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

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.     

lin-35/Rb and ubc-18, an E2 ubiquitin-conjugating enzyme,function redundantly to control pharyngeal morphogenesis in C. elegans

The retinoblastoma gene product has been implicated in the regulation of multiple cellular and developmental processes, including a well-defined role in the control of cell cycle progression. The Caenorhabditis elegans retinoblastoma protein homolog, LIN-35, is also a key regulator of cell cycle entry and, as shown by studies of synthetic multivulval genes, plays an important role in the determination of vulval cell fates. We demonstrate an additional and unexpected function for lin-35 in organ morphogenesis. Using a genetic approach to isolate lin-35 synthetic-lethal mutations, we have identified redundant roles for lin-35 and ubc-18, a gene that encodes an E2 ubiquitin-conjugating enzyme closely related to human UBCH7. lin-35 and ubc-18 cooperate to control one or more steps during pharyngeal morphogenesis. Based on genetic and phenotypic analyses, this role for lin-35 in pharyngeal morphogenesis appears to be distinct from its cell cycle-related functions. lin-35 and ubc-18 may act in concert to regulate the levels of one or more critical targets during C. elegans development.     

p24 proteins and quality control of LIN-12 and GLP-1 trafficking in Caenorhabditis elegans.

Mutations in the Caenorhabditis elegans sel-9 gene elevate the activity of lin-12 and glp-1, which encode members of the LIN-12/NOTCH family of receptors. Sequence analysis indicates SEL-9 is one of several C. elegans p24 proteins. Allele-specific genetic interactions suggest that reducing sel-9 activity increases the activity of mutations altering the extracellular domains of LIN-12 or GLP-1. Reducing sel-9 activity restores the trafficking to the plasma membrane of a mutant GLP-1 protein that would otherwise accumulate within the cell. Our results suggest a role for SEL-9 and other p24 proteins in the negative regulation of transport of LIN-12 and GLP-1 to the cell surface, and favor a role for p24 proteins in a quality control mechanism for endoplasmic reticulum-Golgi transport.     

lin-35/Rb cooperates with the SWI/SNF complex to control Caenorhabditis elegans larval development

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.     

C. elegans LIN-18 is a Ryk ortholog and functions in parallel to LIN-17/Frizzled in Wnt signaling

Wnt proteins are intercellular signals that regulate various aspects of animal development. In Caenorhabditis elegans, mutations in lin-17, a Frizzled-class Wnt receptor, and in lin-18 affect cell fate patterning in the P7.p vulval lineage. We found that lin-18 encodes a member of the Ryk/Derailed family of tyrosine kinase-related receptors, recently found to function as Wnt receptors. Members of this family have nonactive kinase domains. The LIN-18 kinase domain is dispensable for LIN-18 function, while the Wnt binding WIF domain is required. We also found that Wnt proteins LIN-44, MOM-2, and CWN-2 redundantly regulate P7.p patterning. Genetic interactions indicate that LIN-17 and LIN-18 function independently of each other in parallel pathways, and different ligands display different receptor specificities. Thus, two independent Wnt signaling pathways, one employing a Ryk receptor and the other a Frizzled receptor, function in parallel to regulate cell fate patterning in the C. elegans vulva.     

The bromodomain protein LIN-49 and trithorax-related protein LIN-59 affect development and gene expression in Caenorhabditis elegans

We have molecularly characterized the lin-49 and lin-59 genes in C. elegans, and found their products are related to Drosophila trithorax group (trx-G) proteins and other proteins implicated in chromatin remodelling. LIN-49 is structurally most similar to the human bromodomain protein BR140, and LIN-59 is most similar to the Drosophila trx-G protein ASH1. In C. elegans, lin-49 and lin-59 are required for the normal development of the mating structures of the adult male tail, for the normal morphology and function of hindgut (rectum) cells in both males and hermaphrodites and for the maintenance of structural integrity in the hindgut and egg-laying system in adults. Expression of the Hox genes egl-5 and mab-5 is reduced in lin-49 and lin-59 mutants, suggesting lin-49 and lin-59 regulate HOM-C gene expression in C. elegans as the trx-G genes do in Drosophila. lin-49 and lin-59 transgenes are expressed widely throughout C. elegans animals. Thus, in contrast to the C. elegans Polycomb group (Pc-G)-related genes mes-2 and mes-6 that function primarily in the germline, we propose lin-49 and lin-59 function in somatic development similar to the Drosophila trx-G genes.     

