Sin, Olga et al. (2015) International Worm Meeting "Identification of MOAG-2/LIR-3 as a regulator of protein aggregation."

Guryev, Victor, Reinke, Valerie, Sin, Olga, Willinge Prins, Romeo, Wang, Hai Hui, Martineau, Celine, Mata Cabana, Alejandro, Seinstra, Renee, Nollen, Ellen, Kudron, Michelle, de Jong, Tristan

[International Worm Meeting, 2015]

Aging-related protein aggregation is one of the hallmarks of neurodegenerative disorders such as Alzheimer, Parkinson and polyglutamine diseases. The cellular processes that drive protein aggregation in these diseases have remained largely unknown. Using a genetic screen in a C. elegans model for polyglutamine aggregation, we here identified modifier of aggregation 2 (moag-2). Mutation or partial deletion in this gene decreased the number of aggregates in our model. Additionally, moag-2/lir-3 mutants reduce the amount of SDS-resistant aggregates without changing total polyglutamine expression levels. We discovered that the causative gene of moag-2 is lir-3. moag-2/lir-3 encodes a protein with a predicted nuclear localization signal and two non-canonical C2H2 domains, which are homologous to the transcription factor for RNA polymerase III. The molecular function of MOAG-2/LIR-3 is unknown. Chromatin immunoprecipitation followed by sequencing data revealed that MOAG-2/LIR-3 is preferentially bound to promoter regions of non-coding RNA, namely tRNAs and snoRNAs. The consensus DNA sequence to which MOAG-2/LIR-3 is bound to corresponds to Box A and Box B motifs, which are recognized by the RNA Polymerase III machinery to initiate tRNA and snoRNA transcription. In fact, MOAG-2/LIR-3 is positioned in the same binding positions as the RNA Pol III complex, further suggesting a role for LIR-3 in non-coding RNA transcription. We are currently performing total RNA sequencing to reveal whether the aggregation phenotype displayed by the MOAG-2/LIR-3 mutants can be due to imbalanced RNA metabolism. By unraveling the role of moag-2/lir-3 we hope to get insight into how cells cope with aggregation-prone proteins.

Ohnishi, Y. et al. (2017) International Worm Meeting "Analyzing of comparative transcriptomics, SIN-3/HDAC complex is related to epigenome in early embryogenesis of Caenorhabditis elegans."

Ito, M., Ohnishi, Y., Kojima, H., Nagano, T.

[International Worm Meeting, 2017]

Epigenetic regulation by histone acetylation, methylation, and long non-coding genes is proven to have crucial roles during embryogenesis. Since DNA is wrapped around histones forming a specific chromatin structure, several histone modifications provide tight regulation of transcription by changing this arrangement. Different histone modifications compete at the same residues and subsequently become the markers that facilitate induction of other histone modifications. Histone acetylation is catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs); the latter regulate various gene groups and are known to form different complexes centered on transcription corepressors, SIN-3, NuRD, and CoREST. To elucidate the functions of SIN-3/HDAC complex in early embryogenesis, we analyzed SIN-3 encoded by the gene sin-3. Interestingly, embryonic lethality was detected in more than 20% of the sin-3 (tm1276) mutants. qRT-PCR was then used to measure sin-3 mRNA levels at three developmental stages of the N2 strain, including early embryo, larva first (L1), and adult stage. Our results showed that mRNA levels of sin-3 in early embryo stage were higher than that of in L1 stage and adult stage (7.8-fold and 6.5-fold, respectively). Comparative transcriptomics in early embryo stage of the N2 strain and sin-3(tm1276) mutant was also performed. Many essential genes in embryogenesis including polarity mediators (mex-1,mex-3,mex-5,mex-6,,pos-1, spn-4) and cell fate regulators (apx-1,mes-1,mom-1,nos-2,pal-1,pie-1,skn-1) were found to be transcriptionally upregulated by SIN-3. These results suggest that SIN-3 has essential roles in regulation of epigenome in embryogenesis and cell fate determination of C. elegans. Similar to SIN-3/HDAC complex, transcription corepressors NuRD and CoREST may regulate several essential genes during embryogenesis by forming a complex with HDAC. Therefore, it is our future plan to functionally identify other HDACs complexes involving NuRD and CoREST by comparative transcriptomics.

Laha, Michael et al. (2011) International Worm Meeting "Identifying host factors required for Sindbis virus replication using C. elegans."

