not play a critical role in cell-cycle regulation in embryos (Kirienko and Fay 2007).

Figure 5
Suppression of lin-35; fzr-1 hyper-proliferation defects. (A) Percentage of adult animals that display a Shv phenotype, which results from excess divisions of the DTCs. Whereas RNAi of hcf-1 and iff-1 significantly suppresses ectopic divisions of the ...

As a final test of the multi-phenotypic lin-35 suppressors, we assayed suppression of a nonsynthetic lin-35 phenotype. High-temperature arrest (HTA; 26°) has been reported for a number of class B synMuv genes, including lin-35 (Petrella et al. 2011). Notably, RNAi of mes-4 and mrg-1 suppresses the lin-35 HTA phenotype (Petrella et al. 2011). Testing of the 26 lin-35; slr-2 RNAi suppressors confirmed suppression of lin-35(n745) by mes-4 and mrg-1 and also identified hcf-1 as a strong suppressor of lin-35 HTA (Figure S9). These results are consistent with HCF-1, MES-4, and MRG-1 playing a central role in opposing the activities of LIN-35.

Discussion

We have identified a new category of genes that we term the “multi-phenotypic lin-35 suppressors” (MPLS). Our data indicate that these genes play opposing roles to the pocket protein ortholog lin-35/pRb in multiple developmental contexts (Figure 4). To our knowledge, this is the first genetic analysis in C. elegans that has sought to characterize the suppression of multiple, distinct synthetic phenotypes that have in common a single inactivated gene (in our case, lin-35). Our previous analyses of lin-35-synthetic mutants have suggested that unique sets of misregulated genes underlie the diversity of observed phenotypes (Fay et al. 2002, 2003, 2004; Bender et al. 2004, 2007; Cui et al. 2004; Kirienko et al. 2008). We propose that genes capable of suppressing multiple genetically related but phenotypically distinct synthetic phenotypes are likely to act at the level of the shared mutant gene or its core downstream targets. Differences in the spectrum of lin-35-synthetic phenotypes that are suppressed by individual MPLS genes may reflect tissue-specific differences in their expression patterns or requirements for activity (Figure 4). Alternatively, differences in the efficiency of RNAi within distinct tissues may account for the inability of certain MPLS genes to mediate suppression for some phenotypes. In general, the analysis of multi-phenotypic suppressors of synthetic interactions should lead to the identification of genes whose activities are most germane to the direct or central functions of the common mutated gene. This multi-stage screening strategy could be useful for the study of any broadly acting gene for which multiple phenotypically distinct synthetic phenotypes are available.

In addition to suppressing multiple lin-35-synthetic phenotypes, several of the identified suppressors (e.g., mes-4) also suppress phenotypes associated with single mutants in a number of class B synMuv genes, including lin-35. lin-35 is an established negative regulator of the RNAi response, and lin-35 mutants are correspondingly hyper-sensitive to RNAi (Wang et al. 2005; Lehner et al. 2006; Ceron et al. 2007). Interestingly, several suppressors identified in our screen were previously shown to suppress lin-35 hyper-sensitivity to RNAi, including mes-4, sin-3, M03C11.3, ZK1127.3, and zfp-1 (Kim et al. 2005; Wang et al. 2005). lin-35 mutants also exhibit a repetitive-transgene silencing phenotype that is suppressed by mes-4 and ZK1127.3 RNAi (Hsieh et al. 1999; Pirrotta 2002; Wang et al. 2005). Furthermore, lin-35 mutants display ectopic expression of germline-specific genes in somatic tissues, a phenotype that is also suppressed by mes-4 RNAi (Wang et al. 2005; Petrella et al. 2011). Misexpression of germline genes in the soma of synMuv B mutants has been proposed to account for the silencing of somatic transgenes in these mutants, as repetitive transgenes are typically silenced in the germline (Hsieh et al. 1999; Wang et al. 2005; Petrella et al. 2011; Praitis and Maduro 2011).

