S6K links cell fate, cell cycle and nutrient response in C. elegans germline stem/progenitor cells

Coupling of stem/progenitor cell proliferation and differentiation to organismal physiological demands ensures the proper growth and homeostasis of tissues. However, in vivo mechanisms underlying this control are poorly characterized. We investigated the role of ribosomal protein S6 kinase (S6K) at the intersection of nutrition and the establishment of a stem/progenitor cell population using the C. elegans germ line as a model. We find that rsks-1 (which encodes the worm homolog of mammalian p70S6K) is required germline-autonomously for proper establishment of the germline progenitor pool. In the germ line, rsks-1 promotes cell cycle progression and inhibits larval progenitor differentiation, promotes growth of adult tumors and requires a conserved TOR phosphorylation site. Loss of rsks-1 and ife-1 (eIF4E) together reduces the germline progenitor pool more severely than either single mutant and similarly to reducing the activity of let-363 (TOR) or daf-15 (RAPTOR). Moreover, rsks-1 acts in parallel with the glp-1 (Notch) and daf-2 (insulin-IGF receptor) pathways, and does not share the same genetic dependencies with its role in lifespan control. We show that overall dietary restriction and amino acid deprivation cause germline defects similar to a subset of rsks-1 mutant phenotypes. Consistent with a link between diet and germline proliferation via rsks-1, loss of rsks-1 renders the germ line largely insensitive to the effects of dietary restriction. Our studies establish the C. elegans germ line as an in vivo model to understand TOR-S6K signaling in proliferation and differentiation and suggest that this pathway is a key nutrient-responsive regulator of germline progenitors.     

FBF-1 and FBF-2 regulate the size of the mitotic region in the C. elegans germline

In the C. elegans germline, GLP-1/Notch signaling and two nearly identical RNA binding proteins, FBF-1 and FBF-2, promote proliferation. Here, we show that the fbf-1 and fbf-2 genes are largely redundant for promoting mitosis but that they have opposite roles in fine-tuning the size of the mitotic region. The mitotic region is smaller than normal in fbf-1 mutants but larger than normal in fbf-2 mutants. Consistent with gene-specific roles, fbf-2 expression is limited to the distal germline, while fbf-1 expression is broader. The fbf-2 gene, but apparently not fbf-1, is controlled by GLP-1/Notch signaling, and the abundance of FBF-1 and FBF-2 proteins is limited by reciprocal 3'UTR repression. We propose that the divergent fbf genes and their regulatory subnetwork enable a precise control over size of the mitotic region. Therefore, fbf-1 and fbf-2 provide a paradigm for how recently duplicated genes can diverge to fine-tune patterning during animal development.     

CDC-25.1 controls the rate of germline mitotic cell cycle by counteracting WEE-1.3 and by positively regulating CDK-1 in Caenorhabditis elegans

In Caenorhabditis elegans, cdc-25.1 loss-of-function mutants display a lack of germline proliferation. We found that the proliferation defect of cdc-25.1 mutants was suppressed by wee-1.3 RNAi. Further, among the seven cdk and seven cyclin homologs examined, cdk-1 and cyb-3 RNAi treatment caused the most severe germline proliferation defects in an rrf-1 mutant background, which were similar to those of the cdc-25.1 mutants. In addition, while RNAi of cyd-1 and cye-1 caused significant germline proliferation defects, RNAi of cdk-2 and cdk-4 did not. Compared with the number of germ nuclei in wee-1.3(RNAi) worms, the number in wee-1.3(RNAi);cdk-1(RNAi) and wee-1.3(RNAi);cyb-3(RNAi) worms further decreased to the level of cdk-1(RNAi) and cyb-3(RNAi) worms, respectively, indicating that cdk-1 and cyb-3 are epistatic and function downstream of cdc-25.1 and wee-1.3 in the control of the cell cycle. BrdU labeling of adult worms showed that, while 100% of the wild-type germ nuclei in the mitotic region incorporated BrdU when labeled for more than 12 h at 20°C, a small fraction of the cdc-25.1 mutant germ nuclei failed to incorporate BrdU even when labeled for 68 h. These results indicate that CDC-25.1 is required for maintaining proper rate of germline mitotic cell cycle. We propose that CDC-25.1 regulates the rate of germline mitotic cell cycle by counteracting WEE-1.3 and by positively controlling CDK-1, which forms a complex primarily with CYB-3, but also possibly with CYD-1 and CYE-1.     

