Guo, Yiqing et al. (2013) International Worm Meeting "Differential expression of germline genes in the presence/absence of H3K9me2."

Maine, Eleanor, Guo, Yiqing

[International Worm Meeting, 2013]

In C. elegans, indirect immunofluorescence experiments indicate a high level of histone H3 lysine 9 dimethylation (H3K9me2) on any unpaired/unsynapsed chromosomes during first meiotic prophase, particularly in pachytene stage nuclei (Kelly et al 2002; Bean et al 2004). In contrast, H3K9me2 marks are relatively low on paired chromosomes at this time. H3K9me2 marks are typically associated with transcriptional repression. Thus, the elevated level of H3K9me2 on unpaired chromosomes may play a role in meiotic silencing of unpaired chromosomes (MSUC). Germline H3K9me2 marks require activity of MET-2 (Bessler et al 2010), a SET domain protein that is the only C. elegans ortholog of human SETDB1 H3K9 methyltransferase (Schultz et al 2002). To begin to investigate the importance of H3K9me2 marks on unpaired chromosomes, we performed RNA-seq on mRNA isolated from him-8 vs met-2;him-8 adult hermaphrodite gonads. Our data identify genes that are expressed in the gonads and that differentially expressed in met-2(0) and met 2(+). In most cases, the differentially expressed transcripts are elevated in met-2(0), consistent with a role for H3K9me2 in limiting transcription. Although elevated transcripts are not preferentially X-linked, we observe a greater average fold-change for X-linked transcripts than for autosomal transcripts. To evaluate whether the unpaired status of the X contributes to this pattern, we are evaluating additional genotypes. As a complementary approach, we are also investigating the distribution of H3K9me2 on paired vs unpaired chromosomes using a ChIP-seq approach.

Guo, Yiqing et al. (2011) International Worm Meeting "Complementary mechanisms to specify hermaphrodite development during evolution."

Guo, Yiqing, Ellis, Ronald

[International Worm Meeting, 2011]

To learn how novel traits are created, we are studying the origin of self-fertile hermaphrodites in the nematode genus Caenorhabditis. Although most species in this group are male/female, three separate lineages have acquired the ability to make self-fertile hermaphrodites. Throughout the genus, spermatogenesis is initiated by fog-1 and fog-3, which are themselves repressed by the master transcription factor TRA-1. Thus, the activity of TRA-1 has to be limited in XX hermaphrodites to a range that allows fog-1 and fog-3 to be turned on during larval development, and turned off in adults. We have shown that C. briggsae and C. elegans use unrelated F-box proteins to direct hermaphrodite spermatogenesis. The C. briggsae SHE-1 and C. elegans FOG-2 proteins work by distinct mechanisms to control the receptor TRA-2, which regulates TRA-1 activity. But how is TRA-1 activity kept at a level where these genes can alter germ cell fates effectively? In C. elegans, the FEM proteins promote the ubiquitinylation and degradation of TRA-1, and are required for spermatogenesis in both sexes. In C. briggsae, however, mutations in the fem genes do not block spermatogenesis (Hill et al. 2006), except in special genetic backgrounds. We found that trr-1 and other components of the Tip60 Histone Acetyl Transferase complex are essential for spermatogenesis in both sexes of C. briggsae. Moreover, trr-1 acts just upstream of TRA-1, at the same regulatory level as the fem genes. Further, if trr-1 activity is lowered, the FEM proteins become essential for spermatogenesis in C. briggsae. By contrast, mutations in C. elegans trr-1 do not block spermatogenesis. However, if the activity of the C. elegans fem genes is decreased, the animals need TRR-1 to make sperm. Thus, the Tip60 HAT complex and the FEM proteins play partially conserved roles in sex determination. However, each hermaphroditic species has adapted a unique mix of their regulatory activities to control the master transcription factor TRA-1.

