New genes that interact with lin-35 Rb to negatively regulate the let-60 ras pathway in Caenorhabditis elegans

Previous studies have shown that a synthetic multivulva phenotype results from mutations in genes that antagonize the ras-mediated intercellular signaling system responsible for vulval induction in Caenorhabditis elegans. Synthetic multivulva mutations define two classes of genes, A and B, and a mutation in a gene of each class is required to produce the multivulva phenotype. The ectopic vulval tissue in multivulva animals is generated by vulval precursor cells that in the wild type do not generate vulval tissue. One of the class B synthetic multivulva genes, lin-35, encodes a protein similar to the retinoblastoma (Rb) protein. In this article, we describe the isolation and characterization of 50 synthetic multivulva mutations, the identification of new components of both the class A and class B lin-35 Rb pathways, and the cloning of lin-52, a class B gene that may have a conserved role in Rb-mediated signaling.     

Large-scale RNAi screens identify novel genes that interact with the <Emphasis Type="Italic">C. elegans</Emphasis> retinoblastoma pathway as well as splicing-related components with synMuv B activity

Background  

The retinoblastoma tumor suppressor (Rb) acts in a conserved pathway that is deregulated in most human cancers. Inactivation of the single Rb-related gene in Caenorhabditis elegans, lin-35, has only limited effects on viability and fertility, yet causes changes in cell-fate and cell-cycle regulation when combined with inactivation of specific other genes. For instance, lin-35 Rb is a synthetic multivulva (synMuv) class B gene, which causes a multivulva phenotype when inactivated simultaneously with a class A or C synMuv gene.     

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.     

The Caenorhabditis elegans synthetic multivulva genes prevent ras pathway activation by tightly repressing global ectopic expression of lin-3 EGF

The Caenorhabditis elegans class A and B synthetic multivulva (synMuv) genes redundantly antagonize an EGF/Ras pathway to prevent ectopic vulval induction. We identify a class A synMuv mutation in the promoter of the lin-3 EGF gene, establishing that lin-3 is the key biological target of the class A synMuv genes in vulval development and that the repressive activities of the class A and B synMuv pathways are integrated at the level of lin-3 expression. Using FISH with single mRNA molecule resolution, we find that lin-3 EGF expression is tightly restricted to only a few tissues in wild-type animals, including the germline. In synMuv double mutants, lin-3 EGF is ectopically expressed at low levels throughout the animal. Our findings reveal that the widespread ectopic expression of a growth factor mRNA at concentrations much lower than that in the normal domain of expression can abnormally activate the Ras pathway and alter cell fates. These results suggest hypotheses for the mechanistic basis of the functional redundancy between the tumor-suppressor-like class A and B synMuv genes: the class A synMuv genes either directly or indirectly specifically repress ectopic lin-3 expression; while the class B synMuv genes might function similarly, but alternatively might act to repress lin-3 as a consequence of their role in preventing cells from adopting a germline-like fate. Analogous genes in mammals might function as tumor suppressors by preventing broad ectopic expression of EGF-like ligands.     

Multiple levels of redundant processes inhibit Caenorhabditis elegans vulval cell fates

Many mutations cause obvious abnormalities only when combined with other mutations. Such synthetic interactions can be the result of redundant gene functions. In Caenorhabditis elegans, the synthetic multivulva (synMuv) genes have been grouped into multiple classes that redundantly inhibit vulval cell fates. Animals with one or more mutations of the same class undergo wild-type vulval development, whereas animals with mutations of any two classes have a multivulva phenotype. By varying temperature and genetic background, we determined that mutations in most synMuv genes within a single synMuv class enhance each other. However, in a few cases no enhancement was observed. For example, mutations that affect an Mi2 homolog and a histone methyltransferase are of the same class and do not show enhancement. We suggest that such sets of genes function together in vivo and in at least some cases encode proteins that interact physically. The approach of genetic enhancement can be applied more broadly to identify potential protein complexes as well as redundant processes or pathways. Many synMuv genes are evolutionarily conserved, and the genetic relationships we have identified might define the functions not only of synMuv genes in C. elegans but also of their homologs in other organisms.     

