Munoz Jimenez, C. et al. (2017) International Worm Meeting "EMR-1/emerin is involved in tissue-specific anchoring of chromatin to the nuclear envelope and neuromuscular junction activity."

Ayuso, C., Askjaer, P., Munoz Jimenez, C.

[International Worm Meeting, 2017]

The nuclear envelope (NE) regulates transport of macromolecules between the nucleus and the cytoplasm and plays critical roles in nuclear organization and gene expression. Reflecting the importance of its functions, several human diseases are attributed to alterations in NE structure. Most notably are the laminopathies, with one example being Emery-Dreifuss muscular dystrophy (EDMD), which is caused by mutations in the inner nuclear membrane protein emerin or in the nuclear lamina protein lamin A. We have found recently that EMR-1, the Caenorhabditis elegans ortholog of emerin, associates with genes implicated in muscle and nervous system function and regulate their expression. Interestingly, deletion of emr-1 causes local changes in nuclear architecture and hypersensitivity to the cholinesterase inhibitor aldicarb, indicating altered activity at neuromuscular junctions. Although many NE proteins are ubiquitously expressed, laminopathies often affect a single tissue. It is hypothesized that tissue-specific alterations in nuclear organization might be responsible for particular symptoms of laminopathies. For this reason, we have developed novel tools to study changes in interactions between NE proteins and chromatin in a tissue-specific manner in intact animals. We report here the results of DamID experiments based on FLP-mediated recombination to dissect the function of EMR-1 in tissue-specific nuclear organization. Interestingly, EMR-1 interaction profiles in muscles vs. neurons reveal both many common domains and local changes in chromatin organization, with central part of chromosomes interacting more frequently with EMR-1 in neurons than in muscles. We are currently addressing the role of EMR-1 in regulation of gene expression in a tissue-specific way to understand the contribution of this protein to the neuromuscular junction activity and potentially to unravel the etiology of EDMD. It has been reported that NE morphology is prone to suffer from changes during normal aging and in progeria patients. Again, it has been proposed that alterations in nuclear organization through development and aging may have an impact on the aging-associated disorders. By performing FLP-mediated temporal DamID in young vs. old animals we have detected that chromatin accessibility changes dramatically in aged worms. Furthermore, we have identified local changes in EMR-1 interaction profiles in aged vs. young animals, with depletion of EMR-1 in central part of chromosomes in old animals.

Lourenco AB et al. (2015) Biochim Biophys Acta "Analysis of the effect of the mitochondrial prohibitin complex, a context-dependent modulator of longevity, on the C. elegans metabolome."

Munoz-Jimenez C, Artal-Sanz M, Venegas-Caleron M, Lourenco AB

[Biochim Biophys Acta, 2015]

The mitochondrial prohibitin complex, composed of two proteins, PHB-1 and PHB-2, is a context-dependent modulator of longevity. Specifically, prohibitin deficiency shortens the lifespan of otherwise wild type worms, while it dramatically extends the lifespan under compromised metabolic conditions. This extremely intriguingly phenotype has been linked to alterations in mitochondrial function and in fat metabolism. However, the true function of the mitochondrial prohibitin complex remains elusive. Here, we used gas chromatography coupled to a flame ionization detector (GC/FID) and H NMR spectroscopy to gain molecular insights into the effect of prohibitin depletion on the Caenorhabditis elegans metabolome. We analysed the effect of prohibitin deficiency in two different developmental stages and under two different conditions, which result in opposing longevity phenotypes, namely wild type worms and daf-2(e1370) insulin signalling deficient mutants. Prohibitin depletion was shown to alter the fatty acid (GC/FID) and H NMR metabolic profiles of wild type animals both at the fourth larval stage of development (L4) and at the young adult (YA) stage, while being more pronounced at the later stage. Furthermore, wild type and the diapause mutant daf-2(e1370), either expressing or not prohibitin, were clearly distinguishable based on their metabolic profiles, revealing changes in fatty acid composition, as well as in carbohydrate and amino acid metabolism. Moreover, the metabolic data indicate that daf-2(e1370) mutants are more robust than the wild type animals to changes induced by prohibitin depletion. The impact of prohibitin depletion on the C. elegans metabolome will be discussed herein in the scope of its effect on longevity. This article is part of a Special Issue entitled: Mitochondrial Dysfunction in Aging. Guest Editor: Aleksandra Trifunovic.

