Prashant Raghavan et al. (2003) International Worm Meeting "CeUBC9 Is Required for the Repression of MEP-1 function by PIE-1"

Byung-Jae Park, Seung-il Kim, Prashant Raghavan, Tae Ho Shin, Jungsoon Lee

[International Worm Meeting, 2003]

During embryogenesis, the nuclear/cytoplasmic protein PIE-1 promotes proper germline development in part by inducing global transcriptional silencing and by blocking somatic differentiation in the germline. Recent evidence suggests that another important function of PIE-1 is to collaborate with MES proteins and to maintain unique chromatin organization essential for germline development. According to this model, PIE-1 protects germline-specific chromatin state by inhibiting a putative chromatin remodeling complex, the MEP-1 complex, which functions to establish soma-specific chromatin organization. We are investigating a potential importance of CeUBC9, the C. elegans homologue of the sumoylation enzyme UBC9, for the repression of the MEP-1 complex.CeUBC9 is essential for the differentiation suppression function of PIE-1, as CeUBC9(RNAi) causes cell fate transformation defects similar to those caused by the pie-1 mutations. In the CeUBC9(RNAi) embryos, the PIE-1 protein aggregates at 20 to 30 nucleoplasmic foci in the germline nuclei, suggesting that CeUBC9 is required for proper subnuclear localization of PIE-1.Several lines of evidence suggest that the effect of CeUBC9 is mediated through covalent modification of PIE-1 with a small ubiquitin-like peptide, called CeSMT3. In vitro, CeUBC9 binds PIE-1 and conjugates CeSMT3 to Lys68 of PIE-1. A mutant PIE-1 protein containing the Lys68-to-Arg substitution mislocalizes to nuclear speckles in the embryo and is unable to rescue the pie-1 mutant embryo. Furthermore, a C. elegans homologue of PIAS SUMO ligases, CePIAS1, binds PIE-1 and is required for proper PIE-1 localization and function.In order to test whether CeUBC9 ,CeSMT3 and CePIAS1 are also important for the MEP-1 repression function of PIE-1, we impaired endogenous CeSMT3-modificaiton activities by RNAi and by overexpression of a modified CeSMT3 peptide that acts as a dominant interfering activity. Expression of PIE-1 in somatic cells of the lin-15A animal causes ectopic repression of MEP-1 activity, resulting in a characteristic synMuv (synthetic multivulva) phenotype. We found that the penetrance of this phenotype is significantly reduced by RNAi targeted to CeUBC9 ,CeSMT3 or CePIAS1 and also by the expression of the dominant interfering peptide. These and additional data raise the possibility that the binding or subsequent inhibitory effects of PIE-1 on the MEP-1 complex may be regulated by CeSMT3-modification.

Prashant Raghavan et al. (2005) International Worm Meeting "Does the X chromosome play a special role in development?"

Prashant Raghavan, Tae Ho Shin

[International Worm Meeting, 2005]

Previous studies by us and others implicate the LET-418/HDA-1/MEP-1 complex (the LET-418 complex) in multiple developmental processes, including the development of the adult germ cells and gonad, the repression of vulval fate as part of the synMuvB pathway and the repression of a germline-like fate in the somatic cells. We are interested in identifying the specific targets of the LET-418 complex in these processes. Data from the other labs suggests that the X chromosome is partly responsible for the distinction between the germ and somatic cells; the X is specifically silenced in the adult germ cells, and this repression requires the MES group of chromatin regulators. MES-4 is excluded from the X chromosome in a manner dependent on other MES activities, and the mes mutant phenotype (germ cell death) can be exacerbated by increasing the dose of the X. In mep-1 and let-418 mutants,many somatic cells morphologically resemble the germ cells and ectopically express germ granule components. Since this phenotype can be strongly suppressed by removing the MES activities,it raises the interesting possibility that the X may be the common target of the MES proteins and the LET-418 complex. Could it be that the LET-418 complex generally targets the X chromosome for all its functions, including the repression of the vulval fate? If so, we should suspect that the MES proteins regulate vulva induction in a positive way and that other components of the synMuvB pathway antagonize the MES function, presumably on the X, and by doing so repress the germline-like fate. The answers to these questions appear to beyes. Ectopically expressed PIE-1,a germline-specific protein, can partially inhibit the LET-418 complex and cause ectopic vulvae formation. If MES activities are removed in this context, PIE-1 can no longer induce the synMuvB defect. Also, most synMuvB mutants we examined resemble the mep-1 and let-418 mutants in that they derepress germline-specific genes in somatic cells. To further examine the involvement of the X chromosome in vulva repression, we disrupted the dosage compensation machinery and found that the derepression of the X can reduce the synMuvB defect. Furthermore, the duplication of a small region of the left arm of the X is sufficient to cause the suppression of ectopic vulvae in the PIE-1-expressing animals. These data could be taken to mean that the LET-418 complex regulates the vulva potential through a small number of X-linked genes.

