Metabolic enzymes aldo-2 and pdhb-1 as potential epigenetic regulators during C. elegans embryogenesis.

The intersection of metabolic processes and epigenetic regulation during embryogenesis is crucial yet not fully understood. Through a candidate RNAi screen in Caenorhabditis elegans , we identified metabolic enzymes ALDO-2 and PDHB-1 as potential epigenetic regulators. Mild alteration of the chromatin remodeler LET-418 /Mi2 activity rescues embryonic lethality induced by suppressing aldo-2 or pdhb-1 , suggesting a critical role for glucose and pyruvate metabolism in chromatin remodeling during embryogenesis. Given the conservation of central metabolic pathways and chromatin modifiers across species, our findings lay the foundation for future mechanistic investigations into the interplay between epigenetics and metabolism during development and upon disease.

The New Nematicide Cyclobutrifluram Targets the Mitochondrial Succinate Dehydrogenase Complex in Caenorhabditis elegans.

Today, agriculture around the world is challenged by parasitic nematode infections. Plant-parasitic nematodes (PPNs) can cause significant damage and crop loss and are a threat to food security. For a long time, the management of PPN infection has relied on nematicides that impact not only parasitic nematodes but also other organisms. More recently, new nematicides have been developed that appear to specifically target PPN. Cyclobutrifluram belongs to this new category of nematicides. Using the nematode Caenorhabditis elegans as a model organism, we show here that cyclobutrifluram strongly impacts the survival and fertility rates of the worm by decreasing the number of germ cells. Furthermore, using a genetic approach, we demonstrate that cyclobutrifluram functions by inhibiting the mitochondrial succinate dehydrogenase (SDH) complex. Transcriptomic analysis revealed a strong response to cyclobutrifluram exposure. Among the deregulated genes, we found genes coding for detoxifying proteins, such as cytochrome P450s and UDP-glucuronosyl transferases (UGTs). Overall, our study contributes to the understanding of the molecular mode of action of cyclobutrifluram, to the finding of new approaches against nematicide resistance, and to the discovery of novel nematicides. Furthermore, this study confirms that C. elegans is a suitable model organism to study the mode of action of nematicides.

Methylation of the glucocorticoid receptor gene (NR3C1) in dyads mother-child exposed to intimate partner violence in Cameroon: Association with anxiety symptoms.

BACKGROUND: The glucocorticoid receptor (GR), which is encoded by the NR3C1 (Nuclear Receptor Subfamily 3 Group C Member 1) gene plays an important role in the modulation of the hypothalamic-pituitary-adrenal (HPA) axis activity by providing feedback regulation which allows termination of the stress response. Little is known about epigenetic programming at the level of NGFI-A (nerve growth factor-inducible protein A) putative binding site (CpG) of the NR3C1 exon 1F in dyads mother-child exposed to intimate partner violence (IPV) more specifically in an unstudied region such as the sub-Saharan Africa where levels of violence are very high. OBJECTIVE: Examine NR3C1 exon 1F methylation in response to IPV and possible association with cortisol concentration and mental health. METHOD: We recruited 20 mother-child dyads exposed to IPV and a control group of 20 mother-child dyads not exposed to IPV. We administered self-reported questionnaires to measure mother's mental health and collected saliva samples for cortisol dosage and bisulfite sequencing of DNA methylation. RESULTS: Regarding the mothers, our results showed a significant difference in methylation level at CpG 16-21 sites of the NR3C1 exon 1F promoter region between the groups. In the exposed group as compared to the control group, there was a significant positive association between the level of methylation at CpG 16-21 sites and mother's mental health in particular anxiety symptoms. However, we did not find any significant correlation between methylation level and cortisol concentration. In children, we did not find any significant results. CONCLUSION: This study highlights a NGFI-A putative binding site (CpG 16-21) that is more methylated in mothers exposed to IPV and which may have the potential to confer vulnerability for psychopathologies.

The zinc-finger transcription factor LSL-1 is a major regulator of the germline transcriptional program in Caenorhabditis elegans.

Specific gene transcriptional programs are required to ensure the 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 Caenorhabditis 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.

From the Mother to the Child: The Intergenerational Transmission of Experiences of Violence in Mother-Child Dyads Exposed to Intimate Partner Violence in Cameroon.

