Rodriguez, Juan D. et al. (2021) International Worm Meeting "Regulation of embryonic cell specification by histone methylation"

Rodriguez, Juan D., Katz, David

[International Worm Meeting, 2021]

In C. elegans, two epigenetic enzymes, the H3K4me2 demethylase, SPR5, and the H3K9 methyltransferase, MET-2, are maternally deposited into the oocyte and cooperate to reestablish the epigenetic ground state by modifying histone methylation. Progeny of worms lacking spr-5 and met-2 accumulate high levels of H3K4me2 in the subsequent generation. This inappropriate H3K4me2 is associated with a low level of embryonic lethality, as well as a severe developmental delay and sterility in the small number of animals that reach adulthood. In addition, the progeny of spr-5;met-2 mutants improperly express germline genes in somatic tissues. We hypothesized that this improper expression could result in cell lineage defects. To interrogate how reprogramming defects may affect early embryonic development, we are taking advantage of the invariant C. elegans embryonic lineage by performing automated lineage tracing experiments in spr-5;met-2 progeny. Thus far, we observe several stages of defects. First, in early embryogenesis, the shape of the embryos is rounder, with a diameter that is greater than Wild Type, and the mutant cells divide faster than Wild Type. Since these defects are prior to the onset of zygotic transcription, they may be due directly to the decondensed state of chromatin associated with increased H3K4me2, or indirectly due to altered maternal RNAs and proteins. Second, after the onset of zygotic transcription, we observe many changes in cell timing, along with defects in cell migration. These defects may be due to the inappropriate somatic transcription of germline genes that we observe in the progeny of spr-5;met-2 mutants. Interestingly, those defects happened around the 100-cell stage or later. In C. elegans, the maternal to zygotic transition start during the early stages, but the higher peak of transition occurs around the 60 to the 100-cell stage. We hypothesized that the effect at this stage is due to the ectopic expression of germline genes, which is a consequence of the inappropriate inheritance of histone methylation. To address this question, we are currently performing single-cell RNA sequencing in the spr-5;met-2 mutants embryos at different cell stages including the 100-cell stage.

Rodriguez, Juan D. et al. (2019) International Worm Meeting "Regulation of embryonic cell fate decision by histone methylation."

Carpenter, Brandon, Rodriguez, Juan D., Katz, David

[International Worm Meeting, 2019]

Genetic and epigenetic information are transmitted from one generation to the next through the germline. At fertilization, each embryo must reprogram the epigenome and reestablish an epigenetic ground state to allow normal development to proceed. In C. elegans, two epigenetic enzymes, the H3K4me2 demethylase, SPR5, and the H3K9 methyltransferase, MET-2, are maternally deposited into the oocyte and cooperate to reestablish the epigenetic ground state by modifying histone methylation. Progeny of worms lacking spr-5 and met-2 accumulate high levels of H3K4me2, resulting in 15% embryonic lethality, developmental delay, and complete sterility. In addition, these phenotypes are associated with the ectopic expression of ~200 targets of the H3K36 methyltransferase MES-4 in somatic tissues. To determine if this ectopic expression results in any of the phenotypes, we performed mes-4 RNAi on spr-5;met-2 mutants. Knocking down MES-4 rescues the ectopic expression of germline genes and the developmental delay, suggesting that the developmental delay is caused by the ectopic expression of the ~200 MES-4 germline genes. The ectopic expression of germline genes in somatic tissues provides the unique opportunity to determine how the expression of one developmental program affects cells trying to specify a different developmental program. To address this question, we are using live confocal imaging to perform automated lineage tracing on the progeny of spr-5;met-2 mutants. The C. elegans embryonic lineage is normally invariant. By identifying defects in the embryonic lineage, we hope to gain mechanistic insight into the consequences of improper germline/soma identity. We hypothesize that the somatic expression of germline genes, due to the abnormal maintenance of histone methylation, could affect cell fate, cause abnormal cell divisions, and perhaps lead to inappropriate cell death. Results from the initial experiments will be presented.

Rodriguez-Crespo D et al. (2022) Genetics "The zinc-finger transcription factor LSL-1 is a major regulator of the germline transcriptional program in C. elegans."