Van de Walle P et al. (2020) PLoS One "DamID identifies targets of CEH-60/PBX that are associated with neuron development and muscle structure in Caenorhabditis elegans."

Temmerman L, Schoofs L, Van de Walle P, Munoz-Jimenez C, Askjaer P

[PLoS One, 2020]

Transcription factors govern many of the time- and tissue-specific gene expression events in living organisms. CEH-60, a homolog of the TALE transcription factor PBX in vertebrates, was recently characterized as a new regulator of intestinal lipid mobilization in Caenorhabditis elegans. Because CEH-60's orthologs and paralogs exhibit several other functions, notably in neuron and muscle development, and because ceh-60 expression is not limited to the C. elegans intestine, we sought to identify additional functions of CEH-60 through DNA adenine methyltransferase identification (DamID). DamID identifies protein-genome interaction sites through GATC-specific methylation. We here report 872 putative CEH-60 gene targets in young adult animals, and 587 in L2 larvae, many of which are associated with neuron development or muscle structure. In light of this, we investigate morphology and function of ceh-60 expressing AWC neurons, and contraction of pharyngeal muscles. We find no clear functional consequences of loss of ceh-60 in these assays, suggesting that in AWC neurons and pharyngeal muscle, CEH-60 function is likely more subtle or redundant with other factors.

Cabianca DS et al. (2019) Nature "Active chromatin marks drive spatial sequestration of heterochromatin in C. elegans nuclei."

Kalck V, Askjaer P, Seeber A, Cabianca DS, Gasser SM, Padeken J, Munoz-Jimenez C, Gaidatzis D

[Nature, 2019]

The execution of developmental programs of gene expression requires an accurate partitioning of the genome into subnuclear compartments, with active euchromatin enriched centrally and silent heterochromatin at the nuclear periphery¹. The existence of degenerative diseases linked to lamin A mutations suggests that perinuclear binding of chromatin contributes to cell-type integrity^2,3. The methylation of lysine 9 of histone H3 (H3K9me) characterizes heterochromatin and mediates both transcriptional repression and chromatin anchoring at the inner nuclear membrane⁴. In Caenorhabditis elegans embryos, chromodomain protein CEC-4 bound to the inner nuclear membrane tethers heterochromatin through H3K9me^3,5, whereas in differentiated tissues, a second heterochromatin-sequestering pathway is induced. Here we use an RNA interference screen in the cec-4 background and identify MRG-1 as a broadly expressed factorthat is necessary for this second chromatin anchor in intestinal cells. However,MRG-1 is exclusively bound to euchromatin, suggesting that it acts indirectly. Heterochromatin detachment in double mrg-1; cec-4 mutants is rescued by depleting the histone acetyltransferase CBP-1/p300 or the transcription factor ATF-8, a member of the bZIP family (which is known to recruit CBP/p300). Overexpression of CBP-1 in cec-4 mutants is sufficient to delocalize heterochromatin in an ATF-8-dependent manner. CBP-1 and H3K27ac levels increase in heterochromatin upon mrg-1 knockdown, coincident with delocalization. This suggests that the spatial organization of chromatin in C. elegans is regulated both by the direct perinuclear attachment of silent chromatin, and by an active retention of CBP-1/p300 in euchromatin. The two pathways contribute differentially in embryos and larval tissues, with CBP-1 sequestration by MRG-1 having a major role in differentiated cells.

Fragoso-Luna A et al. (2022) Genetics "Expanded FLP toolbox for spatiotemporal protein degradation and transcriptomic profiling in C. elegans."

