lin-35 Rb and cki-1 Cip/Kip cooperate in developmental regulation of G1 progression in C. elegans.

We have investigated the regulation of cell-cycle entry in C. elegans, taking advantage of its largely invariant and completely described pattern of somatic cell divisions. In a genetic screen, we identified mutations in cyd-1 cyclin D and cdk-4 Cdk4/6. Recent results indicated that during Drosophila development, cyclin D-dependent kinases regulate cell growth rather than cell division. However, our data indicate that C. elegans cyd-1 primarily controls G1 progression. To investigate whether cyd-1 and cdk-4 solely act to overcome G1 inhibition by retinoblastoma family members, we constructed double mutants that completely eliminate the function of the retinoblastoma family and cyclin D-Cdk4/6 kinases. Inactivation of lin-35 Rb, the single Rb-related gene in C. elegans, substantially reduced the DNA replication and cell-division defects in cyd-1 and cdk-4 mutant animals. These results demonstrate that lin-35 Rb is an important negative regulator of G1/S progression and probably a downstream target for cyd-1 and cdk-4. However, as the suppression by lin-35 Rb is not complete, cyd-1 and cdk-4 probably have additional targets. An additional level of control over G1 progression is provided by Cip/Kip kinase inhibitors. We demonstrate that lin-35 Rb and cki-1 Cip/Kip contribute non-overlapping levels of G1/S inhibition in C. elegans. Surprisingly, loss of cki-1, but not lin-35, results in precocious entry into S phase. We suggest that a rate limiting role for cki-1 Cip/Kip rather than lin-35 Rb explains the lack of cell-cycle phenotype of lin-35 mutant animals.     

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.     

Regulation of postembryonic G(1) cell cycle progression in Caenorhabditis elegans by a cyclin D/CDK-like complex.

In many organisms, initiation and progression through the G(1) phase of the cell cycle requires the activity of G(1)-specific cyclins (cyclin D and cyclin E) and their associated cyclin-dependent kinases (CDK2, CDK4, CDK6). We show here that the Caenorhabditis elegans genes cyd-1 and cdk-4, encoding proteins similar to cyclin D and its cognate cyclin-dependent kinases, respectively, are necessary for proper division of postembryonic blast cells. Animals deficient for cyd-1 and/or cdk-4 activity have behavioral and developmental defects that result from the inability of the postembryonic blast cells to escape G(1) cell cycle arrest. Moreover, ectopic expression of cyd-1 and cdk-4 in transgenic animals is sufficient to activate a S-phase reporter gene. We observe no embryonic defects associated with depletion of either of these two gene products, suggesting that their essential functions are restricted to postembryonic development. We propose that the cyd-1 and cdk-4 gene products are an integral part of the developmental control of larval cell proliferation through the regulation of G(1) progression.     

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.     

Caenorhabditis elegans lin-35/Rb, efl-1/E2F and other synthetic multivulva genes negatively regulate the anaphase-promoting complex gene mat-3/APC8

Retinoblastoma (Rb)/E2F complexes repress expression of many genes important for G(1)-to-S transition, but also appear to regulate gene expression at other stages of the cell cycle. In C. elegans, lin-35/Rb and other synthetic Multivulva (SynMuv) group B genes function redundantly with other sets of genes to regulate G(1)/S progression, vulval and pharyngeal differentiation, and other unknown processes required for viability. Here we show that lin-35/Rb, efl-1/E2F, and other SynMuv B genes negatively regulate a component of the anaphase-promoting complex or cyclosome (APC/C). The APC/C is a multisubunit complex that promotes metaphase-to-anaphase progression and G(1) arrest by targeting different substrates for ubiquitination and proteasome-mediated destruction. The C. elegans APC/C gene mat-3/APC8 has been defined by temperature-sensitive embryonic lethal alleles that strongly affect germline meiosis and mitosis but only weakly affect somatic development. We describe severe nonconditional mat-3 alleles and a hypomorphic viable allele (ku233), all of which affect postembryonic cell divisions including those of the vulval lineage. The ku233 lesion is located outside of the mat-3 coding region and reduces mat-3 mRNA expression. Loss-of-function alleles of lin-35/Rb and other SynMuv B genes suppress mat-3(ku233) defects by restoring mat-3 mRNA to wild-type levels. Therefore, Rb/E2F complexes appear to repress mat-3 expression.     

The coordinate regulation of pharyngeal development in C. elegans by lin-35/Rb, pha-1, and ubc-18

Organ development is a complex process involving the coordination of cell proliferation, differentiation, and morphogenetic events. Using a screen to identify genes that function coordinately with lin-35/Rb during animal development, we have isolated a weak loss-of-function (LOF) mutation in pha-1. lin-35; pha-1 double mutants are defective at an early step in pharyngeal morphogenesis leading to an abnormal pharyngeal architecture. pha-1 is also synthetically lethal with other class B synthetic multivulval (SynMuv) genes including the C. elegans E2F homolog, efl-1. Reporter analyses indicate that pha-1 is broadly expressed during embryonic development and that its functions reside in the cytoplasm. We also provide genetic and phenotypic evidence to support the model that PHA-1, a novel protein, and UBC-18, a ubiquitin-conjugating enzyme that we have previously shown to function with lin-35 during pharyngeal development, act in parallel pathways to regulate the activity of a common cellular target.     

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.     

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.     

mir-35 is involved in intestine cell G1/S transition and germ cell proliferation in C. elegans

MicroRNA (miRNA) regulates gene expression in many cellular events, yet functions of only a few miRNAs are known in C. elegans. We analyzed the function of mir-35-41 unique to the worm, and show here that mir-35 regulates the G1/S transition of intestinal cells and germ cell proliferation. Loss of mir-35 leads to a decrease of nuclei numbers in intestine and distal mitotic gonad, while re-introduction of mir-35 rescues the mutant phenotypes. Genetic analysis indicates that mir-35 may act through Rb/E2F and SCF pathways. Further bioinformatic and functional analyses demonstrate that mir-35 targets evolutionally conserved lin-23 and gld-1. Together, our study reveals a novel function of mir-35 family in cell division regulation.     

The C. elegans F-box/WD-repeat protein LIN-23 functions to limit cell division during development

In multicellular eukaryotes, a complex program of developmental signals regulates cell growth and division by controlling the synthesis, activation and degradation of G(1) cell cycle regulators. Here we describe the lin-23 gene of Caenorhabditis elegans, which is required to restrain cell proliferation in response to developmental cues. In lin-23 null mutants, all postembryonic blast cells undergo extra divisions, creating supernumerary cells that can differentiate and function normally. In contrast to the inability to regulate the extent of blast cell division in lin-23 mutants, the timing of initial cell cycle entry of blast cells is not affected. lin-23 encodes an F-box/WD-repeat protein that is orthologous to the Saccharomyces cerevisiae gene MET30, the Drosophila melanogaster gene slmb and the human gene betaTRCP, all of which function as components of SCF ubiquitin-ligase complexes. Loss of function of the Drosophila slmb gene causes the growth of ectopic appendages in a non-cell autonomous manner. In contrast, lin-23 functions cell autonomously to negatively regulate cell cycle progression, thereby allowing cell cycle exit in response to developmental signals.     

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.