A novel nuclear receptor subfamily enlightens the origin of heterodimerization.

BACKGROUND: Nuclear receptors are transcription factors of central importance in human biology and associated diseases. Much of the knowledge related to their major functions, such as ligand and DNA binding or dimerization, derives from functional studies undertaken in classical model animals. It has become evident, however, that a deeper understanding of these molecular functions requires uncovering how these characteristics originated and diversified during evolution, by looking at more species. In particular, the comprehension of how dimerization evolved from ancestral homodimers to a more sophisticated state of heterodimers has been missing, due to a too narrow phylogenetic sampling. Here, we experimentally and phylogenetically define the evolutionary trajectory of nuclear receptor dimerization by analyzing a novel NR7 subgroup, present in various metazoan groups, including cnidarians, annelids, mollusks, sea urchins, and amphioxus, but lost in vertebrates, arthropods, and nematodes. RESULTS: We focused on NR7 of the cephalochordate amphioxus B. lanceolatum. We present a complementary set of functional, structural, and evolutionary analyses that establish that NR7 lies at a pivotal point in the evolutionary trajectory from homodimerizing to heterodimerizing nuclear receptors. The crystal structure of the NR7 ligand-binding domain suggests that the isolated domain is not capable of dimerizing with the ubiquitous dimerization partner RXR. In contrast, the full-length NR7 dimerizes with RXR in a DNA-dependent manner and acts as a constitutively active receptor. The phylogenetic and sequence analyses position NR7 at a pivotal point, just between the basal class I nuclear receptors that form monomers or homodimers on DNA and the derived class II nuclear receptors that exhibit the classical DNA-independent RXR heterodimers. CONCLUSIONS: Our data suggest that NR7 represents the "missing link" in the transition between class I and class II nuclear receptors and that the DNA independency of heterodimer formation is a feature that was acquired during evolution. Our studies define a novel paradigm of nuclear receptor dimerization that evolved from DNA-dependent to DNA-independent requirements. This new concept emphasizes the importance of DNA in the dimerization of nuclear receptors, such as the glucocorticoid receptor and other members of this pharmacologically important oxosteroid receptor subfamily. Our studies further underline the importance of studying emerging model organisms for supporting cutting-edge research.

Ultra-structural time-course study in the C. elegans model for Duchenne muscular dystrophy highlights a crucial role for sarcomere-anchoring structures and sarcolemma integrity in the earliest steps of the muscle degeneration process.

Duchenne muscular dystrophy (DMD) is a genetic disease characterized by progressive muscle degeneration due to mutations in the dystrophin gene. In spite of great advances in the design of curative treatments, most patients currently receive palliative therapies with steroid molecules such as prednisone or deflazacort thought to act through their immunosuppressive properties. These molecules only slightly slow down the progression of the disease and lead to severe side effects. Fundamental research is still needed to reveal the mechanisms involved in the disease that could be exploited as therapeutic targets. By studying a Caenorhabditis elegans model for DMD, we show here that dystrophin-dependent muscle degeneration is likely to be cell autonomous and affects the muscle cells the most involved in locomotion. We demonstrate that muscle degeneration is dependent on exercise and force production. Exhaustive studies by electron microscopy allowed establishing for the first time the chronology of subcellular events occurring during the entire process of muscle degeneration. This chronology highlighted the crucial role for dystrophin in stabilizing sarcomeric anchoring structures and the sarcolemma. Our results suggest that the disruption of sarcomeric anchoring structures and sarcolemma integrity, observed at the onset of the muscle degeneration process, triggers subcellular consequences that lead to muscle cell death. An ultra-structural analysis of muscle biopsies from DMD patients suggested that the chronology of subcellular events established in C. elegans models the pathogenesis in human. Finally, we found that the loss of sarcolemma integrity was greatly reduced after prednisone treatment suggesting a role for this molecule in plasma membrane stabilization.

Reliable CRISPR/Cas9 Genome Engineering in Caenorhabditis elegans Using a Single Efficient sgRNA and an Easily Recognizable Phenotype.

CRISPR/Cas9 genome engineering strategies allow the directed modification of the Caenorhabditis elegans genome to introduce point mutations, generate knock-out mutants, and insert coding sequences for epitope or fluorescent tags. Three practical aspects, however, complicate such experiments. First, the efficiency and specificity of single-guide RNAs (sgRNA) cannot be reliably predicted. Second, the detection of animals carrying genome edits can be challenging in the absence of clearly visible or selectable phenotypes. Third, the sgRNA target site must be inactivated after editing to avoid further double-strand break events. We describe here a strategy that addresses these complications by transplanting the protospacer of a highly efficient sgRNA into a gene of interest to render it amenable to genome engineering. This sgRNA targeting the dpy-10 gene generates genome edits at comparatively high frequency. We demonstrate that the transplanted protospacer is cleaved at the same time as the dpy-10 gene. Our strategy generates scarless genome edits because it no longer requires the introduction of mutations in endogenous sgRNA target sites. Modified progeny can be easily identified in the F1 generation, which drastically reduces the number of animals to be tested by PCR or phenotypic analysis. Using this strategy, we reliably generated precise deletion mutants, transcriptional reporters, and translational fusions with epitope tags and fluorescent reporter genes. In particular, we report here the first use of the new red fluorescent protein mScarlet in a multicellular organism. wrmScarlet, a C. elegans-optimized version, dramatically surpassed TagRFP-T by showing an eightfold increase in fluorescence in a direct comparison.

Identification, evolution and expression of an insulin-like peptide in the cephalochordate Branchiostoma lanceolatum.