Decker, Hugo, Bradley, Ellen, Laha, Michael, Rice, Charles, MacDonald, Margaret, Shaham, Shai

[International Worm Meeting, 2011]

Members of the Alphavirus genus infect equine and human hosts and cause symptoms such as rash, arthritis, fever, encephalitis and in some cases death. Of the Alphaviruses, Sindbis virus (SIN) is the most extensively studied and viral factors required for SIN genome replication have been characterized. While proteomic approaches have uncovered host factors that bind viral proteins, the absence of a genetic model has hindered progress in uncovering the functional roles of such factors. Although a natural C. elegans virus has not been described, a number of viruses have been reported to replicate in C. elegans, suggesting that this animal may provide a suitable setting to study host factors required for SIN replication. To study SIN replication, we generated transgenic C. elegans harboring a SIN replicon in which the structural proteins open reading frame was replaced with GFP, and whose expression is controlled by the heat-shock promoter (HSP). GFP expression from similar SIN replicons in other systems is dependent on viral replication and expression of a subgenomic viral RNA. We found that upon heat shock, transgenic animals expressed low and variable levels of GFP. Introducing the SIN transgene into rde-4 RNAi-defective mutants greatly enhanced expression as assessed by GFP and by Northern analysis, suggesting that the double-stranded RNA SIN replication intermediate is likely inactivated by the RNAi machinery. Although HIP::mCherry co-injected with the HIP::SIN transgene was expressed in all cells upon heat shock, GFP expression was primarily detected in C. elegans muscle cells. This result suggests that SIN preferentially replicates in this cell type. Similar muscle-cell replication tropism has been described for SIN in murine models, and suggests that C. elegans may indeed be an appropriate setting for uncovering host factors required for SIN replication. To uncover such host factors, we performed EMS mutagenesis and isolated 12 mutants with reduced GFP expression. We are using SNP mapping, whole genome sequencing and fosmid rescue experiments to identify the relevant genes. Once identified, genes with obvious mammalian homologs will be studied for roles in established cell culture systems. Our studies may provide insight into the complicated host-Alphavirus interplay that leads to infection and disease.

KONWAR, CHANDRIKA et al. (2021) International Worm Meeting "Apoptosis in the context of autophagy and lifespan in C. elegans"

Pandey, Renu, Sharma, Meenakshi, Saluja, Daman, KONWAR, CHANDRIKA

[International Worm Meeting, 2021]

Apoptosis is a genetically orchestrated execution of cells that acts as a toll-gate mechanism for the development, aging, and homeostasis of the cell. In order to study cellular processes and related diseases, it is essential to understand the role of eukaryotic gene expression in the advent or evasion of cell death. Caenorhabditis elegans can be used to rapidly assess the functional impact of gene mutations to understand the dynamics of gene regulation. Sin-3, a co-repressor protein that controls gene transcription by acting as a scaffold for the multiprotein Sin-3/HDAC complex, is known to be involved in multiple cellular processes. Experimental results from our laboratory showed that sin-3 mutation in C. elegans leads to an increase in ROS-mediated autophagy along with a sharp decline in life span. In addition, sin-3 deletion abrogates normal development and growth in worms, resulting in cuticle defects, diminished muscle function, more susceptibility to stress, and extensive DNA damage surmounting to cell death. Therefore, Sin-3 may play a crucial role in the modulation of autophagy and longevity by regulating apoptosis. This was studied in our laboratory by an in silico analysis of the C. elegans Sin-3 protein interactome and it was found that certain Sin-3 interactors were involved in the regulation of cell death. In addition, sin-3 mutants showed increased germline apoptosis. The major cell signaling pathways that might get upregulated in sin-3 mutation background in C. elegans are - DNA damage response (DDR), extracellular signal-regulated protein kinase (ERK), p38 mitogen-activated protein kinase (PMK), c-Jun N-terminal kinase (JNK) pathways. Our observations suggest the regulation of insulin/IGF-1 signaling (daf-16), and JNK pathway (mek-1, jnk-1) in C. elegans by Sin-3 may play a pivotal role.