synMuv B proteins antagonize germline fate in the intestine and ensure C. elegans survival

Previous studies demonstrated that a subset of synMuv B mutants ectopically misexpress germline-specific P-granule proteins in their somatic cells, suggesting a failure to properly orchestrate a soma/germline fate decision. Surprisingly, this fate confusion does not affect viability at low to ambient temperatures. Here, we show that, when grown at high temperature, a majority of synMuv B mutants irreversibly arrest at the L1 stage. High temperature arrest (HTA) is accompanied by upregulation of many genes characteristic of germ line, including genes encoding components of the synaptonemal complex and other meiosis proteins. HTA is suppressed by loss of global regulators of germline chromatin, including MES-4, MRG-1, ISW-1 and the MES-2/3/6 complex, revealing that arrest is caused by somatic cells possessing a germline-like chromatin state. Germline genes are preferentially misregulated in the intestine, and necessity and sufficiency tests demonstrate that the intestine is the tissue responsible for HTA. We propose that synMuv B mutants fail to erase or antagonize an inherited germline chromatin state in somatic cells during embryonic and early larval development. As a consequence, somatic cells gain a germline program of gene expression in addition to their somatic program, leading to a mixed fate. Somatic expression of germline genes is enhanced at elevated temperature, leading to developmentally compromised somatic cells and arrest of newly hatched larvae.     

Golgi-associated RhoBTB3 targets Cyclin E for ubiquitylation and promotes cell cycle progression

Cyclin E regulates the cell cycle transition from G1 to S phase and is degraded before entry into G2 phase. Here we show that RhoBTB3, a Golgi-associated, Rho-related ATPase, regulates the S/G2 transition of the cell cycle by targeting Cyclin E for ubiquitylation. Depletion of RhoBTB3 arrested cells in S phase, triggered Golgi fragmentation, and elevated Cyclin E levels. On the Golgi, RhoBTB3 bound Cyclin E as part of a Cullin3 (CUL3)-dependent RING–E3 ubiquitin ligase complex comprised of RhoBTB3, CUL3, and RBX1. Golgi association of this complex was required for its ability to catalyze Cyclin E ubiquitylation and allow normal cell cycle progression. These experiments reveal a novel role for a Ras superfamily member in catalyzing Cyclin E turnover during S phase, as well as an unexpected, essential role for the Golgi as a ubiquitylation platform for cell cycle control.     

C. elegans DAF-18/PTEN mediates nutrient-dependent arrest of cell cycle and growth in the germline

The molecular pathways that link nutritional cues to developmental programs are poorly understood. Caenorhabditis elegans hatchlings arrest in a dormant state termed "L1 diapause" until food is supplied. However, little is known about what signal transduction pathways mediate nutritional status to control arrest and initiation of postembryonic development. We report that C. elegans embryonic germline precursors undergo G2 arrest with condensed chromosomes and remain arrested throughout L1 diapause. Loss of the DAF-18/PTEN tumor suppressor bypasses this arrest, resulting in inappropriate germline growth dependent on the AGE-1/PI-3 and AKT-1/PKB kinases. DAF-18 also regulates an insulin/IGF-like pathway essential for longevity and dauer larva formation. However, DAF-16/FoxO, which is repressed by this pathway, is not required for germline arrest in L1 diapause. Thus, these findings indicate that quiescence of germline development during L1 diapause is not a passive consequence of nutrient deprivation, but rather is actively maintained by DAF-18 through a pathway distinct from that which regulates longevity and dauer formation.     