C. elegans class B synthetic multivulva genes act in G(1) regulation

The single C. elegans member of the retinoblastoma gene family, lin-35 Rb, was originally identified as a synthetic Multivulva (synMuv) gene [1]. These genes form two redundant classes, A and B, that repress ectopic vulval cell fate induction. Recently, we demonstrated that lin-35 Rb also acts as a negative regulator of G(1) progression and likely is the major target of cyd-1 Cyclin D and cdk-4 CDK4/6. Here, we describe G(1) control functions for several other class B synMuv genes. We found that efl-1 E2F negatively regulates cell cycle entry, while dpl-1 DP appeared to act both as a positive and negative regulator. In addition, we identified a negative G(1) regulatory function for lin-9 ALY, as well as lin-15B and lin-36, which encode novel proteins. Inactivation of lin-35 Rb, efl-1, or lin-36 allowed S phase entry in the absence of cyd-1/cdk-4 and increased ectopic cell division when combined with cki-1 Cip/Kip RNAi. These data are consistent with lin-35 Rb, efl-1, and lin-36 acting in a common pathway or complex that negatively regulates G(1) progression. In contrast, lin-15B appeared to act in parallel to lin-35. Our results demonstrate the potential for genetic identification of novel G(1) regulators in C. elegans.     

Repression of germline RNAi pathways in somatic cells by retinoblastoma pathway chromatin complexes

The retinoblastoma (Rb) tumor suppressor acts with a number of chromatin cofactors in a wide range of species to suppress cell proliferation. The Caenorhabditis elegans retinoblastoma gene and many of these cofactors, called synMuv B genes, were identified in genetic screens for cell lineage defects caused by growth factor misexpression. Mutations in many synMuv B genes, including lin-35/Rb, also cause somatic misexpression of the germline RNA processing P granules and enhanced RNAi. We show here that multiple small RNA components, including a set of germline-specific Argonaute genes, are misexpressed in the soma of many synMuv B mutant animals, revealing one node for enhanced RNAi. Distinct classes of synMuv B mutants differ in the subcellular architecture of their misexpressed P granules, their profile of misexpressed small RNA and P granule genes, as well as their enhancement of RNAi and the related silencing of transgenes. These differences define three classes of synMuv B genes, representing three chromatin complexes: a LIN-35/Rb-containing DRM core complex, a SUMO-recruited Mec complex, and a synMuv B heterochromatin complex, suggesting that intersecting chromatin pathways regulate the repression of small RNA and P granule genes in the soma and the potency of RNAi. Consistent with this, the DRM complex and the synMuv B heterochromatin complex were genetically additive and displayed distinct antagonistic interactions with the MES-4 histone methyltransferase and the MRG-1 chromodomain protein, two germline chromatin regulators required for the synMuv phenotype and the somatic misexpression of P granule components. Thus intersecting synMuv B chromatin pathways conspire with synMuv B suppressor chromatin factors to regulate the expression of small RNA pathway genes, which enables heightened RNAi response. Regulation of small RNA pathway genes by human retinoblastoma may also underlie its role as a tumor suppressor gene.     

The Multivulva Phenotype of Certain Caenorhabditis Elegans Mutants Results from Defects in Two Functionally Redundant Pathways

We previously identified Caenorhabditis elegans mutants in which certain of the six vulval precursor cells adopt fates normally expressed by other vulval precursor cells. These mutants define genes that appear to function in the response to an intercellular signal that induces vulval development. The multivulva (Muv) phenotype of one such mutant, CB1322, results from an interaction between two unlinked mutations, lin-8(n111) II and lin-9(n112) III. In this paper, we identify 18 new mutations, which are alleles of eight genes, that interact with either lin-8(n111) or lin-9(n112) to generate a Muv phenotype. None of these 20 mutations alone causes any vulval cell lineage defects. The "silent Muv" mutations fall into two classes; hermaphrodites carrying a mutation of each class are Muv, while hermaphrodites carrying two mutations of the same class have a wild-type vulval phenotype. Our results indicate that the Muv phenotype of these mutants results from defects in two functionally-redundant pathways, thereby demonstrating that redundancy can occur at the level of gene pathways as well as at the level of gene families.     