Sarah SW Choy et al. (2003) International Worm Meeting "Characterization of a sin-3 like corepressor in sensory ray identity determination"

S Hong Ho, Y Ming Wong, King L Chow, Sarah SW Choy

[International Worm Meeting, 2003]

The male tail of C. elegans is a complex structure composed of nine pairs of sensory rays embedded in a cuticular fan. Among the regulatory genes that define the patterning of these organ structures, mab-21 is required for the specification of ray 6 identity. Since MAB-21 can shuttle between subcellular compartments, such relocation may be facilitated by other cellular components interacting with MAB-21. To test this hypothesis and isolate these components, a yeast two-hybrid screen using a full-length MAB-21 bait was employed to isolate the interacting partner of MAB-21. Several candidate partners were identified, among which include SIN-3, a protein capable of eliciting transcriptional repression through association with different transcription factors and histone deacetylase. We showed that ectopic expression of mab-21 by sin-3 promoter was sufficient to rescue mab-21 mutant efficiently, where anti-sense blocking of sin-3 gene and expression of dominant negative truncated SIN-3 protein induced ray phenotypes similar to that of mab-21. All these results support the biological relevance of their interaction during ray 6 identity determination. In addition, sin-3 was found to be expressed in the ray structural cells, which make the interaction of SIN-3 with MAB-21 possible during ray differentiation. The same reporter transgene also revealed that this gene was expressed from young L1 stage through adulthood in some head neurons, ventral nerve cord and tail ganglia. cis-acting regulating elements of sin-3 promoter are being defined by promoter deletion and reporter assays. Elements controlling the sin-3 expression in ray structural cell would provide insight about the overlapping spatial and temporal expression of mab-21 and sin-3 gene in the determination of ray 6 identity. Moreover, we have characterized the interaction of sin-3 product and MAB-21 using full length and truncated products with two-hybrid system and in vitro binding assay. The PAH domain present in the truncated SIN-3 isolated clone, which can interact with a number of transcription repressors, will be tested for its role in MAB-21 association. This dissection of their molecular interaction will shed light on the subcellular function of MAB-21 in neural differentiation.

Siu Wah Choy et al. (2004) East Asia Worm Meeting "Characterization of a transcriptional repressor complex in male sensory ray patterning of C. elegans"

Siu Wah Choy, King L Chow, Yau Ming Wong

[East Asia Worm Meeting, 2004]

SIN-3, a histone deacetylase corepressor associated molecule, has longed been identified as a repression mediator in transcription. Yet, few studies have evaluated its role as a developmental regulator. The isolation of C. elegans SIN-3 through yeast two-hybrid screen as an interacting partner of MAB-21 provides the first clue to this notion. Among the regulatory genes that pattern sensory ray structure, mab-21 is required for the specification of the ray 6 identity. To evaluate the developmental requirement of sin-3 , expression analysis with fluorescent reporter was examined. Result revealed that sin-3 was not turned on ubiquitously but was found only in a subset of cells. It was first detected at the L1 stage in some head neurons. Expression could be seen in the ventral nerve cord, ray structural cells from ray 1 to ray 6 and neurons in the tail and the head region in adults. Most importantly, overlapping expression of mab-21 with sin-3 in the ray 6 structural cell lends strong support to the temporal spatial requirement of these two molecules to interact during ray 6 differentiation. Furthermore, Trichostatin-A treatment blocking the histone deacetylase activity in worms resulted in a plethora of ray phenotypes, indicating that histone deacetylase is critical for ray cells identity determination. Yeast two-hybrid system was further employed to dissect this C. elegans corepressor complex components. Results suggested that there was an important link between BMP signaling pathway and transcriptional repressor in ray patterning. Moreover, novel SIN-3 associated corepressor components in C. elegans were identified through the two-hybrid screen using SIN-3 as a bait. Among several positive clones obtained, some appeared to be required for ray patterning and displayed various ray phenotypes in RNAi knockdown animals. The results suggest that these novel interacting partners are indeed required specifically for ray patterning. Their properties as well as their role in ray cell identity determination will be discussed.

S W Choy et al. (2005) International Worm Meeting "Characterization of a transcriptional repressor complex in male sensory ray patterning of C. elegans"

Y M Wong, S W Choy, K L Chow

[International Worm Meeting, 2005]