Guo, Yiqing et al. (2009) International Worm Meeting "The Tip60/NuA4 HAT complex promotes spermatogenesis in C. briggsae."

Guo, Yiqing, Ellis, Ronald E

[International Worm Meeting, 2009]

In a general screen for female mutants in C.briggsae, we found two alleles of a new gene required for spermatogenesis in both hermaphrodites and males. We named this gene cb-fog-4 because of its Fog phenotype. Epistasis studies showed that fog-4 acts downstream of tra-2, and upstream of or in parallel to tra-1. Genetic mapping located cb-fog-4 on Chromosome II, and fine scale mapping placed it in a 160kb region between the SNPs cb43333 and cb43262. There are only 25 predicted genes in this region. One of these genes, cb-trr-1, is fog-4, since cb-trr-1(RNAi) animals are Fog, and both mutants have lesions in cb-trr-1. TRR-1 is a homolog of TRRAP in humans. Since both mutations are missense alleles, we set up a non-complementation screen using TMP/UV to isolate null alleles, and found 3 Fog alleles that have deletions in cb-trr-1. TRRAP is highly conserved. It is a common subunit of several HAT complexes, including Tip60/NuA4 and PCAF/GCN5. RNAi against cb-mys-1, the catalytic subunit of Tip60/NuA4, also causes a Fog phenotype, but RNAi against cb-pcaf-1, the catalytic subunit of PCAF1/GCN5, does not. Furthermore, RNAi targeting four other components of the Tip60/NuA4 complex, gfl-1, yl-1, epc-1, and ssl-1, also causes a Fog phenotype. Thus, we propose that the Tip60/NuA4 complex controls spermatogenesis in C. briggsae. In C. elegans, trr-1 mutations cause a weakly penetrant vulval defect on their own, as well as slow growth and sterility, but do not create Fog animals. RNAi against either cb-trr-1 or cb-mys-1 in a fem-2 mutant background caused embryonic lethality and L1 arrest. Moreover, RNAi showed that epc-1 and ssl-1 are also essential for the viability of embryos. By contrast, cb-trr-1; cb-fem-3 animals produce normal oocytes. These results suggest that the Tip60/NuA4 complex is essential for embryonic development and is redundant with FEM-2. We propose that cb-trr-1 might provide a model for the recruitment of chromatin regulators into a development pathway.

Guo, Yiqing et al. (2009) International Worm Meeting "Independent Recruitment of F-box Genes to Specify Hermaphrodite Development."

Ellis, Ronald E, Guo, Yiqing

[International Worm Meeting, 2009]

Although sexual reproduction is found throughout the animal kingdom, the mechanisms that control it change rapidly. To elucidate how sex-determination pathways evolve, we are comparing two related nematodes, C. elegans and C. briggsae. Both species make XX hermaphrodites, which are female in appearance, but produce sperm during larval development that can be used for self-fertilization. Phylogenies suggest that each species evolved hermaphroditism independently. In C. elegans, fog-2 is essential for XX animals to become hermaphrodites. However, the Schedl group showed that FOG-2 is a novel F-box protein with no homolog in C. briggsae. This result implied that C. briggsae must use another mechanism to specify hermaphrodite development. Thus, we screened for C. briggsae mutants that became female rather than hermaphrodite. Four mutations created XX females but did not affect males, and all failed to complement each other, so they define the new gene she-1, for spermless hermaphrodites. Two alleles isolated in non-complementation screens have the same phenotype. Genetic tests place she-1 upstream of tra-2 in the sex-determination pathway, at the same position that fog-2 occupies in C. elegans. Furthermore, both she-1 and fog-2 are sensitive to changes in tra-2 dosage. These results imply that tra-2 acts at a key point in the sex-determination pathway, and that two species have evolved hermaphroditism by modulating its activity. Positional cloning revealed that SHE-1 is an F-box protein that is unrelated in structure to FOG-2. Since SHE-1 can bind SKR-1, a component of the E3 ubiquitin ligase complex, and a missense mutation in the F-box domain inactivates SHE-1, it is likely to promote hermaphrodite development by regulating the stability of a target protein. We are currently using the yeast two-hybrid system to find the target. Thus, two different species have recruited novel members of the F-box family of genes into the sex-determination pathway to create hermaphrodites. Since FOG-2 regulates the translation of tra-2 messages by interacting with GLD-1, and SHE-1 does not bind GLD-1, these proteins act by different molecular mechanisms.