gon-14 functions with class B and class C synthetic multivulva genes to control larval growth in Caenorhabditis elegans

Previous work showed that C. elegans gon-14 is required for gonadogenesis. Here we report that gon-14 encodes a protein with similarity to LIN-15B, a class B synMuv protein. An extensive region of GON-14 contains blocks of sequence similarity to transposases of the hAT superfamily, but key residues are not conserved, suggesting a distant relationship. GON-14 also contains a putative THAP DNA-binding domain. A rescuing gon-14::GON-14::VENUS reporter is broadly expressed during development and localizes to the nucleus. Strong loss-of-function and predicted null gon-14 alleles have pleiotropic defects, including multivulval (Muv) defects and temperature-sensitive larval arrest. Although the gon-14 Muv defect is not enhanced by synMuv mutations, gon-14 interacts genetically with class B and class C synMuv genes, including lin-35/Rb, let-418/Mi-2beta, and trr-1/TRRAP. The gon-14; synMuv double mutants arrest as larvae when grown under conditions supporting development to adulthood for the respective single mutants. The gon-14 larval arrest is suppressed by loss of mes-2/E(Z), mes-6/ESC, or mes-4, which encodes a SET domain protein. Additionally, gon-14 affects expression of pgl-1 and lag-2, two genes regulated by the synMuv genes. We suggest that gon-14 functions with class B and class C synMuv genes to promote larval growth, in part by antagonizing MES-2,3,6/ESC-E(z) and MES-4.     

Loss of LIN-35, the Caenorhabditis elegans ortholog of the tumor suppressor p105Rb, results in enhanced RNA interference

Background

Genome-wide RNA interference (RNAi) screening is a very powerful tool for analyzing gene function in vivo in Caenorhabditis elegans. The effectiveness of RNAi varies from gene to gene, however, and neuronally expressed genes are largely refractive to RNAi in wild-type worms.

Results

We found that C. elegans strains carrying mutations in lin-35, the worm ortholog of the tumor suppressor gene p105Rb, or a subset of the genetically related synMuv B family of chromatin-modifying genes, show increased strength and penetrance for many germline, embryonic, and post-embryonic RNAi phenotypes, including neuronal RNAi phenotypes. Mutations in these same genes also enhance somatic transgene silencing via an RNAi-dependent mechanism. Two genes, mes-4 and zfp-1, are required both for the vulval lineage defects resulting from mutations in synMuv B genes and for RNAi, suggesting a common mechanism for the function of synMuv B genes in vulval development and in regulating RNAi. Enhanced RNAi in the germline of lin-35 worms suggests that misexpression of germline genes in somatic cells cannot alone account for the enhanced RNAi observed in this strain.

Conclusion

A worm strain with a null mutation in lin-35 is more sensitive to RNAi than any other previously described single mutant strain, and so will prove very useful for future genome-wide RNAi screens, particularly for identifying genes with neuronal functions. As lin-35 is the worm ortholog of the mammalian tumor suppressor gene p105Rb, misregulation of RNAi may be important during human oncogenesis.     

Caenorhabditis elegans lin-13, a member of the LIN-35 Rb class of genes involved in vulval development, encodes a protein with zinc fingers and an LXCXE motif

The SynMuv genes appear to be involved in providing a signal that inhibits vulval precursor cells from adopting vulval fates in Caenorhabditis elegans. One group of SynMuv genes, termed class B, includes genes encoding proteins related to the tumor suppressor Rb and RbAp48, a protein that binds Rb. Here, we provide genetic evidence that lin-13 behaves as a class B SynMuv gene. We show that null alleles of lin-13 are temperature sensitive and maternally rescued, resulting in phenotypes ranging in severity from L2 arrest (when both maternal and zygotic activities are removed at 25 degrees ), to sterile Multivulva (when only zygotic activity is removed at 25 degrees ), to sterile non-Multivulva (when both maternal and zygotic activities are removed at 15 degrees ), to wild-type/class B SynMuv (when only zygotic activity is removed at 15 degrees ). We also show that LIN-13 is a nuclear protein that contains multiple zinc fingers and a motif, LXCXE, that has been implicated in Rb binding. These results together suggest a role for LIN-13 in Rb-mediated repression of vulval fates.