The CRL2LRR-1 ubiquitin ligase regulates cell cycle progression during C. elegans development

The molecular mechanisms that regulate cell cycle progression in a developmental context are poorly understood. Here, we show that the leucine-rich repeat protein LRR-1 promotes cell cycle progression during C. elegans development, both in the germ line and in the early embryo. Our results indicate that LRR-1 acts as a nuclear substrate-recognition subunit of a Cullin 2-RING E3 ligase complex (CRL2(LRR-1)), which ensures DNA replication integrity. LRR-1 contains a typical BC/Cul-2 box and binds CRL2 components in vitro and in vivo in a BC/Cul-2 box-dependent manner. Loss of lrr-1 function causes cell cycle arrest in the mitotic region of the germ line, resulting in sterility due to the depletion of germ cells. Inactivation of the DNA replication checkpoint signaling components ATL-1 and CHK-1 suppresses this cell cycle arrest and, remarkably, restores lrr-1 mutant fertility. Likewise, in the early embryo, loss of lrr-1 function induces CHK-1 phosphorylation and a severe cell cycle delay in P lineage division, causing embryonic lethality. Checkpoint activation is not constitutive in lrr-1 mutants but is induced by DNA damage, which may arise due to re-replication of some regions of the genome as evidenced by the accumulation of single-stranded DNA-replication protein A (ssDNA-RPA-1) nuclear foci and the increase in germ cell ploidy in lrr-1 and lrr-1; atl-1 double mutants, respectively. Collectively, these observations highlight a crucial function of the CRL2(LRR-1) complex in genome stability via maintenance of DNA replication integrity during C. elegans development.     

Caenorhabditis elegans atx-2 promotes germline proliferation and the oocyte fate

In the Caenorhabditis elegans germline, proliferation is induced by Notch-type signaling. Entry of germ cells into meiosis is triggered by activity of the GLD-1 and GLD-2 pathways, which function redundantly to promote meiosis and/or inhibit proliferation. Activation of the germline Notch-type receptor, GLP-1, ultimately inhibits the activities of the GLD-1 and GLD-2 pathways. We previously identified several ego (enhancer of glp-1) genes that promote germline proliferation and interact genetically with the GLP-1 signaling pathway. Here, we show that atx-2 is an ego gene. Our data suggest that ATX-2 is not a positive regulator of the GLP-1 signaling pathway and GLP-1 signaling is not the sole positive regulator of ATX-2 activity. Moreover, our data indicate that GLP-1 must have an additional function, which may be to repress activity of a third meiotic entry pathway that would work in parallel with the GLD-1 and GLD-2 pathways. In addition to its role in proliferation, ATX-2 acts downstream of FOG-2 to promote the female germline fate.     

Cyclin D1 promotes mitogen-independent cell cycle progression in hepatocytes.

Cyclin D1 is widely believed to regulate progression through G1 phase of the cell cycle, and previous studies have shown that this protein is induced during hepatocyte proliferation in culture and in vivo. In this study, the role of cyclin D1 in the cell cycle of primary rat hepatocytes was further examined. Following epidermal growth factor stimulation, cyclin D1 was upregulated at time points corresponding to the mitogen restriction point, and this was associated with enhanced cyclin D1-associated kinase activity. To test whether cyclin D1 expression was sufficient to promote mitogen-independent progression through the G1-S transition, we constructed a replication-defective adenovirus that overexpressed human cyclin D1. Transfection with the cyclin D1 vector but not a control vector resulted in hepatocyte DNA synthesis in the absence of growth factor that was similar to that seen in mitogen-treated cells. Furthermore, cyclin D1 transfection led to activation of downstream biochemical events, including cyclin A and proliferating cell nuclear antigen expression and cyclin E- and cyclin A-associated kinase activation. These results suggest that cyclin D1 expression is sufficient to promote progression of hepatocytes through the G1 restriction point.     