Most recently, a study by Petrella et al. (2011) revealed that lin-35, along with several other class B synMuv mutants, undergoes L1 larval arrest at 26°, a temperature at which wild-type worms are viable. Furthermore, HTA is likely due to the misexpression of one or more classes of genes within the intestine (Petrella et al. 2011). Interestingly, RNAi of mes-4, mrg-1, hcf-1, and isw-1, four genes that suppress multiple lin-35-synthetic phenotypes, strongly suppressed HTA (Figure S9) (Petrella et al. 2011). Notably, genes that were upregulated in lin-35 single mutants at 26° returned to levels that were more similar to those of wild type in lin-35; mes-4(RNAi) larvae, a corrective phenomenon not observed for genes that were downregulated in lin-35 single mutants at 26°. Moreover, these mutual targets of LIN-35 and MES-4 included many intestine-associated genes along with germline genes. Given our findings that lin-35; slr-2 mutants have defective intestines because of the misexpression of many intestinal genes and that lin-35; slr-2 arrest is suppressed by mes-4(RNAi), we can suggest that genes that are specifically upregulated in the intestines of lin-35; slr-2 mutants lead to intestinal malfunction. This conclusion is supported by our observation that blunt inhibition of protein synthesis either by cycloheximide or G418 treatment (Figure 2) or by depletion of ribosome biogenesis genes (Figure 1) also leads to the suppression of lin-35; slr-2 and dpl-1; slr-2 larval arrest.

Work in multiple organisms has demonstrated that pRb complex members play a primarily repressive role in transcription, a function that is also conserved in C. elegans (Chi and Reinke 2006; Kirienko and Fay 2007). This is consistent with the majority of MPLS genes functioning in the opposite role—as transcriptional activators (Figure 4; Table 1). In fact, the broadest-acting lin-35 suppressors encode orthologs of proteins present in several established transcriptional activating complexes. The evolutionarily conserved suppressors hcf-1, set-2, F52B11.1, and mes-4 are orthologous to members of the Set1/COMPASS H3K4 methylation complex (Stec et al. 1998; Strahl et al. 2002; Wysocka et al. 2003; Yokoyama et al. 2004; Bender et al. 2006; Nimura et al. 2009; Mohan et al. 2011). SET-2 is an active H3K4 methyltransferase in C. elegans and F52B11.1 is a homolog of yeast Cps40, a factor that promotes H3K4 methylation, suggesting that a Set1/COMPASS-like complex is antagonistic to LIN-35 (Fisher et al. 2010; Xiao et al. 2011). Interestingly, although homologous to Set1, MES-4 is a well-characterized H3K36 methyltransferase in C. elegans and flies (Bell et al. 2007; Rechtsteiner et al. 2010; Xiao et al. 2011). Furthermore, MES-4 does not require colocalization with RNA polymerase II, a feature of its well-studied yeast homolog, Set2 (Furuhashi et al. 2010; Rechtsteiner et al. 2010). Currently, the role H3K36 methylation in regulating ongoing transcription, as well as the possible mechanism by which loss of this activity would suppress lin-35-synthetic phenotypes, remains unclear. The functions of hcf-1 orthologs have been primarily studied in systems other than C. elegans. In HeLa cells, HCF1 physically interacts with multiple E2Fs and recruits the Set1 complex to E2F promoter regions (Tyagi et al. 2007). Furthermore, pRb complex members interact with the Set1/COMPASS methyltransferase complex in human cell culture to control the expression of cell-cycle genes (Reilly et al. 2002; Delehouzee et al. 2005). mrg-1 and ZK1127.3 orthologs are members of multiple histone acetyltransferase complexes, including the NuA4 acetylation complex in budding yeast and the related Tip60 HAT complex in humans (Ceol and Horvitz 2004; Pena et al. 2011). Moreover, yeast two-hybrid analysis indicates that mrg-1 and ZK1127.3 physically interact in C. elegans, whereas the human mrg-1 ortholog MRG15 physically interacts with the ZK1127.3 ortholog MRGBP in HeLa cells (Cai et al. 2003; Li et al. 2004). Additionally, MRG15 physically interacts with pRb and has been proposed to play antagonistic roles in controlling E2F target regulation (Leung et al. 2001).