Prashant Raghavan et al. (2004) East Coast Worm Meeting "Components of the dosage compensation machinery antagonize the MEP-1 complex function"

Prashant Raghavan, Tae Ho Shin, Byun-Jae Park, Seun-il Kim, Jungsoon Lee, Keunhee Park

[East Coast Worm Meeting, 2004]

We previously proposed that the germline-specific PIE-1 protein inhibits the activity of the MEP-1 chromatin remodeling complex, which promotes somatic differentiation, and thus prevents the implementation of soma-specific, pro-differentiation chromatin structures, in place of the default germline-specific chromatin organization. In order to investigate this model further, we are examining the potential involvement of known and presumed chromatin regulators in the MEP-1-dependent developmental processes. MEP-1 and its binding partners LET-418 and HDA-1 are known to participate in vulva specification as a component of the genetically redundant process known as the synthetic multivulva B (synMuv B) pathway. Loss-of-function mutations in the mep-1, let-418 or hda-1 gene cause ectopic vulval specification in synMuv A mutant backgrounds. Similarly, the forced expression of PIE-1 in the somatic cells mimics the effect of synMuv B mutations. Using this phenotype as readout, we found that reducing the activity of the dosage compensation machinery increases the repression of the primary vulval fate in the vulva equivalent cells. For example, dpy-30(RNAi) reduces the occurrence of ectopic vulvae from ~85% to ~10% in animals ectopically expressing PIE-1. Similarly, RNAi directed against dpy-27 , dpy-28 and sdc-2 result in significant suppression of the synMuv phenotype caused by ectopic PIE-1 expression and also by mep-1(RNAi) , though for unknown reasons, the suppression in the latter case is much less dramatic. In addition, the dosage compensation machinery appears to be important for the derepression of the vulval fate when other synMuv B genes such as lin-35 and lin-36 are inactivated. Previous studies indicate that the MEP-1 complex antagonizes the function of MES proteins, a group of Polycomb group-related chromatin regulators, and that the effect of MES proteins is mediated, at least partly, by the repression of the X chromosome in the germline nuclei. One possible interpretation of these results is that activity levels of the X chromosome directly influence the developmental decision between the germline and somatic fates. The analogous antagonistic relationship between the MEP-1 complex and the X repression mechanism in the somatic cells raise an intriguing possibility that the major function of the MEP-1 complex may be to regulate the X chromosome, which in turn is consistent with the model that the X inactivity corresponds to the germline fate.

Li, Zao et al. (2013) International Worm Meeting "Necrotic Cells Share a Similar Mechanism with Apoptotic Cells in being Recognized by Engulfing Cells in C. elegans."

Venegas, Victor, Li, Zao, Raghavan, Prashant, Nakanishi, Yoshinobu, Zhou, Zheng

[International Worm Meeting, 2013]

Necrosis is the premature death of cells caused by external factors, such as acute cell injury or trauma. In contrast to apoptosis, the programmed cell death, necrosis is caspase-independent and necrotic cells are morphologically distinct from apoptotic cells. Although these two categories of cell deaths are genetically different, it has been suggested that necrotic cell corpses are actively removed by the same set of genes required in apoptotic cell removal indicating they might share a similar clearance mechanism. In the nematode Caenorhabditis elegans, gain-of-function mutations in certain ion channel subunits result in necrotic-like cell death of six touch neurons. Necrotic touch neurons are subsequently engulfed and degraded inside engulfing cells. However, it is unclear how necrotic cells are recognized by phagocytes. Phosphatidylserine (PS) is an important apoptotic cell surface signal that attracts engulfing cells. Using ectopically expressed MFG-E8, a high-affinity PS-binding protein, we observed that PS was actively present on the surface of necrotic touch neurons. In addition, phagocytic receptor CED-1, whose function is needed for the efficient clearance of apoptotic cells, also acts as a phagocytic receptor for necrotic cells. We demonstrate that necrotic cells, like apoptotic cells, rely on cell-surface PS as an "eat me" signal to attract CED-1. We further found CED-7, the worm homolog of mouse ABC1 transporter, was necessary for PS-exposure on necrotic cell surfaces. Moreover, we discovered another protein contributing to the presentation of PS on necrotic cell surfaces and acting in a parallel pathway to CED-7. Our findings suggest between two distinct cell deaths, a conserved mechanism may exist for the recognition of cell corpse.