Ayuso C, Cases I, Munoz-Jimenez C, Romero-Bueno R, Benes V, Askjaer P, Fragoso-Luna A, Eibl M

[Genetics, 2022]

Control of gene expression in specific tissues and/or at certain stages of development allows the study and manipulation of gene function with high precision. Site-specific genome recombination by the Flippase (FLP) and Cre enzymes has proven particularly relevant. Joint efforts of many research groups have led to the creation of efficient FLP and Cre drivers to regulate gene expression in a variety of tissues in Caenorhabditis elegans. Here, we extend this toolkit by the addition of FLP lines that drive recombination specifically in distal tip cells, the somatic gonad, coelomocytes and the epithelial P lineage. In some cases, recombination-mediated gene knockouts do not completely deplete protein levels due to persistence of long-lived proteins. To overcome this, we developed a spatiotemporally regulated degradation system for GFP fusion proteins (GFPdeg) based on FLP-mediated recombination. Using two stable nuclear pore proteins, MEL-28/ELYS and NPP-2/NUP85 as examples, we report the benefit of combining tissue-specific gene knockout and protein degradation to achieve complete protein depletion. We also demonstrate that FLP-mediated recombination can be utilized to identify transcriptomes in a C. elegans tissue of interest. We have adapted RNA polymerase DamID (RAPID) for the FLP toolbox and by focusing on a well-characterized tissue, the hypodermis, we show that the vast majority of genes identified by RAPID are known to be expressed in this tissue. These tools allow combining FLP activity for simultaneous gene inactivation and transcriptomic profiling, thus enabling the inquiry of gene function in various complex biological processes.

Ayuso, Cristina et al. (2019) MicroPubl Biol

Ayuso, Cristina, Askjaer, Peter

[MicroPubl Biol, 2019]

Several techniques are available for spatiotemporal control of genome recombination and gene expression in the nematode Caenorhabditis elegans. Here we report a novel tool to combine the powerful FLP-Frt and GAL4-UAS systems to increase their versality and to offer additional levels of control.FLP is an enzyme that catalyzes recombination between two short Frt DNA sequences and is frequently used to excise genomic fragments flanked by Frt sites, thereby either activating or knocking out gene expression, depending on the experimental design (Hubbard, 2014). Recently, we generated multiple strains that stably express FLP in different somatic tissues from single-copy transgenes and demonstrated that they in most cases induce recombination in ~100% of the cells of the expected tissue (Munoz-Jimenez et al., 2017). We subsequently constructed a strain for germline recombination to permanently knock out Frt-flanked genes or exons (Macas-Len and Askjaer, 2018).The GAL4-UAS system is based on the Saccharomyces cerevisiae Gal4p transcription factor and its cognate DNA target called upstream activating sequence (UAS). Typically, this bipartite system includes a series of driver strains expressing GAL4 in specific tissues and one or several strains with an effector gene downstream of UAS repeats. Wang and colleagues from the Sternberg laboratory recently optimized the GAL4-UAS system for C. elegans (cGAL) and reported several tissue-specific cGAL drivers (Wang et al., 2017). Moreover, they have developed a split cGAL toolkit where the DNA binding and activation domains are expressed as individual polypeptides, thereby enabling further fine-tuning of spatiotemporal control: only when and where the two components are co-expressed they will activate the UAS::effector transgene (Wang et al., 2018).

Harr JC et al. (2020) Genes Dev "Loss of an H3K9me anchor rescues laminopathy-linked changes in nuclear organization and muscle function in an Emery-Dreifuss muscular dystrophy model."

Harr JC, Schmid CD, Mattout A, Askjaer P, Munoz-Jimenez C, Gonzalez-Sandoval A, Gasser SM, Padeken J, Romero-Bueno R, Hauer MH, Kalck V

[Genes Dev, 2020]

Mutations in the nuclear structural protein lamin A produce rare, tissue-specific diseases called laminopathies. The introduction of a human Emery-Dreifuss muscular dystrophy (EDMD)-inducing mutation into the <i>C. elegans</i> lamin (LMN-Y59C), recapitulates many muscular dystrophy phenotypes, and correlates with hyper-sequestration of a heterochromatic array at the nuclear periphery in muscle cells. Using muscle-specific emerin Dam-ID in worms, we monitored the effects of the mutation on endogenous chromatin. An increased contact with the nuclear periphery along chromosome arms, and an enhanced release of chromosomal centers, coincided with the disease phenotypes of reduced locomotion and compromised sarcomere integrity. The coupling of the LMN-Y59C mutation with the ablation of CEC-4, a chromodomain protein that anchors H3K9-methylated chromatin at the nuclear envelope (NE), suppressed the muscle-associated disease phenotypes. Deletion of <i>cec-4</i> also rescued LMN-Y59C-linked alterations in chromatin organization and some changes in transcription. Sequences that changed position in the LMN-Y59C mutant, are enriched for E2F (EFL-2)-binding sites, consistent with previous studies suggesting that altered Rb-E2F interaction with lamin A may contribute to muscle dysfunction. In summary, we were able to counteract the dominant muscle-specific defects provoked by LMNA mutation by the ablation of a lamin-associated H3K9me anchor, suggesting a novel therapeutic pathway for EDMD.