The C. elegans SoxC protein SEM-2 opposes differentiation factors to promote a proliferative blast cell fate in the postembryonic mesoderm

The proper development of multicellular organisms requires precise regulation and coordination of cell fate specification, cell proliferation and differentiation. Abnormal regulation and coordination of these processes could lead to disease, including cancer. We have examined the function of the sole C. elegans SoxC protein, SEM-2, in the M lineage, which produces the postembryonic mesoderm. We found that SEM-2/SoxC is both necessary and sufficient to promote a proliferating blast cell fate, the sex myoblast fate, over a differentiated striated bodywall muscle fate. A number of factors control the specific expression of sem-2 in the sex myoblast precursors and their descendants. This includes direct control of sem-2 expression by a Hox-PBC complex. The crucial nature of the HOX/PBC factors in directly enhancing expression of this proliferative factor in the C. elegans M lineage suggests a possible more general link between Hox-PBC factors and SoxC proteins in regulating cell proliferation.     

Cyclin E2, the cycle continues

The eukaryotic cell cycle is regulated by a family of serine/threonine protein kinases known as cyclin-dependent kinases (CDKs). The activation of a CDK is dependent on its association with a cyclin regulatory subunit. The formation of distinct cyclin-CDK complexes controls the progression through the first gap phase (G(1)) and initiation of DNA synthesis (S phase). These complexes are in turn regulated by protein phosphorylation and cyclin-dependent kinase inhibitors (CKIs). Cyclin E2 has emerged as the second member of the E-type cyclin family. Cyclin E2-associated kinase activity is regulated in a cell cycle dependent manner with peak activity at the G(1) to S transition. Ectopic expression of cyclin E2 in human cells accelerates G(1), suggesting that cyclin E2 is rate limiting for G(1) progression. Although the pattern and level of cyclin E2 expression in some primary tumor and normal tissue RNAs are distinct from cyclin E1, both E-type cyclins appear to have inherent functional redundancies. This functional redundancy has facilitated the rapid characterization of cyclin E2 and uncovered unique features associated with each E-type cyclin.     

Zyg-11 and cul-2 regulate progression through meiosis II and polarity establishment in C. elegans

The mechanisms that ensure coupling between meiotic cell cycle progression and subsequent developmental events, including specification of embryonic axes, are poorly understood. Here, we establish that zyg-11 and the cullin cul-2 promote the metaphase-to-anaphase transition and M phase exit at meiosis II in Caenorhabditis elegans. Our results indicate that ZYG-11 acts with a CUL-2-based E3 ligase that is essential at meiosis II and that functions redundantly with the anaphase-promoting complex/cyclosome at meiosis I. Our data also indicate that delayed M phase exit in zyg-11(RNAi) embryos is due to accumulation of the B type cyclin CYB-3. We demonstrate that PAR proteins and P granules become polarized in an inverted manner during the meiosis II delay resulting from zyg-11 or cul-2 inactivation, and that zyg-11 and cul-2 can regulate polarity establishment independently of a role in cell cycle progression. Furthermore, we find that microtubules appear dispensable for ectopic polarity during the meiosis II delay in zyg-11(RNAi) embryos, as well as for AP polarity during the first mitotic cell cycle in wild-type embryos. Our findings suggest a model in which a CUL-2-based E3 ligase promotes cell cycle progression and prevents polarity establishment during meiosis II, and in which the centrosome acts as a cue to polarize the embryo along the AP axis after exit from the meiotic cell cycle.     