Intimate partner violence (IPV) is a widespread social problem with serious consequences for the health of both women and their children. However, little is known about the combined effect of maternal childhood abuse and current exposure to IPV with respect to the psychopathological symptoms of the mother-child dyad. In a Cameroonian cultural setting, where IPV affects more than half of women, we aimed to better understand how mother's childhood abuse and current IPV co-occur to lead to psychopathological symptoms in the mother-child dyad. With the help of a non-governmental organization in Cameroon, we recruited 49 mother-child dyads exposed to IPV, along with 25 mother-child dyads who had not been exposed, and who functioned as a control group. All mothers completed a set of questionnaires, including the Revised Conflict Tactics Scale to assess IPV; the Child Trauma Questionnaire to examine their childhood trauma; the Child Behavior Checklist to assess their children's psychopathological traits; the Hospital Anxiety and Depression Scale; and the Symptom Checklist. We found that physical abuse experienced by mothers during childhood was associated with IPV in adulthood, and specifically sexual abuse, p = .001. In addition, we found that the accumulation of maternal childhood abuse and current IPV was related to anxiety and depression symptoms in mothers, all R2 ≥ .18, all ps ≤ .015, as well as to externalized symptoms in children, all R2 ≥ .27, all ps ≤ .017. Our results suggest the intergenerational transmission of experiences of childhood abuse and current IPV, which calls for the development of interventions and care strategies for the mother-child dyad.

HPA-axis activity and the moderating effect of self-esteem in the context of intimate partner violence in Cameroon.

Background: The experience of intimate partner violence (IPV) is stressful. One objective way to monitor it is to assess victims' stress response by measuring the concentration of their salivary cortisol, the major stress hormone released by the hypothalamic-pituitary-adrenal axis. Objective: We investigated how the IPV experienced by women in Cameroon affects their stress levels and those of their children. Method: We recruited 50 mother-child dyads exposed to IPV and a control group of 25 mother-child dyads. All mothers completed questionnaires, including the Revised Conflict Tactics Scale to assess IPV, the Sense of Coherence Scale, and the Self-Esteem Scale, to assess their psychological resources. Mothers were asked to collect 3 saliva samples from themselves and 3 from their children on a single weekday: immediately after waking up, 30 minutes after waking up, and 45 minutes after waking up. The total cortisol secretion over the first hour after awakening was determined by calculating the area under the curve with respect to the ground (AUCg). Results: Mothers exposed to IPV exhibited higher total post-awakening cortisol concentrations compared with those in the control group. However, no significant difference was found between exposed and non-exposed children. In addition, higher IPV, specifically injuries, was significantly and positively associated with greater AUCg among mothers exhibiting lower self-esteem. When self-esteem was high, however, no significant effect of IPV on AUCg was observed. Conclusions: Of particular clinical significance is that self-esteem can modulate the stress levels of women exposed to IPV, a valuable insight into the development of effective psychosocial interventions to support IPV victims in sub-Saharan Africa.

Antecedentes: La experiencia de violencia de pareja (VIP) es estresante. Una forma objetiva de monitorearla es evaluar la respuesta al estrés de las víctimas midiendo la concentración de su cortisol salival, la principal hormona del estrés liberada por el eje hipotalámico-pituitario-adrenal.Objetivo: Investigamos cómo la VIP que experimentan las mujeres en Camerún afecta sus niveles de estrés y la de sus hijos.Método: Reclutamos 50 díadas madre-hijo expuestas a VIP y un grupo de control de 25 díadas madre-hijo. Todas las madres completaron cuestionarios, incluida la Escala de Tácticas de Conflicto revisada para evaluar la VIP, la Escala de Sentido de Coherencia y la Escala de Autoestima, para evaluar sus recursos psicológicos. Se pidió a las madres que recogieran 3 muestras de saliva de ellas mismas y 3 de sus hijos en un solo día de la semana: inmediatamente después de despertarse, 30 minutos después de despertarse y 45 minutos después de despertarse. La secreción total de cortisol durante la primera hora después del despertar se determinó calculando el área bajo la curva con respecto a la base (AUCg).Resultados: Las madres expuestas a VIP exhibieron concentraciones de cortisol total, después del despertar, más altas en comparación con las del grupo de control. Sin embargo, no se encontraron diferencias significativas entre niños expuestos y no expuestos. Además, una mayor VIP, específicamente las lesiones, se asociaron significativa y positivamente con un mayor AUCg entre las madres que mostraban una menor autoestima. Sin embargo, cuando la autoestima era alta, no se observaron efectos significativos de la VIP sobre el AUCg.Conclusiones: Que la autoestima pueda modular los niveles de estrés de las mujeres expuestas a VIP es de particular importancia clínica, una valiosa información sobre el desarrollo de intervenciones psicosociales efectivas para apoyar a las víctimas de VIP en África subsahariana.