Mab21 is required in mouse embryonic turning, neural tube closure, mid-hind brain and eye differentiation. This gene is highly conserved in both vertebrate and invertebrate, and is required for the specification of the ray 6 identity and patterning of sensory rays. Characterization of this gene in C. elegans would therefore allow us to dissect critical molecular events in the organ developmental process of vertebrates. Epitasis analysis showed that mab-21 acts downstream of the BMP pathway to maintain the ray 6 features. SIN-3, a histone deacetylase associated molecule has been identified as a MAB-21 interacting partner. Mutant sin-3 animals displayed sensory ray abnormality, suggesting its biological relevance in ray differentiation. SIN-3 corepressor complex is highly conserved in vertebrate and invertebrate organisms. Using pharmacological and RNAi approaches, SIN-3 corepressor complex was found to be required in the patterning and formation of ray. Genetic interaction between mab-21 and sin-3 was demonstrated in double heterozygous and homozygous mutants. Moreover, SIN-3 interacting partners in C. elegans controlling the repression specificity were identified via a yeast two-hybrid screen. Characterization of these proteins is ongoing and their roles in ray cell identity will be discussed. In a parallel study, conservation of a role of Mab21 in eye development was observed. Our genetic characterization of a number of components relevant to eye formation suggests that although there is no analogous light sensing organ in worm, genetic components and their regulatory interaction in controlling sensory rays patterning are highly similar to those of vertebrate eye development. Therefore, delineation of the genetic pathway in the sensory ray patterning would help us to understand the developmental regulation of eye formation. In the future, we will study these corresponding homologs through genetic and molecular approaches. Results in these experiments may shed light on our understanding of disease states of sensory organ function. (This study is supported by Research Grants Council, Hong Kong.)

Pandey, Renu et al. (2017) International Worm Meeting "Mechanistic insights into SIN-3 regulated autophagy and modulation of Caenorhabditis elegans lifespan."

Sharma, Meenakshi, Saluja, Daman, Pandey, Renu

[International Worm Meeting, 2017]

Statement of Purpose: Any change in the environmental steady state, or homeostasis at the cellular and genetic levels has direct implications on fecundity and the lifespan of an organism. Reactive oxygen species (ROS) long reflected to be mere toxic spin-offs of oxidative metabolism have now been established to be essential mediators in tightly controlled signalling cascades. Understanding the part played by ROS and redox dynamics in stress response, development and aging requires concurrent evaluation in an appropriate living system and Caenorhabditis elegans becomes an obvious choice. SIN-3 (Swi-independent) first isolated in a yeast genetic screen as a negative regulator is now established to be a master transcriptional scaffolding agent and a co-repressor manifesting itself via the Histone Deacetylase Complex (HDACs). With many functions being attributed to SIN-3 from yeast to humans and the paucity of work on the model system C. elegans in this regard led us to study this protein in the model system. The objective of our study was to understand the dynamics of aging regulated through the SIN-3 network. Methods: The methodology employed to gain insights into our study includes- culture of C. elegans strains, gene expression analysis, biochemical analysis of various oxidative stress markers like catalase activity, lipid peroxidation, ATP assay, NADPH redox status and microscopy to visualise autophagic events Summary: Our results indicate a reduction in the mean lifespan of the mutant sin-3(tm1276) worms as compared to the wild type. Both the mitochondrial and overall ROS levels were found to be elevated in the mutant sin-3(tm1276) strain. The mRNA expression analysis through quantitative real time PCR showed differential regulation of ROS associated genes like sod(s), hif-1a, dct-1and hcf-1. Staining with DCFDA also exhibited higher ROS in mutant worms as compared to the control. Other hall marks of excessive ROS like lipid peroxidation and levels of other enzymatic anitoxidants were found to be significantly elevated in the mutant strain. Autophagy has long been associated with cell survival and rescue in stress. Our data demonstrates a ROS dependent increase in autophagy. Hence providing extensive insights into the sin-3 mediated regulation of lifespan .

Maine, Eleanor et al. (2009) International Worm Meeting "Regulators of meiotic silencing."

Fedotov, Alex, Xu, Xia, Maine, Eleanor, She, Xingyu

[International Worm Meeting, 2009]