Cyclin E and CDK2 repress the terminal differentiation of quiescent cells after asymmetric division in C. elegans

Coordination between cell proliferation and differentiation is important in normal development and oncogenesis. These processes usually have an antagonistic relationship, in that differentiation is blocked in proliferative cells, and terminally differentiated cells do not divide. In some instances, cyclins, cyclin-dependent kinases (CDKs) and their inhibitors (CKIs) play important roles in this antagonistic regulation. However, it is unknown whether CKIs and cyclin/CDKs regulate the uncommitted state in quiescent cells where CDK activities are likely to be low. Here, we show in C. elegans that cye-1/cyclin E and cdk-2/CDK2 repress terminal differentiation in quiescent cells. In cye-1 mutants and cdk-2(RNAi) animals, after asymmetric division, certain quiescent cells adopted their sister cells' phenotype and differentiated at some frequency. In contrast, in cki-1(RNAi) animals, these cells underwent extra divisions, while, in cki-1(RNAi); cdk-2(RNAi) or cki-1(RNAi); cye-1 animals, they remained quiescent or differentiated. Therefore, in wild-type animals, CKI-1/CKI in these cells maintained quiescence by inhibiting CYE-1/CDK-2, while sufficient CYE-1/CDK-2 remained to repress the terminal differentiation. The difference between sister cells is regulated by the Wnt/MAP kinase pathway, which causes asymmetric expression of CYE-1 and CKI-1. Our results suggest that the balance between the levels of CKI and cyclin E determines three distinct cell states: terminally differentiated, quiescent and uncommitted, and proliferating.     

The C. elegans Mi-2 chromatin-remodelling proteins function in vulval cell fate determination

The Mi-2 protein is the central component of the recently isolated NuRD nucleosome remodelling and histone deacetylase complex. Although the NuRD complex has been the subject of extensive biochemical analyses, little is known about its biological function. Here we show that the two C. elegans Mi-2 homologues, LET-418 and CHD-3, play essential roles during development. The two proteins possess both shared and unique functions during vulval cell fate determination, including antagonism of the Ras signalling pathway required for vulval cell fate induction and the proper execution of the 2 degrees cell fate of vulval precursor cells, a process under the control of LIN-12 Notch signalling.     

Cyclin E and Cdk2 control GLD-1, the mitosis/meiosis decision, and germline stem cells in Caenorhabditis elegans

Coordination of the cell cycle with developmental events is crucial for generation of tissues during development and their maintenance in adults. Defects in that coordination can shift the balance of cell fates with devastating clinical effects. Yet our understanding of the molecular mechanisms integrating core cell cycle regulators with developmental regulators remains in its infancy. This work focuses on the interplay between cell cycle and developmental regulators in the Caenorhabditis elegans germline. Key developmental regulators control germline stem cells (GSCs) to self-renew or begin differentiation: FBF RNA-binding proteins promote self-renewal, while GLD RNA regulatory proteins promote meiotic entry. We first discovered that many but not all germ cells switch from the mitotic into the meiotic cell cycle after RNAi depletion of CYE-1 (C. elegans cyclin E) or CDK-2 (C. elegans Cdk2) in wild-type adults. Therefore, CYE-1/CDK-2 influences the mitosis/meiosis balance. We next found that GLD-1 is expressed ectopically in GSCs after CYE-1 or CDK-2 depletion and that GLD-1 removal can rescue cye-1/cdk-2 defects. Therefore, GLD-1 is crucial for the CYE-1/CDK-2 mitosis/meiosis control. Indeed, GLD-1 appears to be a direct substrate of CYE-1/CDK-2: GLD-1 is a phosphoprotein; CYE-1/CDK-2 regulates its phosphorylation in vivo; and human cyclin E/Cdk2 phosphorylates GLD-1 in vitro. Transgenic GLD-1(AAA) harbors alanine substitutions at three consensus CDK phosphorylation sites. GLD-1(AAA) is expressed ectopically in GSCs, and GLD-1(AAA) transgenic germlines have a smaller than normal mitotic zone. Together these findings forge a regulatory link between CYE-1/CDK-2 and GLD-1. Finally, we find that CYE-1/CDK-2 works with FBF-1 to maintain GSCs and prevent their meiotic entry, at least in part, by lowering GLD-1 abundance. Therefore, CYE-1/CDK-2 emerges as a critical regulator of stem cell maintenance. We suggest that cyclin E and Cdk-2 may be used broadly to control developmental regulators.