The Chromatin Remodeler LET-418/Mi2 is Required Cell Non-Autonomously for the Post-Embryonic Development of Caenorhabditis elegans.

Chromatin condition is crucial for the cells to respond to their environment. In C. elegans, post-embryonic development is accompanied by the exit of progenitor cells from quiescence in response to food. The chromatin protein LET-418/Mi2 is required for this transition in development indicating that proper chromatin structure in cells of the freshly hatched larvae is important to respond to food. However, the identity of the tissue or cells where LET-418/Mi2 is required, as well as the developmental signals that it is modulating have not been elucidated. By restoring the activity of LET-418/Mi2 in specific tissues, we demonstrate that its activity in the intestine and the hypodermis is able to promote in a cell non-autonomous manner the exit of blast cells from quiescence and further development. Furthermore, we identify the IIS (insulin/insulin-like growth factor signaling) pathway to be one of the signaling pathways that is conveying LET-418/Mi2 cell non-autonomous effect on development.

The Bright Fluorescent Protein mNeonGreen Facilitates Protein Expression Analysis In Vivo.

The Green Fluorescent Protein (GFP) has been tremendously useful in investigating cell architecture, protein localization, and protein function. Recent developments in transgenesis and genome editing methods now enable working with fewer transgene copies and, consequently, with physiological expression levels. However, lower signal intensity might become a limiting factor. The recently developed mNeonGreen protein is a brighter alternative to GFP in vitro The goal of the present study was to determine how mNeonGreen performs in vivo in Caenorhabditis elegans-a model used extensively for fluorescence imaging in intact animals. We started with a side-by-side comparison between cytoplasmic forms of mNeonGreen and GFP expressed in the intestine, and in different neurons, of adult animals. While both proteins had similar photostability, mNeonGreen was systematically 3-5 times brighter than GFP. mNeonGreen was also used successfully to trace endogenous proteins, and label specific subcellular compartments such as the nucleus or the plasma membrane. To further demonstrate the utility of mNeonGreen, we tested transcriptional reporters for nine genes with unknown expression patterns. While mNeonGreen and GFP reporters gave overall similar expression patterns, low expression tissues were detected only with mNeonGreen. As a whole, our work establishes mNeonGreen as a brighter alternative to GFP for in vivo imaging in a multicellular organism. Furthermore, the present research illustrates the utility of mNeonGreen to tag proteins, mark subcellular regions, and describe new expression patterns, particularly in tissues with low expression.

A Network of Chromatin Factors Is Regulating the Transition to Postembryonic Development in Caenorhabditis elegans.

Mi2 proteins are evolutionarily conserved, ATP-dependent chromatin remodelers of the CHD family that play key roles in stem cell differentiation and reprogramming. In Caenorhabditis elegans, the let-418 gene encodes one of the two Mi2 homologs, which is part of at least two chromatin complexes, namely the Nucleosome Remodeling and histone Deacetylase (NuRD) complex and the MEC complex, and functions in larval development, vulval morphogenesis, lifespan regulation, and cell fate determination. To explore the mechanisms involved in the action of LET-418/Mi2, we performed a genome-wide RNA interference (RNAi) screen for suppressors of early larval arrest associated with let-418 mutations. We identified 29 suppressor genes, of which 24 encode chromatin regulators, mostly orthologs of proteins present in transcriptional activator complexes. The remaining five genes vary broadly in their predicted functions. All suppressor genes could suppress multiple aspects of the let-418 phenotype, including developmental arrest and ectopic expression of germline genes in the soma. Analysis of available transcriptomic data and quantitative PCR revealed that LET-418 and the suppressors of early larval arrest are regulating common target genes. These suppressors might represent direct competitors of LET-418 complexes for chromatin regulation of crucial genes involved in the transition to postembryonic development.

Fine-tuning of chromatin composition and Polycomb recruitment by two Mi2 homologues during C. elegans early embryonic development.