Non-coding (nc) RNAs have been implicated in transcriptional gene silencing in a variety of contexts. During first meiotic prophase in many organisms, including C. elegans, unpaired chromosomes (e.g., the male X) become silenced. Meiotic silencing is hypothesized to function in maintaining genome integrity. In C. elegans, the silencing of unpaired chromatin involves enrichment of the histone modification, H3K9me2 [Kelly et al. (2002)]. We find that mutations in several proteins implicated in the biogenesis and/or function of ncRNAs disrupt the normal pattern of H3K9me2 accumulation, suggesting that it may be regulated (at least in part) via an RNA-mediated mechanism. Meiotic H3K9me2 distribution depends on activity of EGO-1 (RdRP), CSR-1 (Argonaute), EKL-1 (a Tudor domain protein), and DRH-3 (Dicer-related helicase), all components of a functional pathway defined by Rocheleau et al. (2008). H3K9me2 accumulation on the male X chromosome is severely reduced in animals lacking EGO-1 [Maine et al. (2005)]. In contrast, H3K9me2 inappropriately spreads to the autosomes in males lacking CSR-1, EKL-1, or DRH-3 function [She et al.]. H3K9me2 distribution appears normal in XX mutants but becomes abnormal if a chromosomal duplication or unpaired chromosomes (that should be targeted for H3K9me2 enrichment) is introduced. In addition to the spreading effect, the H3K9me2 level is variably reduced on unpaired chromatin in csr-1, ekl-1, and drh-3 mutants. Mutations in these four genes cause other meiotic defects, which we are characterizing in order to determine if/how they are related to the altered H3K9me2 pattern. We have identified a mutation in a fifth gene of this phenotypic class, ego(om57), that abolishes H3K9me2 enrichment on unpaired chromatin. We are now working to clone this gene. We also find that SIN-3 activity promotes H3K9me2 accumulation on unpaired chromatin in some contexts. SIN-3 is the C. elegans ortholog of Sin3, known in other systems to assemble histone deacetylase complexes and to interact with other chromatin proteins. While SIN-3 has no known functions in ncRNA-mediated processes, a human histone deacetylase called HDAC-1 has been implicated in transcriptional gene silencing [Hawkins & Morris (2008)]. We are working to distinguish between alternative models for how these factors regulate H3K9me2 accumulation. Do they participate directly in the regulation of the histone-modifying machinery or, instead, regulate the expression of genes responsible for distinguishing between paired and unpaired chromatin?

Xingyu She et al. (2008) Development & Evolution Meeting "Localized H3K9me2 Accumulation During Meiosis Appears To Be Regulated By Multiple Mechanisms"

Xingyu She, Eleanor Maine, Xia Xu

[Development & Evolution Meeting, 2008]

Meiotic Silencing of Unpaired Chromatin (MSUC) is thought to be important for maintaining a healthy and stable genome, and for preparing the genome for recombination and subsequent meiotic events (see Kelly & Aramayo, 2007). One hallmark of meiotic silencing in C. elegans is the enrichment of histone H3 lysine 9 dimethylation on unpaired chromatin regions (Kelly et al. 2002). We have previously shown that this process requires the germline specific RNA-directed RNA polymerase, EGO-1 (Maine et al., 2005). In our candidate gene survey for additional genes that affect the MSUC process, we found that the loss of csr-1, drh-3 or ekl-1 function causes ectopic H3K9me2 accumulation on paired chromatin. We observe ectopic accumulation only in meiotic nuclei that contain unpaired chromosomes/chromosomal regions, suggesting that unpaired chromatin triggers the accumulation of ectopic silencing marks. EGO-1 activity is not required for H3K9me2 accumulation in the absence of CSR-1, DRH-3, or EKL-1 function. We find that csr-1, drh-3, and ekl-1 mutants resemble ego-1 mutants in several respects. The mutations cause similar germ line developmental defects and sterility, enhance glp-1(ts), and are enhanced by him-17. Mutations in all four genes disrupt RNAi (Smardon et al., 2000; Kim et al., 2005; Duchaine et al., 2006; Yigit et al., 2006) and several other processes (Robert et al., 2005; Rocheleau et al. 2008; J. Ahringer, p.c.). We hypothesize that these proteins function together in a common pathway. In mouse, the SIN3B deacetylase complex is associated with the XY body and may function in MSUC (Costa et al. 2006). In human cell lines, the inhibition of histone deacetylase activity reduces H3K9 methylation in gene promoters (Wu et al., 2008). SIN-3/PQN-28 is the sole C. elegans SIN-3-like protein. It may recruit histone deacetylase as well as interact with other regulatory proteins (Choy et al. 2007). Our preliminary data suggest that SIN-3 is required for the enrichment of H3K9me2 on chromatin regions that are normally active (e.g., unpaired Xs in him-8 hermaphrodites) but not on chromatin regions that have low acetylation levels (e.g., the male X). Our results suggest that H3K9me2 levels may be regulated by multiple complementary mechanisms to ensure extensive accumulation on unpaired chromatin and limit accumulation on paired chromatin.