Rajopadhye S, Wicky C, Rodriguez-Crespo D, Nanchen M

[Genetics, 2022]

Specific gene transcriptional programs are required to ensure proper proliferation and differentiation processes underlying the production of specialized cells during development. Gene activity is mainly regulated by the concerted action of transcription factors and chromatin proteins. In the nematode C. elegans, mechanisms that silence improper transcriptional programs in germline and somatic cells have been well studied, however, how are tissue specific sets of genes turned on is less known. LSL-1 is herein defined as a novel crucial transcriptional regulator of germline genes in C. elegans. LSL-1 is first detected in the P4 blastomere and remains present at all stages of germline development, from primordial germ cell proliferation to the end of meiotic prophase. lsl-1 loss-of-function mutants exhibit many defects including meiotic prophase progression delay, a high level of germline apoptosis, and production of almost no functional gametes. Transcriptomic analysis and ChIP-seq data show that LSL-1 binds to promoters and acts as a transcriptional activator of germline genes involved in various processes, including homologous chromosome pairing, recombination, and genome stability. Furthermore, we show that LSL-1 functions by antagonizing the action of the heterochromatin proteins HPL-2/HP1 and LET-418/Mi2 known to be involved in the repression of germline genes in somatic cells. Based on our results, we propose LSL-1 to be a major regulator of the germline transcriptional program during development.

Munro E (2017) Dev Cell "Protein Clustering Shapes Polarity Protein Gradients."

Munro E

[Dev Cell, 2017]

In this issue of Developmental Cell, Dickinson etal. (2017) and Rodriguez etal. (2017), along with Wang etal. (2017) in Nature Cell Biology, show how PAR protein oligomerization can dynamically couple protein diffusion and transport by cortical flow to control kinase activity gradients and polarity in the C.elegans zygote.

Sakoguchi H et al. (2016) Biosci Biotechnol Biochem "Screening of biologically active monosaccharides: growth inhibitory effects of d-allose, d-talose, and l-idose against the nematode Caenorhabditis elegans."

Izumori K, Yoshihara A, Sakoguchi H, Sato M

[Biosci Biotechnol Biochem, 2016]

We compared the growth inhibitory effects of all aldohexose stereoisomers against the model animal Caenorhabditis elegans. Among the tested compounds, the rare sugars d-allose (d-All), d-talose (d-Tal), and l-idose (l-Ido) showed considerable growth inhibition under both monoxenic and axenic culture conditions. 6-Deoxy-d-All had no effect on growth, which suggests that C6-phosphorylation by hexokinase is essential for inhibition by d-All.

Sakoguchi H et al. (2016) Bioorg Med Chem Lett "Growth inhibitory effect of d-arabinose against the nematode Caenorhabditis elegans: Discovery of a novel bioactive monosaccharide."

Shintani T, Izumori K, Sato M, Sakoguchi H, Okuma K, Yoshihara A

[Bioorg Med Chem Lett, 2016]

Biological activities of unusual monosaccharides (rare sugars) have largely remained unstudied until recently. We compared the growth inhibitory effects of aldohexose stereoisomers against the animal model Caenorhabditis elegans cultured in monoxenic conditions with Escherichia coli as food. Among these stereoisomers, the rare sugar d-arabinose (d-Ara) showed particularly strong growth inhibition. The IC50 value for d-Ara was estimated to be 7.5mM, which surpassed that of the potent glycolytic inhibitor 2-deoxy-d-glucose (19.5mM) used as a positive control. The inhibitory effect of d-Ara was also observed in animals cultured in axenic conditions using a chemically defined medium; this excluded the possible influence of E. coli. To our knowledge, this is the first report of biological activity of d-Ara. The d-Ara-induced inhibition was recovered by adding either d-ribose or d-fructose, but not d-glucose. These findings suggest that the inhibition could be induced by multiple mechanisms, for example, disturbance of d-ribose and d-fructose metabolism.

Yoshihara A et al. (2019) Bioorg Med Chem Lett "Growth inhibition by 1-deoxy-d-allulose, a novel bioactive deoxy sugar, screened using Caenorhabditis elegans assay."