BACKGROUND: The nucleosome remodeling and deacetylase complex promotes cell fate decisions throughout embryonic development. Its core enzymatic subunit, the SNF2-like ATPase and Helicase Mi2, is well conserved throughout the eukaryotic kingdom and can be found in multiple and highly homologous copies in all vertebrates and some invertebrates. However, the reasons for such duplications and their implications for embryonic development are unknown. RESULTS: Here we studied the two C. elegans Mi2 homologues, LET-418 and CHD-3, which displayed redundant activities during early embryonic development. At the transcriptional level, these two Mi2 homologues redundantly repressed the expression of a large gene population. We found that LET-418 physically accumulated at TSS-proximal regions on transcriptionally active genomic targets involved in growth and development. Moreover, LET-418 acted redundantly with CHD-3 to block H3K4me3 deposition at these genes. Our study also revealed that LET-418 was partially responsible for recruiting Polycomb to chromatin and for promoting H3K27me3 deposition. Surprisingly, CHD-3 displayed opposite activities on Polycomb, as it was capable of moderating its LET-418-dependent recruitment and restricted the amount of H3K27me3 on the studied target genes. CONCLUSION: Although closely homologous, LET-418 and CHD-3 showed both redundant and opposite functions in modulating the chromatin environment at developmental target genes. We identified the interplay between LET-418 and CHD-3 to finely tune the levels of histone marks at developmental target genes. More than just repressors, Mi2-containing complexes appear as subtle modulators of gene expression throughout development. The study of such molecular variations in vertebrate Mi2 counterparts might provide crucial insights to our understanding of the epigenetic control of early development.

Specific NuRD components are required for fin regeneration in zebrafish.

BACKGROUND: Epimorphic regeneration of a missing appendage in fish and urodele amphibians involves the creation of a blastema, a heterogeneous pool of progenitor cells underneath the wound epidermis. Current evidence indicates that the blastema arises by dedifferentiation of stump tissues in the vicinity of the amputation. In response to tissue loss, silenced developmental programs are reactivated to form a near-perfect copy of the missing body part. However, the importance of chromatin regulation during epimorphic regeneration remains poorly understood. RESULTS: We found that specific components of the Nucleosome Remodeling and Deacetylase complex (NuRD) are required for fin regeneration in zebrafish. Transcripts of the chromatin remodeler chd4a/Mi-2, the histone deacetylase hdac1/HDAC1/2, the retinoblastoma-binding protein rbb4/RBBP4/7, and the metastasis-associated antigen mta2/MTA were specifically co-induced in the blastema during adult and embryonic fin regeneration, and these transcripts displayed a similar spatial and temporal expression patterns. In addition, chemical inhibition of Hdac1 and morpholino-mediated knockdown of chd4a, mta2, and rbb4 impaired regenerative outgrowth, resulting in reduction in blastema cell proliferation and in differentiation defects. CONCLUSION: Altogether, our data suggest that specialized NuRD components are induced in the blastema during fin regeneration and are involved in blastema cell proliferation and redifferentiation of osteoblast precursor cells. These results provide in vivo evidence for the involvement of key epigenetic factors in the cellular reprogramming processes occurring during epimorphic regeneration in zebrafish.

LET-418/Mi2 and SPR-5/LSD1 cooperatively prevent somatic reprogramming of C. elegans germline stem cells.

Throughout their journey to forming new individuals, germline stem cells must remain totipotent, particularly by maintaining a specific chromatin structure. However, the place epigenetic factors occupy in this process remains elusive. So far, "sensitization" of chromatin by modulation of histone arrangement and/or content was believed to facilitate transcription-factor-induced germ cell reprogramming. Here, we demonstrate that the combined reduction of two epigenetic factors suffices to reprogram C. elegans germ cells. The histone H3K4 demethylase SPR-5/LSD1 and the chromatin remodeler LET-418/Mi2 function together in an early process to maintain germ cell status and act as a barrier to block precocious differentiation. This epigenetic barrier is capable of limiting COMPASS-mediated H3K4 methylation, because elevated H3K4me3 levels correlate with germ cell reprogramming in spr-5; let-418 mutants. Interestingly, germ cells deficient for spr-5 and let-418 mainly reprogram as neurons, suggesting that neuronal fate might be the first to be derepressed in early embryogenesis.

The Caenorhabditis elegans LET-418/Mi2 plays a conserved role in lifespan regulation.

The evolutionarily conserved nucleosome-remodeling protein Mi2 is involved in transcriptional repression during development in various model systems, plays a role in embryonic patterning and germ line development, and participates in DNA repair and cell cycle progression. It is the catalytic subunit of the nucleosome remodeling and histone deacetylase (NuRD) complex, a key determinant of differentiation in mammalian embryonic stem cells. In addition, the Drosophila and C. elegans Mi2 homologs participate in another complex, the MEC complex, which also plays an important developmental role in these organisms. Here we show a new and unexpected feature of the C. elegans Mi2 homolog, LET-418/Mi2. Lack of LET-418/Mi2 results in longevity and enhanced stress resistance, a feature that we found to be conserved in Drosophila and in Arabidopsis. The fact that depletion of other components of the NuRD and the MEC complexes did not result in longevity suggests that LET-418 may regulate lifespan in a different molecular context. Genetic interaction studies suggest that let-418 could act in the germ-cell-loss pathway, downstream of kri-1 and tcer-1. On the basis of our data and on previous findings showing a role for let-418 during development, we propose that LET-418/Mi2 could be part of a system that drives development and reproduction with concomitant life-reducing effects later in life.

