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.     

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.     

The C. elegans Homolog of RBBP6 (RBPL-1) Regulates Fertility through Controlling Cell Proliferation in the Germline and Nutrient Synthesis in the Intestine

RBBP6 (retinoblastoma binding protein 6, also known as PACT or P2P-R in humans) is a multi-domain protein that functions in multiple processes, such as mitosis, cell differentiation, and cell apoptosis. RBBP6 is evolutionarily conserved and is present in unicellular organisms to mammals. Studies of RBBP6 have mostly focused on its RB- and p53-binding domains, which are found exclusively in mammals. Here, we investigated the C. elegans homolog of RBBP6 to explore the functional roles of its other domains. We found that RBPL-1, the homolog of RBBP6 in C. elegans, is indispensable for worm development. RNAi silencing of rbpl-1 led to embryonic lethality, as well as defects in oocyte production and intestine development. rbpl-1 RNAi worms showed defects in germ cell proliferation, suggesting that RBPL-1 regulates mitosis. Moreover, RNAi silencing of rbpl-1 inhibited nutrient synthesis in the worm intestine. RBPL-1, as a nucleolus protein, was found to be expressed in diverse tissues and necessary for both germline and soma development. Using microarray analysis, we identified ≈700 genes whose expression levels were changed at least 10-fold in rbpl-1 worms. We propose that RBPL-1, like its yeast homolog, may regulate gene expression as an mRNA cleavage and polyadenylation factor. Taken together, the findings from this study reveal that RBPL-1 plays a pivotal role in C. elegans germline and soma development, suggesting that the functions of RBBP6 are conserved in diverse eukaryotic species.     

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.     

Tissue-specific direct targets of Caenorhabditis elegans Rb/E2F dictate distinct somatic and germline programs

Background

The tumor suppressor Rb/E2F regulates gene expression to control differentiation in multiple tissues during development, although how it directs tissue-specific gene regulation in vivo is poorly understood.

Results

We determined the genome-wide binding profiles for Caenorhabditis elegans Rb/E2F-like components in the germline, in the intestine and broadly throughout the soma, and uncovered highly tissue-specific binding patterns and target genes. Chromatin association by LIN-35, the C. elegans ortholog of Rb, is impaired in the germline but robust in the soma, a characteristic that might govern differential effects on gene expression in the two cell types. In the intestine, LIN-35 and the heterochromatin protein HPL-2, the ortholog of Hp1, coordinately bind at many sites lacking E2F. Finally, selected direct target genes contribute to the soma-to-germline transformation of lin-35 mutants, including mes-4, a soma-specific target that promotes H3K36 methylation, and csr-1, a germline-specific target that functions in a 22G small RNA pathway.

Conclusions

In sum, identification of tissue-specific binding profiles and effector target genes reveals important insights into the mechanisms by which Rb/E2F controls distinct cell fates in vivo.     

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.     

A Pre- and Co-Knockdown of RNAseT Enzyme,Eri-1, Enhances the Efficiency of RNAi Induced Gene Silencing in Caenorhabditis elegans

Background

The approach of RNAi mediated gene knockdown, employing exogenous dsRNA, is being beneficially exploited in various fields of functional genomics. The immense utility of the approach came to fore from studies with model system C. elegans, but quickly became applicable with varied research models ranging from in vitro to various in vivo systems. Previously, there have been reports on the refractoriness of the neuronal cells to RNAi mediated gene silencing following which several modulators like eri-1 and lin-15 were described in C. elegans which, when present, would negatively impact the gene knockdown.

Methodology/Principal Findings

Taking a clue from these findings, we went on to screen hypothesis-driven- methodologies towards exploring the efficiency in the process of RNAi under various experimental conditions, wherein these genes would be knocked down preceding to, or concurrently with, the knocking down of a gene of interest. For determining the efficiency of gene knockdown, we chose to study visually stark phenotypes of uncoordinated movement, dumpy body morphology and blistered cuticle obtained by knocking down of genes unc-73, dpy-9 and bli-3 respectively, employing the RNAi-by-feeding protocol in model system C. elegans.

Conclusions/Significance

Our studies led to a very interesting outcome as the results reveal that amongst various methods tested, pre-incubation with eri-1 dsRNA synthesizing bacteria followed by co-incubation with eri-1 and gene-of-interest dsRNA synthesizing bacteria leads to the most efficient gene silencing as observed by the analysis of marker phenotypes. This provides an approach for effectively employing RNAi induced gene silencing while working with different genetic backgrounds including transgenic and mutant strains.     