Robida-Stubbs, Stacey et al. (2011) International Worm Meeting "TOR signaling and rapamycin modulate aging by regulating SKN-1- and DAF-16-driven transcription."

Mizunuma, Masaki, Blackwell, TK, Robida-Stubbs, Stacey, Glover-Cutter, Kira, Raghavan, Prashant, Operana, Theresa

[International Worm Meeting, 2011]

The TOR (target of rapamycin) kinase is central to growth regulation, and influences aging in diverse species. In the context of the TORC1 complex, TOR promotes protein synthesis in response to nutrient and growth signals. Less is understood about the related TOR-containing complex TORC2. TORC1 signaling has been implicated in the effects of dietary restriction on longevity, and inhibition of TOR through genetics or treatment with rapamycin, a clinically used TORC1 inhibitor, extends lifespan from yeast to mice. It is a central challenge in the aging and growth signaling fields to understand how TOR influences longevity and resistance to stresses. Previous C. elegans studies showed that reduction of TOR kinase activity extends lifespan independently of DAF-16/FOXO, suggesting that TOR influences aging through different mechanisms from the insulin/IGF-1 signaling (IIS) and germline stem cell (GSC) pathways. We have determined that specific inhibition of TORC1 increases longevity and stress resistance by acting through both the SKN-1/Nrf and DAF-16 transcription factors. Adulthood knockdown of individual TORC1 complex genes increases longevity dependent upon both skn-1 and daf-16. This longevity increase does not involve the GSC pathway, which we show also inhibits SKN-1 as well as DAF-16. In contrast to specific TORC1 inhibition, rapamycin treatment confers skn-1-dependent, daf-16-independent longevity. This appears to involve inhibition of both TORC1 and TORC2, because we find that TORC2 inhibition increases lifespan independently of daf-16. The daf-16-independence of TORC2-associated longevity can explain the previously observed daf-16-independence of the effects of TOR kinase inhibition on aging. Importantly, both SKN-1 and DAF-16 upregulate transcription of protective downstream target genes in response to either TORC1 inhibition or rapamycin treatment. We conclude that the TOR, IIS, and GSC pathways each influence aging by acting through distinct mechanisms to inhibit gene activation by SKN-1 and DAF-16. The data have important implications for understanding effects of TOR-based therapies, and relationships between nutrient availability, growth regulation, and aging.

Steinbaugh, Michael J. et al. (2013) International Worm Meeting "Regulation of SKN-1/Nrf by the germline longevity pathway."

Robida-Stubbs, Stacey, Operana, Theresa, Steinbaugh, Michael J., Raghavan, Prashant, Narasimhan, Sri Devi, Blackwell, T. Keith

[International Worm Meeting, 2013]

In C. elegans, a reduction in germline stem cell (GSC) number increases lifespan and stress resistance through a longevity pathway that seems to be distinct from those related to nutrient sensing and metabolism (e.g. insulin/IGF-1 signaling, the TOR pathway, mitochondrial function, and dietary restriction). The GSC pathway is a tissue non-autonomous regulator of longevity that is known to involve the transcription factors DAF-16, DAF-12, and NHR-80 in the intestine. We recently determined that the transcription factor SKN-1 (Nrf in mammals) is also regulated by this pathway. The germline can be removed through laser ablation or genetic disruption of the Notch ortholog GLP-1. Using skn-1(zu135), skn-1 RNAi, and temperature-sensitive glp-1(bn18) mutants, we determined that skn-1 is required for lifespan extension associated with GSC removal. In contrast to DAF-16, SKN-1 is also required for the associated increase in oxidative stress resistance (e.g. sodium arsenite, tert-butylhydroperoxide). We found that SKN-1 accumulates in intestinal nuclei when GSC number is reduced, and that this is partially dependent upon KRI-1, an ankryin repeat protein implicated in regulation of DAF-16 by this pathway. Using qRT-PCR and analysis of transcriptional reporters, we determined that multiple SKN-1 target genes (e.g. gst-4, gcs-1, nit-1, and F20D6.11) are transcriptionally upregulated in germline-deficient animals in a SKN-1-dependent manner. This distinctive longevity pathway provides an exciting example of environmental sensing and signaling from the reproductive stem cell niche. Current efforts are aimed at understanding how the GSC pathway regulates SKN-1, and identifying processes controlled by SKN-1 in this context.

Li, Zao et al. (2015) International Worm Meeting "Necrotic Cells Actively Attract Phagocytes through the Collaborative Action of Two Distinct PS-exposure Mechanisms."