Yiqing Guo et al. (2006) Development & Evolution Meeting "glf-1 Controls Hermaphrodite Development in C. briggsae"

Yiqing Guo, Ronald Ellis

[Development & Evolution Meeting, 2006]

In C. elegans, XX animals are self-fertile hermaphrodites, and XO animals are males. The only other male/hermaphrodite species in this genus is C. briggsae. However, our phylogeny shows that these species are not sisters. Furthermore, work by Eric Haag and Dave Pilgrim indicates that the fem genes do not regulate germ cell fates in C. briggsae, although they are essential in C. elegans. Thus, it seems likely that hermaphroditism evolved separately in these species. How did this happen? And why did it happen twice within this genus? To answer these questions, we are taking two approaches. Here, we describe a genetic analysis of C. briggsae, to identify the genes that control hermaphrodite development. In a separate abstract, C. Baldi and R. Ellis describe what changes would be needed in the male/female species C. remanei to create hermaphrodites. By screening for mutations that transform XX animals into females, we identified these genes: glf-1 IV. GLF-1 is required for XX animals to develop as hermaphrodites, but plays no role in males. Thus, it acts like fog-2 does in C. elegans. However, Tim Schedl could not identify a homolog of fog-2 in C. briggsae, so GLF-1 probably acts by a novel mechanism. The original allele, v35, is temperature-sensitive, and we have isolated several suppressors. We also found two more alleles in our screen for XX females, and four alleles in a non-complementation screen. All are ts, and all affect only XX animals. Three alleles on LGI that cause both sexes to produce oocytes. We suspect that two are alleles of fog-1, and that one is an allele of fog-3, and are testing this hypothesis by sequencing DNA from each strain. At least five alleles on LGII that cause both sexes to produce oocytes. These mutations fail to complement each other, and a new gene, glf-2. At least one allele of tra-1 on LGIII that causes oogenesis in both sexes, perhaps like rare tra-1(gf) alleles do in C. elegans. Taken together, these results suggest that some parts of the sex-determination pathway are similar in both C. elegans and C. briggsae, like the roles of fog-1, fog-3 and tra-1. Other parts, in particular the functions of glf-1 and glf-2, are likely to be novel. Since glf-1 appears to be the key to hermaphroditic development in C. briggsae, we have been using SNP mapping to clone it. We have narrowed its location down to one of two finger-printed supercontigs on LGIV, and are using fine-scale mapping to identify the locus.

Yiqing Guo et al. (2007) International Worm Meeting "The F-box protein GLF-1 is required for hermaphrodites development in C. briggsae."

Yiqing Guo, XIANGMEI CHEN, Ronald Ellis

[International Worm Meeting, 2007]