Different Mi-2 complexes for various developmental functions in Caenorhabditis elegans.

Biochemical purifications from mammalian cells and Xenopus oocytes revealed that vertebrate Mi-2 proteins reside in multisubunit NuRD (Nucleosome Remodeling and Deacetylase) complexes. Since all NuRD subunits are highly conserved in the genomes of C. elegans and Drosophila, it was suggested that NuRD complexes also exist in invertebrates. Recently, a novel dMec complex, composed of dMi-2 and dMEP-1 was identified in Drosophila. The genome of C. elegans encodes two highly homologous Mi-2 orthologues, LET-418 and CHD-3. Here we demonstrate that these proteins define at least three different protein complexes, two distinct NuRD complexes and one MEC complex. The two canonical NuRD complexes share the same core subunits HDA-1/HDAC, LIN-53/RbAp and LIN-40/MTA, but differ in their Mi-2 orthologues LET-418 or CHD-3. LET-418 but not CHD-3, interacts with the Krüppel-like protein MEP-1 in a distinct complex, the MEC complex. Based on microarrays analyses, we propose that MEC constitutes an important LET-418 containing regulatory complex during C. elegans embryonic and early larval development. It is required for the repression of germline potential in somatic cells and acts when blastomeres are still dividing and differentiating. The two NuRD complexes may not be important for the early development, but may act later during postembryonic development. Altogether, our data suggest a considerable complexity in the composition, the developmental function and the tissue-specificity of the different C. elegans Mi-2 complexes.

Inactivation of the autophagy gene bec-1 triggers apoptotic cell death in C. elegans.

Programmed cell death (PCD) is an essential and highly orchestrated process that plays a major role in morphogenesis and tissue homeostasis during development. In humans, defects in regulation or execution of cell death lead to diabetes, neurodegenerative disorders, and cancer. Two major types of PCD have been distinguished: the caspase-mediated process of apoptosis and the caspase-independent process involving autophagy. Although apoptosis and autophagy are often activated together in response to stress, the molecular mechanisms underlying their interplay remain unclear. Here we show that BEC-1, the C. elegans ortholog of the yeast and mammalian autophagy proteins Atg6/Vps30 and Beclin 1, is essential for development. We demonstrate that BEC-1 is necessary for the function of the class III PI3 kinase LET-512/Vps34, an essential protein required for autophagy, membrane trafficking, and endocytosis. Furthermore, BEC-1 forms a complex with the antiapoptotic protein CED-9/Bcl-2, and its depletion triggers CED-3/Caspase-dependent PCD. Based on our results, we propose that bec-1 represents a link between autophagy and apoptosis, thus supporting the view that the two processes act in concerted manner in the cell death machinery.

Multiple genetic pathways involving the Caenorhabditis elegans Bloom's syndrome genes him-6, rad-51, and top-3 are needed to maintain genome stability in the germ line.

Bloom's syndrome (BS) is an autosomal-recessive human disorder caused by mutations in the BS RecQ helicase and is associated with loss of genomic integrity and an increased incidence of cancer. We analyzed the mitotic and the meiotic roles of Caenorhabditis elegans him-6, which we show to encode the ortholog of the human BS gene. Mutations in him-6 result in an enhanced irradiation sensitivity, a partially defective S-phase checkpoint, and in reduced levels of DNA-damage induced apoptosis. Furthermore, him-6 mutants exhibit a decreased frequency of meiotic recombination that is probably due to a defect in the progression of crossover recombination. In mitotically proliferating germ cells, our genetic interaction studies, as well as the assessment of the number of double-strand breaks via RAD-51 foci, reveal a complex regulatory network that is different from the situation in yeast. Although the number of double-strand breaks in him-6 and top-3 single mutants is elevated, the combined depletion of him-6 and top-3 leads to mitotic catastrophe concomitant with a massive increase in the level of double-strand breaks, a phenotype that is completely suppressed by rad-51. him-6 and top-3 are thus needed to maintain low levels of double-strand breaks in normally proliferating germ cells, and both act in partial redundant pathways downstream of rad-51 to prevent mitotic catastrophy. Finally, we show that topoisomerase IIIalpha acts independently during a late stage of meiotic recombination.