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.     

sel-11 and cdc-42, Two Negative Modulators of LIN-12/Notch Activity in C. elegans

Background

LIN-12/Notch signaling is important for cell-cell interactions during development, and mutations resulting in constitutive LIN-12/Notch signaling can cause cancer. Loss of negative regulators of lin-12/Notch activity has the potential for influencing cell fate decisions during development and the genesis or aggressiveness of cancer.

Methodology/Principal Findings

We describe two negative modulators of lin-12 activity in C. elegans. One gene, sel-11, was initially defined as a suppressor of a lin-12 hypomorphic allele; the other gene, cdc-42, is a well-studied Rho GTPase. Here, we show that SEL-11 corresponds to yeast Hrd1p and mammalian Synoviolin. We also show that cdc-42 has the genetic properties consistent with negative regulation of lin-12 activity during vulval precursor cell fate specification.

Conclusions/Significance

Our results underscore the multiplicity of negative regulatory mechanisms that impact on lin-12/Notch activity and suggest novel mechanisms by which constitutive lin-12/Notch activity might be exacerbated in cancer.     

Exploring systemic RNA interference in insects: a genome-wide survey for RNAi genes in Tribolium

Background

RNA interference (RNAi) is a highly conserved cellular mechanism. In some organisms, such as Caenorhabditis elegans, the RNAi response can be transmitted systemically. Some insects also exhibit a systemic RNAi response. However, Drosophila, the leading insect model organism, does not show a robust systemic RNAi response, necessitating another model system to study the molecular mechanism of systemic RNAi in insects.

Results

We used Tribolium, which exhibits robust systemic RNAi, as an alternative model system. We have identified the core RNAi genes, as well as genes potentially involved in systemic RNAi, from the Tribolium genome. Both phylogenetic and functional analyses suggest that Tribolium has a somewhat larger inventory of core component genes than Drosophila, perhaps allowing a more sensitive response to double-stranded RNA (dsRNA). We also identified three Tribolium homologs of C. elegans sid-1, which encodes a possible dsRNA channel. However, detailed sequence analysis has revealed that these Tribolium homologs share more identity with another C. elegans gene, tag-130. We analyzed tag-130 mutants, and found that this gene does not have a function in systemic RNAi in C. elegans. Likewise, the Tribolium sid-like genes do not seem to be required for systemic RNAi. These results suggest that insect sid-1-like genes have a different function than dsRNA uptake. Moreover, Tribolium lacks homologs of several genes important for RNAi in C. elegans.

Conclusion

Although both Tribolium and C. elegans show a robust systemic RNAi response, our genome-wide survey reveals significant differences between the RNAi mechanisms of these organisms. Thus, insects may use an alternative mechanism for the systemic RNAi response. Understanding this process would assist with rendering other insects amenable to systemic RNAi, and may influence pest control approaches.     

The Wnts

Background

The eukaryotic ubiquitin-conjugation system sets the turnover rate of many proteins and includes activating enzymes (E1s), conjugating enzymes (UBCs/E2s), and ubiquitin-protein ligases (E3s), which are responsible for activation, covalent attachment and substrate recognition, respectively. There are also ubiquitin-like proteins with distinct functions, which require their own E1s and E2s for attachment. We describe the results of RNA interference (RNAi) experiments on the E1s, UBC/E2s and ubiquitin-like proteins in Caenorhabditis elegans. We also present a phylogenetic analysis of UBCs.

Results

The C. elegans genome encodes 20 UBCs and three ubiquitin E2 variant proteins. RNAi shows that only four UBCs are essential for embryogenesis: LET-70 (UBC-2), a functional homolog of yeast Ubc4/5p, UBC-9, an ortholog of yeast Ubc9p, which transfers the ubiquitin-like modifier SUMO, UBC-12, an ortholog of yeast Ubc12p, which transfers the ubiquitin-like modifier Rub1/Nedd8, and UBC-14, an ortholog of Drosophila Courtless. RNAi of ubc-20, an ortholog of yeast UBC1, results in a low frequency of arrested larval development. A phylogenetic analysis of C. elegans, Drosophila and human UBCs shows that this protein family can be divided into 18 groups, 13 of which include members from all three species. The activating enzymes and the ubiquitin-like proteins NED-8 and SUMO are required for embryogenesis.

Conclusions

The number of UBC genes appears to increase with developmental complexity, and our results suggest functional overlap in many of these enzymes. The ubiquitin-like proteins NED-8 and SUMO and their corresponding activating enzymes are required for embryogenesis.