Chantal Wicky

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.

New telomere formation after developmentally regulated chromosomal breakage during the process of chromatin diminution in ascaris lumbricoides

During the process of chromatin diminution, which takes place in all presomatic cells of the early Ascaris embryo, the heterochromatic termini of the chromosomes are lost. Here we show that the newly formed ends of the reduced somatic chromosomes carry tandem repeats of the telomeric sequence TTAGGC. Comparison of a cloned somatic telomere with the corresponding germline chromosomal region revealed that these telomeric repeats are not present at or near the chromosomal breakage site. They are most likely added by a telomerase-mediated event. Chromosomal breakage, which precedes the telomere addition process, takes place within a short, specific chromosomal region (CBR); however, it does not occur at a single locus, but rather at many different sites. Altogether, our data show that chromatin diminution in Ascaris is a complex molecular process that includes site-specific chromosomal breakage, new telomere formation, and DNA degradation.

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

ABSTRACT Blooms 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 IIIα acts independently during a late stage of meiotic recombination.

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.

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.

LET-418/Mi2 and SPR-5/LSD1 Cooperatively Prevent Somatic Reprogramming of C. elegans Germline Stem Cells

Summary 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.

Specific NuRD components are required for fin regeneration in zebrafish

BackgroundEpimorphic 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.ResultsWe 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.ConclusionAltogether, 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.

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.

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

BackgroundThe 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.ResultsHere 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.ConclusionAlthough 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.

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.