M Mercedes Perez-Jimenez et al. (2008) European Worm Meeting "Characterization and identification of the long-lived mutant liv-7(pv17)"

Jose M Monje, M Mercedes Perez-Jimenez, Manuel J Munoz

[European Worm Meeting, 2008]

Do not add objects such as pictures, boxes, headers, footers, footnotes,. etc.. Using a positive selection for thermotolerant mutants (Munoz et al.. Genetics, 2003. 163: 171-80) we have isolated liv-7(pv17), a long-lived. mutant that does not show any visible phenotype.. We have done a double mutant with the temperature sensitive allele daf-2. (e1370)III, which is also long-lived and encodes to the only insulin. receptor homologue that exists in C. elegans. The double daf-2 (e1370);. liv-7 (pv17), shows a phenotype different to any of the single mutants, an. L1 arrest at 25degrees. Besides this phenotype we noticed that liv-7 (pv17). enhances other phenotype characteristics of daf-2 mutants in different. alleles like e1370, m577 or e979.. The longevity of the mutants of the Insulin/IGF pathway depends of the. transcription factor daf-16, in the same way the longevity of liv-7 (pv17). is suppressed by daf-16. This result allows us to think that liv-7 is a. negative regulator of the transcription factor daf-16.. We are now actively working in the mapping of liv-7 for its molecular. identification.

Zhao Y et al. (2007) BMC Genomics "Poly-G/poly-C tracts in the genomes of Caenorhabditis."

Zhao Y, O'Neil NJ, Rose AM

[BMC Genomics, 2007]

ABSTRACT: BACKGROUND: In the genome of Caenorhabditis elegans, homopolymeric poly-G/poly-C tracts (G/C tracts) exist at high frequency and are maintained by the activity of the DOG-1 protein. The frequency and distribution of G/C tracts in the genomes of C. elegans and the related nematode, C. briggsae were analyzed to investigate possible biological roles for G/C tracts. RESULTS: In C. elegans, G/C tracts are distributed along every chromosome in a non-random pattern. Most G/C tracts are within introns or are close to genes. Analysis of SAGE data showed that G/C tracts correlate with the levels of regional gene expression in C. elegans. G/C tracts are over-represented and dispersed across all chromosomes in another Caenorhabditis species, C. briggase. However, the positions and distribution of G/C tracts in C. briggsae differ from those in C. elegans. Furthermore, the C. briggsae dog-1 ortholog CBG19723 can rescue the mutator phenotype of C. elegans dog-1 mutants. CONCLUSIONS: The abundance and genomic distribution of G/C tracts in C. elegans, the effect of G/C tracts on regional transcription levels, and the lack of positional conservation of G/C tracts in C. briggsae suggest a role for G/C tracts in chromatin structure but not in the transcriptional regulation of specific genes.

Bhagwati P Gupta et al. (2002) West Coast Worm Meeting "Genetic analysis of the vulval mutants in C. briggsae"

Shahla Gharib, Bhagwati P Gupta, Paul W Sternberg, Jennifer X Li

[West Coast Worm Meeting, 2002]

To understand the evolution of developmental mechanisms, we are doing a comparative analysis of vulval patterning in C. elegans and C. briggsae. C. briggsae is closely related to C. elegans and has identical looking vulval morphology. However, recent studies have indicated subtle differences in the underlying mechanisms of development. The recent completion of C. briggsae genome sequence by the C. elegans Sequencing Consortium is extremely valuable in identifying the conserved genes between C. elegans and C. briggsae.