In a general screen for XX females in C. briggsae, we found three mutants that cause hermaphrodites to develop as females, but do not affect males. These alleles define a single gene, glf-1. We isolate another four alleles in a non-complementation screen. Because all 7 alleles are temperature sensitive, we believe that loss of glf-1 reveals an underlying temperature-sensitivity in sex determination. Since glf-1 is only required for XX animals to develop as hermaphrodites, but plays no role in males, it functions much like fog-2 in C. elegans. In C. elegans, FOG-2 contains both an F-box and an FTH domain, which is required to bind GLD-1, and GLD-1 binds the 3UTR of tra-2 mRNA to inhibit translation. However, Tim Schedls group show that there is no a homolog of fog-2 in C. briggsae, and that gld-1 RNAi in C. briggsae has an unexpected Mog phenotype. Thus, glf-1 probably controls germ cell fates by a novel mechanism. Since glf-1 appears to be the key to hermaphroditic development in C. briggsae, we have been using SNP mapping to clone it. We narrowed its location down to a region between cb17324 and cb17358 on cb25.fpc2501. Three F-box genes are included in this region, RNAi against CBG11663 produce 95% Fog animals at 25C, but only 26% Fog at 15C. Furthermore, this RNAi phenotype can be suppressed by tra-2(lf)/+, just like glf-1 mutations. Thus, CBG11663 is likely to be glf-1. We have sequenced cDNA and genomic DNA from glf-1 mutants, and found a C to T transition in v35 that causes a missense mutation, and another C to T transition in v49 that creates a TAA stop condon. Both lesions occur in the F-box coding region. We cloned CBG11663 transcripts, and found that they are smaller than predicted by the Genome Sequencing Consortium, and that its 5-end is trans-spliced to SL1. Northern blots and RT-PCR show that glf-1 expression level increases during development, and is strongest in adults, even though it is only required in L4 larvae. We propose that GLF-1 binds other proteins and catalyzes their degrading to down-regulate TRA-2 to allow spermatogenesis in XX L4 larvae.

Yiqing Guo et al. (2008) Development & Evolution Meeting "The F-box Protein GLF-1 Is Required for Hermaphrodites Development in C. briggsae"

Ronald Ellis, Yiqing Guo, XIANGMEI CHEN

[Development & Evolution Meeting, 2008]

In a general screen for XX females in C. briggsae, we found four mutants that cause hermaphrodites to develop as females, but do not affect males. These alleles define a single gene, glf-1. We isolated three more glf-1 mutations in a non-complement screen. Because all seven alleles are temperature sensitive, we believe that loss of glf-1 reveals an underlying temperature-sensitivity in sex determination. Since glf-1 appears to be the key to hermaphroditic development in C. briggsae, we used SNP mapping to clone it. We narrowed its location down to a region between cb17324 and cb17358 on Chromosome IV. RNAi targeting one of the genes in this region produced 95% Fog animals at 25 degC, but only 26% Fog at 15 degC. Furthermore, this RNAi phenotype could be suppressed by tra-2(lf)/+, just like glf-1 mutations. Finally, we sequenced cDNA and genomic DNA from six glf-1 mutants, and found that all had lesions in this gene. Northern blots and RT-PCR show that glf-1 expression increases during development, and is strongest in adults, even though it is only required in L4 larvae. glf-1 encodes an F-box protein, and since missense mutations in the F-box behave like null alleles, the F-box domain is important for its function. Thus, we used the Yeast Two-Hybridization to look for proteins that interact with GLF-1. We found that GLF-1 binds SKP-1, and have identified several other candidates we have not characterized yet. In Conclusion, we propose that GLF-1 binds other proteins and catalyzes their degrading to down-regulate TRA-2 to allow spermatogenesis in XX L4 larvae.

da Silveira TL et al. (2019) Neuroscience "Guanosine Prevents against Glutamatergic Excitotoxicity in C. elegans."

da Silveira TL, Arantes LP, Tassi CL, Zamberlan DC, Soares FAA, Machado ML, Obetine FBB, Cordeiro LM, da Silva AF

[Neuroscience, 2019]