Identification of cis-regulatory elements from the C. elegans Hox gene lin-39 required for embryonic expression and for regulation by the transcription factors LIN-1, LIN-31 and LIN-39

Expression of the Caenorhabditis elegans Hox gene lin-39 begins in the embryo and continues in multiple larval cells, including the P cell lineages that generate ventral cord neurons (VCNs) and vulval precursor cells (VPCs). lin-39 is regulated by several factors and by Wnt and Ras signaling pathways; however, no cis-acting sites mediating lin-39 regulation have been identified. Here, we describe three elements controlling lin-39 expression: a 338-bp upstream fragment that directs embryonic expression in P5-P8 and their descendants in the larva, a 247-bp intronic region sufficient for VCN expression, and a 1.3-kb upstream cis-regulatory module that drives expression in the VPC P6.p in a Ras-dependent manner. Three trans-acting factors regulate expression via the 1.3-kb element. A single binding site for the ETS factor LIN-1 mediates repression in VPCs other than P6.p; however, loss of LIN-1 decreases expression in P6.p. Therefore, LIN-1 acts both negatively and positively on lin-39 in different VPCs. The Forkhead domain protein LIN-31 also acts positively on lin-39 in P6.p via this module. Finally, LIN-39 itself binds to this element, suggesting that LIN-39 autoregulates its expression in P6.p. Therefore, we have begun to unravel the cis-acting sites regulating lin-39 Hox gene expression and have shown that lin-39 is a direct target of the Ras pathway acting via LIN-1 and LIN-31.     

Identification of residues of the Caenorhabditis elegans LIN-1 ETS domain that are necessary for DNA binding and regulation of vulval cell fates

LIN-1 is an ETS domain protein. A receptor tyrosine kinase/Ras/mitogen-activated protein kinase signaling pathway regulates LIN-1 in the P6.p cell to induce the primary vulval cell fate during Caenorhabditis elegans development. We identified 23 lin-1 loss-of-function mutations by conducting several genetic screens. We characterized the molecular lesions in these lin-1 alleles and in several previously identified lin-1 alleles. Nine missense mutations and 10 nonsense mutations were identified. All of these lin-1 missense mutations affect highly conserved residues in the ETS domain. These missense mutations can be arranged in an allelic series; the strongest mutations eliminate most or all lin-1 functions, and the weakest mutation partially reduces lin-1 function. An electrophoretic mobility shift assay was used to demonstrate that purified LIN-1 protein has sequence-specific DNA-binding activity that required the core sequence GGAA. LIN-1 mutant proteins containing the missense substitutions had dramatically reduced DNA binding. These experiments identify eight highly conserved residues of the ETS domain that are necessary for DNA binding. The identification of multiple mutations that reduce the function of lin-1 as an inhibitor of the primary vulval cell fate and also reduce DNA binding suggest that DNA binding is essential for LIN-1 function in an animal.     

Novel heterochronic functions of the Caenorhabditis elegans period-related protein LIN-42

LIN-42, the Caenorhabditis elegans homolog of the Period (Per) family of circadian rhythm proteins, functions as a member of the heterochronic pathway, regulating temporal cell identities. We demonstrate that lin-42 acts broadly, timing developmental events in the gonad, vulva, and sex myoblasts, in addition to its well-established role in timing terminal differentiation of the hypodermis. In the vulva, sex myoblasts, and hypodermis, lin-42 activity prevents stage-specific cell division patterns from occurring too early. This general function of timing stage-appropriate cell division patterns is shared by the majority of heterochronic genes; their mutation temporally alters stage-specific division patterns. In contrast, lin-42 function in timing gonad morphogenesis is unique among the known heterochronic genes: inactivation of lin-42 causes the elongating gonad arms to reflex too early, a phenotype which implicates lin-42 in temporal regulation of cell migration. Three additional isoforms of lin-42 are identified that expand our view of the lin-42 locus and significantly extend the homology between LIN-42 and other PER family members. We show that, similar to PER proteins, LIN-42 has a dynamic expression pattern; its levels oscillate relative to the molts during postembryonic development. Transformation rescue studies indicate lin-42 is bipartite with respect to function. Intriguingly, the hallmark PAS domain is dispensable for LIN-42 function in transgenic animals.     

sel-7, a positive regulator of lin-12 activity, encodes a novel nuclear protein in Caenorhabditis elegans

Suppressor genetics in C. elegans has identified key components of the LIN-12/Notch signaling pathway. Here, we describe a genetic and molecular characterization of the suppressor gene sel-7. We show that reducing or eliminating sel-7 activity suppresses the effects of constitutive lin-12 activity, enhances the effects of partially reduced lin-12 activity, and causes a synthetic Lin-12(0) phenotype when combined with a null mutation in the sel-12 presenilin gene. These observations suggest that sel-7 is a positive regulator of lin-12 activity. We also show that SEL-7 encodes a novel nuclear protein. Through yeast two-hybrid screening, we identified an apparent interaction partner, K08E3.8, that also interacts with SEL-8, a known component of the nuclear complex that forms upon LIN-12 activation. Our data suggest potential roles for SEL-7 in the assembly or function of this nuclear complex.