The above results suggest that both the Set1/COMPASS and NuA4/Tip60 complexes coregulate a set of conserved and overlapping targets with pRb and E2F family members. To examine this, we analyzed data from an HCF-1 ChIP-seq study carried out in mouse embryonic stem cells (Dejosez et al. 2010). Of the 305 HCF-1-bound genes identified, 151 had annotated orthologs in C. elegans and 23 of these were previously shown to be targets of LIN-35 by transcriptome analysis (Kirienko and Fay 2007). This represents an ~1.5 enrichment of HCF-1-bound genes among LIN-35 targets (P = 0.05, chi square test). Thus, LIN-35 and HCF-1 may possibly coregulate conserved targets across species. Moreover, greater overlap of LIN-35/pRb and HCF-1 targets might be expected in parallel ChiP-seq experiments carried out in the same species or cell type. Intriguingly, cell-cycle arrest in HCF-1 mutants in human tissue culture cells can be suppressed by loss of pRb activity, indicating that both the regulatory framework and the genetic suppressor relationship between these genes have been maintained (Reilly et al. 2002).

In addition to MPLS genes, other identified suppressors may enhance our understanding of the lin-35; slr-2 arrest phenotype. For example, the ability of lin-35; slr-2 mutants to reach adulthood following inhibition of the prohibitin genes phb-1 and phb-2 indicates that intestinal malfunction is not the direct cause of the observed L1 arrest. Rather, our findings suggest that lin-35; slr-2 mutants arrest because of the activation of an L1 metabolic checkpoint, which can be overridden by increasing energy availability through the mobilization and utilization of fat stores. A similar effect was also observed in inx-6 mutants following inactivation of phb-1 or phb-2, which are nutrient deprived because of impairment in food uptake (Figure 3). Thus, our studies have revealed a possible novel mechanism to suppress at least some aspects of nutrient deprivation-induced L1 diapause arrest.

In addition, it is perhaps surprising that gross inhibition of ribosome biogenesis and protein translation would prove to be an effective means to suppress a phenotype caused by the overexpression of a specific subset of proteins. Rather, if protein expression were to be uniformly reduced, imbalances in relative protein levels should persist. This might indicate that inhibition of protein synthesis in our suppressed strains is not uniform but may correlate with mRNA levels or translation rates, a phenomenon previously reported for cycloheximide-treated cells infected with virus (David 1976; Jen et al. 1978). We also note that inhibition of ribosome biogenesis was not effective at suppressing other lin-35-synthetic phenotypes. This finding may appear counterintuitive, given that suppression of most lin-35-associated phenotypes by SET1 and NuA4 complex members indicates that these phenotypes also result from the increased expression of LIN-35 targets. However, differences in the ability of the protein translation factors to suppress individual lin-35-synthetic phenotypes may depend more on specific changes in the secondary targets of LIN-35 and its synthetic lethal protein counterparts. For example, upregulated intestinal genes in lin-35; slr-2 mutants are not thought to be direct targets of a LIN-35EFL-1DPL-1 complex (Kirienko et al. 2008). Thus, in the case of other lin-35-synthetic mutants, the proximal cause of the observed defects may not be due to the strongly elevated expression of multiple loci, and therefore these phenotypes may not be effectively alleviated by decreased protein synthesis. Nevertheless, our findings do suggest that phenotypes caused by multi-gene overexpression may be generally suppressible through the inhibition of protein synthesis.

Supplementary Material

Supporting Information:

Acknowledgments

We thank Amy Fluet, John Yochem, and Aleksandra Kuzmanov for critically reading the manuscript and Evguenia Karina, Olivia Wolpert, James Polek, and Aya Murakami for aid with RNAi screening. We also thank the Caenorhabditis elegans Genetics Center, the U.S./Canadian C. elegans knockout consortium, and the National Bioresource Project for the Experimental Animal C. elegans (Japan). This work was supported by grant GM066868 from the National Institutes of Health.

Footnotes

Communicating editor: P. Sengupta

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