Glutamatergic neurotransmission is present in most mammalian excitatory synapses and plays a key role in central nervous system homeostasis. When over-activated, it can induce excitotoxicity, which is present in several neuropathologies. The nucleoside guanosine (GUO), is a guanine-based purine known to have neuroprotective effects by modulating glutamatergic system during glutamate excitotoxicity in mammals. However, GUO action in Caenorhabditis elegans, as well as on C. elegans glutamatergic excitotoxicity model, is not known. The GUO effects on behavioral parameters in Wild Type (WT) and knockouts worms for glutamate transporters (GLT-3, GLT-1), glutamate vesicular transporter (EAT-4), and NMDA and non-NMDA receptors were used to evaluate the GUO modulatory effects. The GUO tested concentrations did not alter the animals' development, but GUO reduced pharyngeal pumps in WT animals in a dose-dependent manner. The same effect was observed in pharyngeal pumps, when the animals were treated with 4mM of GUO in glr-1, nmr-1 and eat-4, but not in glt-3 and glt-3;glt-1 knockouts. The double mutant glt-3; glt-1 for GluTs had decreased body bends and an increased number of reversions. This effect was reverted after treatment with GUO. Furthermore, GUO did not alter the sensory response in worms with altered glutamatergic signaling. Thus, GUO seems to modulate the worm's glutamatergic system in situations of exacerbated glutamatergic signaling, which are represented by knockout strains to glutamate transporters. However, in WT animals, GUO appears to reinforce glutamatergic signaling in specific neurons. Our findings indicate that C. elegans strains are useful models to study new compounds that could be used in glutamate-associated neurodegenerative diseases.

Guo S et al. (1994) Worm Breeder's Gazette "Identification of par-1 Gene by Injecting in vitro-transcribed anti-sense RNA"

Guo S, Kemphues KJ

[Worm Breeder's Gazette, 1994]

Identification of par-l gene by injecting in vitro- transcribed anti-sense RNA Su Guo, Kenneth Kemphues, Dept. of Genetics & Development, Cornell University, Ithaca, NY 14853

Chen, Xiangmei et al. (2009) International Worm Meeting "Defining the pathway that controls hermaphrodite development in C. briggsae ."

Ellis, Ronald, Chen, Xiangmei, Guo, Yiqing

[International Worm Meeting, 2009]

Phylogenetic studies suggest that hermaphroditism might have evolved independently in C. elegans and C. briggsae. In C. briggsae, she-1 is a novel gene that promotes spermatogenesis in XX animals. Surprisingly, null alleles of she-1 are temperature sensitive, which implies that other genes help control hermaphroditic development. To identify these genes, we isolated 12 she-1 suppressors. The dominant suppressors v93 and v98 are tra-2 alleles. Homozygous XX animals are males, just like null allele of tra-2. Furthermore, a null allele of tra-2 also suppresses she-1. Thus, she-1 acts upstream of tra-2 and is very sensitive to changes in tra-2 dosage. The mutations v94, v99, v100, v102 and v103 are dominant. Even after extensive backcrossing, two of them have additional phenotypes. Homozygous she-1; v100 animals are sterile and have very small germlines, and homozygous v103 mutants are dead. We mapped v99 to a region between 7 Mbp and 11 Mbp on LG III, and v100 to a region between 2 Mbp and 7 Mbp on LG I. Five suppressors v92, v95, v96, v97 and v101, are recessive; they restore she-1(lf) XX self-fertility, but do not appear to affect the soma. We mapped v95 and v101 to LG I, since they complement to each other, which indicates that they define two different genes. We also mapped v97 to LG V, v92 to a region between 2 Mbp and 8 Mbp on LG II, and v95 to a region between 0.4 Mbp and 7 Mbp on LG I. Mutations that suppress she-1 might promote spermatogenesis in all situations, or might specifically correct the defect caused by loss of she-1 activity. To distinguish between these possibilities, we built strains that lacked the she-1 mutation. The brood size of cby-7 mip-10; v92 is 30% lower than that of cby-7 mip-10, and the brood size of cby-7 mip-10; v99 is 55% lower than that of cby-7 mip-10, suggesting that neither suppressors increase the spermatogenesis. Finally cby-7 mip-10; v100 XX animals are sterile with a very small germline, just like cby-7 she-1(v35) mip-10; v100 animals, so v100 is likely to define an essential gene. We are now using RNAi to see if any suppressors also affect other mutations in the sex-determination pathway.