Li, Zao, Nagaoka, Yuji, Zhou, Zheng, Morino, Eri, Venegas, Victor, Audhya, Anjon, Raghavan, Prashant, Nakanishi, Yoshinobu

[International Worm Meeting, 2015]

Necrosis is the premature death of cells caused by cell injury and genetic alternations and is closely related to human diseases including neuron degeneration, stroke, and cancer. Like apoptotic cells, necrotic cell also need to be swiftly removed from the organisms to prevent inflammatory and autoimmune responses. However, unlike apoptosis, the programmed cell death, necrosis is a caspase-independent cellular event and necrotic cells show morphologically distinct features from the button-like apoptotic cells. In the nematode Caenorhabditis elegans, gain-of-function mutations in certain ion channel subunits result in necrotic-like cell death of six touch neurons. Necrotic touch neurons are subsequently engulfed and degraded inside engulfing cells. However, it is unclear of how necrotic cells are recognized by phagocytes. Phosphatidylserine (PS) is an important apoptotic cell surface signal that attracts engulfing cells. Using ectopically expressed MFG-E8, a high-affinity PS-binding protein, we observed that PS was actively present on the surface of necrotic touch neurons. In addition, we found that CED-1, the phagocytic receptor could also recognize necrotic cells by associating with PS. We further found CED-7, the worm homolog of mouse ABC1 transporter, was necessary for PS-exposure on necrotic cell surfaces. In addition to CED-7, ANOH-1, the C. elegans homolog of the mammalian Ca2+-dependent phospholipid scramblase TMEM16F, plays an independent role in promoting PS exposure on necrotic cells. The combined activities from CED-7 and ANOH-1 ensure sufficient exposure of PS on necrotic cells to attract their phagocytes. Our work indicates that cells killed by different mechanisms (necrosis or apoptosis) expose a common "eat me" signal to attract their phagocytic receptor(s); furthermore, unlike what was previously believed, necrotic cells actively present PS on their outer surfaces through two distinct molecular mechanisms rather than leaking out PS passively.

Jungsoon Lee et al. (2005) International Worm Meeting "A him-8 mutation suppresses the PIE-1-induced synMuv defect"

Jungsoon Lee, Tae Ho Shin

[International Worm Meeting, 2005]

PIE-1, a nuclear protein enriched in germline, binds and inhibits a NuRD-like complex, consisting of at least three proteins, the zinc-finger protein MEP-1, the Mi-2 homologue LET-418 and the histone deacetylase HDA-1. This putative chromatin remodeling complex is involved in multiple developmental processes, including the maintenance of somatic potential during larval development and the repression of the vulval fate as a part of the synthetic multivulva (synMuv) B pathway. Animals ectopically expressing PIE-1 in the somatic cells mimic loss-of-function mutations in the mep-1 and let-418 and show highly penetrant multivulva phenotype in the presence of synMuv A mutation, consistent with its proposed role in inhibiting the MEP-1/LET-418/HDA-1 activity. In order to understand how PIE-1 represses the MEP-1/LET-418/HDA-1 complex, we are carrying out a genetic screen for suppressors of the synMuv phenotype induced by the ectopic expression of PIE-1. In the course of this study, we found that him-8 mutations strongly suppress the PIE-1-induced synMuv phenotype and that this suppression occurs without apparent changes in the level or the localization of the PIE-1 protein. The him-8 alleles that suppress the PIE-1-induced synMuv phenotype include e1489 and mn253. All other him mutations we have tested (i.e. those in the him-3, him-4 and him-5 genes) fail to do so. him-8(e1489) does not suppress the synMuv defect caused by mep-1(RNAi), raising the possibility that him-8 may mediate the repressive function of PIE-1. him-8 mutants frequently display (a) the failure to form stable synapsis between the X homologues during meiosis and (b) meiotic non-disjunction of the X chromosome. Recent studies indicate that this unsynapsed X chromosome accumulates high levels of methyl-Lys-9 histone 3, raising the possibility that a loss of proper synapsis in him-8 mutants causes the repression of an X-linked gene(s), which in turn leads to the suppression of the PIE-1 ectopic expression phenotype. However, the presence of unsynapsed X chromosomes due either to other him mutations or to the deletion of the X pairing center do not appreciably suppress the PIE-1-induced phenotype. Our data are consistent with the possibility that alterations in the level of X-linked transcription modulate the synMuv defect caused by ectopic expression of PIE-1 (see also Abstract by Raghavan et al.), but suggest that the him-8 mutation may affect the X-linked transcription in a more complex manner.