Loss of an H3K9me anchor rescues laminopathy-linked changes in nuclear organization and muscle function in an Emery-Dreifuss muscular dystrophy model.

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 C. elegans 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 cec-4 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.

Active chromatin marks drive spatial sequestration of heterochromatin in C. elegans nuclei.

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 integrity2,3. 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 H3K9me3,5, 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 factor that 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.

Analysis of the effect of the mitochondrial prohibitin complex, a context-dependent modulator of longevity, on the C. elegans metabolome.

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 ¹H 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 ¹H 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.

Expanded FLP toolbox for spatiotemporal protein degradation and transcriptomic profiling in Caenorhabditis elegans.

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 cyclization recombination (Cre) enzymes has proved 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 green fluorescent fusion proteins based on FLP-mediated recombination. Using 2 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 for the FLP toolbox and by focusing on a well-characterized tissue, the hypodermis, we show that the vast majority of genes identified by RNA polymerase DamID 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.

DamID identifies targets of CEH-60/PBX that are associated with neuron development and muscle structure in Caenorhabditis elegans.

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.

An Efficient FLP-Based Toolkit for Spatiotemporal Control of Gene Expression in Caenorhabditis elegans.

Site-specific recombinases are potent tools to regulate gene expression. In particular, the Cre (cyclization recombination) and FLP (flipase) enzymes are widely used to either activate or inactivate genes in a precise spatiotemporal manner. Both recombinases work efficiently in the popular model organism Caenorhabditis elegans, but their use in this nematode is still only sporadic. To increase the utility of the FLP system in C. elegans, we have generated a series of single-copy transgenic strains that stably express an optimized version of FLP in specific tissues or by heat induction. We show that recombination efficiencies reach 100% in several cell types, such as muscles, intestine, and serotonin-producing neurons. Moreover, we demonstrate that most promoters drive recombination exclusively in the expected tissues. As examples of the potentials of the FLP lines, we describe novel tools for induced cell ablation by expression of the PEEL-1 toxin and a versatile FLP-out cassette for generation of GFP-tagged conditional knockout alleles. Together with other recombinase-based reagents created by the C. elegans community, this toolkit increases the possibilities for detailed analyses of specific biological processes at developmental stages inside intact animals.