Sakoguchi H, Fleet GWJ, Izumori K, Shintani T, Sato M, Yoshihara A

[Bioorg Med Chem Lett, 2019]

The biological activities of deoxy sugars (deoxy monosaccharides) have remained largely unstudied until recently. We compared the growth inhibition by all 1-deoxyketohexoses using the animal model Caenorhabditis elegans. Among the eight stereoisomers, 1-deoxy-d-allulose (1d-d-Alu) showed particularly strong growth inhibition. The 50% inhibition of growth (GI₅₀) concentration by 1d-d-Alu was estimated to be 5.4mM, which is approximately 10 times lower than that of d-allulose (52.7mM), and even lower than that of the potent glycolytic inhibitor, 2-deoxy-d-glucose (19.5mM), implying that 1d-d-Alu has a strong growth inhibition. In contrast, 5-deoxy- and 6-deoxy-d-allulose showed no growth inhibition of C. elegans. The inhibition by 1d-d-Alu was alleviated by the addition of d-ribose or d-fructose. Our findings suggest that 1d-d-Alu-mediated growth inhibition could be induced by the imbalance in d-ribose metabolism. To our knowledge, this is the first report of biological activity of 1d-d-Alu which may be considered as an antimetabolite drug candidate.

Katane M et al. (2020) Biochim Biophys Acta Proteins Proteom "Biochemical characterization of d-aspartate oxidase from Caenorhabditis elegans: Its potential use in the determination of free D-glutamate in biological samples."

Katane M, Sekine M, Nakayama K, Homma H, Kuwabara H, Saitoh Y, Miyamoto T

[Biochim Biophys Acta Proteins Proteom, 2020]

d-Aspartate oxidase (DDO) is a flavin adenine dinucleotide (FAD)-containing flavoprotein that stereospecifically acts on acidic D-amino acids (i.e., free d-aspartate and D-glutamate). Mammalian DDO, which exhibits higher activity toward d-aspartate than D-glutamate, is presumed to regulate levels of d-aspartate in the body and is not thought to degrade D-glutamate in vivo. By contrast, three DDO isoforms are present in the nematode Caenorhabditis elegans, DDO-1, DDO-2, and DDO-3, all of which exhibit substantial activity toward D-glutamate as well as d-aspartate. In this study, we optimized the Escherichia coli culture conditions for production of recombinant C. elegans DDO-1, purified the protein, and showed that it is a flavoprotein with a noncovalently but tightly attached FAD. Furthermore, C. elegans DDO-1, but not mammalian (rat) DDO, efficiently and selectively degraded D-glutamate in addition to d-aspartate, even in the presence of various other amino acids. Thus, C. elegans DDO-1 might be a useful tool for determining these acidic D-amino acids in biological samples.

Shintani T et al. (2019) J Appl Glycosci (1999) "D-Allose, a Stereoisomer of D-Glucose, Extends the Lifespan of Caenorhabditis elegans via Sirtuin and Insulin Signaling."

Izumori K, Sakoguchi H, Yoshihara A, Shintani T, Sato M

[J Appl Glycosci (1999), 2019]

D-Allose (D-All), C-3 epimer of D-glucose, is a rare sugar known to suppress reactive oxygen species generation and prevent hypertension. We previously reported that D-allulose, a structural isomer of D-All, prolongs the lifespan of the nematode Caenorhabditis elegans. Thus, D-All was predicted to affect longevity. In this study, we provide the first empirical evidence that D-All extends the lifespan of C. elegans. Lifespan assays revealed that a lifespan extension was induced by 28 mM D-All. In particular, a lifespan extension of 23.8 % was achieved (p< 0.0001). We further revealed that the effects of D-All on lifespan were dependent on the insulin gene daf-16 and the longevity gene sir-2.1, indicating a distinct mechanism from those of other hexoses, such as D-allulose, with previously reported antiaging effects.

Sato M et al. (2008) J Nat Med "Potential anthelmintic: D-psicose inhibits motility, growth and reproductive maturity of L1 larvae of Caenorhabditis elegans."

Izumori K, Yamasaki T, Kurose H, Sato M

[J Nat Med, 2008]

No anthelmintic sugars have yet been identified. Eight ketohexose stereoisomers (D- and L-forms of psicose, fructose, tagatose and sorbose), along with D-galactose and D-glucose, were examined for potency against L1 stage Caenorhabditis elegans fed Escherichia coli. Of the sugars, D-psicose specifically inhibited the motility, growth and reproductive maturity of the L1 stage. D-Psicose probably interferes with the nematode nutrition. The present results suggest that D-psicose, one of the rare sugars, is a potential anthelmintic.