Insulin is one of the most studied proteins since it is central to the regulation of carbohydrate and fat metabolism in vertebrates and its expression and release are disturbed in diabetes, the most frequent human metabolic disease worldwide. However, the evolution of the function of the insulin protein family is still unclear. In this study, we present a phylogenetic and developmental analysis of the Insulin Like Peptide (ILP) in the cephalochordate amphioxus. We identified an ILP in the European amphioxus Branchiostoma lanceolatum that displays structural characteristics of both vertebrate insulin and Insulin-like Growth Factors (IGFs). Our phylogenetic analysis revealed that amphioxus ILP represents the sister group of both vertebrate insulin and IGF proteins. We also characterized both temporal and spatial expression of ILP in amphioxus. We show that ilp is highly expressed in endoderm and paraxial mesoderm during development, and mainly expressed in the gut of both the developing embryo and adult. We hypothesize that ILP has critical implications in both developmental processes and metabolism and could display IGF- and insulin-like functions in amphioxus supporting the idea of a common ancestral protein.

ZYX-1, the unique zyxin protein of Caenorhabditis elegans, is involved in dystrophin-dependent muscle degeneration.

In vertebrates, zyxin is a LIM-domain protein belonging to a family composed of seven members. We show that the nematode Caenorhabditis elegans has a unique zyxin-like protein, ZYX-1, which is the orthologue of the vertebrate zyxin subfamily composed of zyxin, migfilin, TRIP6, and LPP. The ZYX-1 protein is expressed in the striated body-wall muscles and localizes at dense bodies/Z-discs and M-lines, as well as in the nucleus. In yeast two-hybrid assays ZYX-1 interacts with several known dense body and M-line proteins, including DEB-1 (vinculin) and ATN-1 (α-actinin). ZYX-1 is mainly localized in the middle region of the dense body/Z-disk, overlapping the apical and basal regions containing, respectively, ATN-1 and DEB-1. The localization and dynamics of ZYX-1 at dense bodies depend on the presence of ATN-1. Fluorescence recovery after photobleaching experiments revealed a high mobility of the ZYX-1 protein within muscle cells, in particular at dense bodies and M-lines, indicating a peripheral and dynamic association of ZYX-1 at these muscle adhesion structures. A portion of the ZYX-1 protein shuttles from the cytoplasm into the nucleus, suggesting a role for ZYX-1 in signal transduction. We provide evidence that the zyx-1 gene encodes two different isoforms, ZYX-1a and ZYX-1b, which exhibit different roles in dystrophin-dependent muscle degeneration occurring in a C. elegans model of Duchenne muscular dystrophy.

The cephalochordate amphioxus: a key to reveal the secrets of nuclear receptor evolution.

The members of the nuclear receptor (NR) superfamily are transcription factors characterized by a particular mode of function, which is related to the conserved nature of their molecular structure. NR proteins usually contain a DNA-binding domain (DBD) and a ligand-binding domain (LBD) allowing them to directly bind to DNA and regulate target gene expression in a ligand-dependent manner. In this review, we are summarizing our current understanding of the NR diversity in the cephalochordate amphioxus, which represents the best available proxy for the last common chordate ancestor both in terms of morphology and genome organization. The amphioxus genome encodes 33 NRs, which is more than expected based on its phylogenetic position, with at least one representative of all major NR groups, excepting NR1E and NR1I/J. This elevated number of receptor genes shows that the amphioxus NR complement has experienced some secondary modifications that are most evident in the NR1H group, which is characterized by three members in humans and ten representatives in amphioxus. By highlighting specific examples of the NR repertoire, including the receptors for retinoic acid, thyroid hormone, estrogen and steroids as well as the bile acid and oxysterol receptors of the NR1H group, we are illustrating the functional diversity of these receptors in amphioxus. We conclude that the amphioxus NRs are valuable models for assessing the evolutionary interplay between receptors and their ligands and that more integrative and comparative approaches are required for assessment of the evolutionary plasticity of receptor-ligand interactions revealed by the studies of amphioxus NRs.

DYC-1, a protein functionally linked to dystrophin in Caenorhabditis elegans is associated with the dense body, where it interacts with the muscle LIM domain protein ZYX-1.

In Caenorhabditis elegans, mutations of the dystrophin homologue, dys-1, produce a peculiar behavioral phenotype (hyperactivity and a tendency to hypercontract). In a sensitized genetic background, dys-1 mutations also lead to muscle necrosis. The dyc-1 gene was previously identified in a genetic screen because its mutation leads to the same phenotype as dys-1, suggesting that the two genes are functionally linked. Here, we report the detailed characterization of the dyc-1 gene. dyc-1 encodes two isoforms, which are expressed in neurons and muscles. Isoform-specific RNAi experiments show that the absence of the muscle isoform, and not that of the neuronal isoform, is responsible for the dyc-1 mutant phenotype. In the sarcomere, the DYC-1 protein is localized at the edges of the dense body, the nematode muscle adhesion structure where actin filaments are anchored and linked to the sarcolemma. In yeast two-hybrid assays, DYC-1 interacts with ZYX-1, the homologue of the vertebrate focal adhesion LIM domain protein zyxin. ZYX-1 localizes at dense bodies and M-lines as well as in the nucleus of C. elegans striated muscles. The DYC-1 protein possesses a highly conserved 19 amino acid sequence, which is involved in the interaction with ZYX-1 and which is sufficient for addressing DYC-1 to the dense body. Altogether our findings indicate that DYC-1 may be involved in dense body function and stability. This, taken together with the functional link between the C. elegans DYC-1 and DYS-1 proteins, furthermore suggests a requirement of dystrophin function at this structure. As the dense body shares functional similarity with both the vertebrate Z-disk and the costamere, we therefore postulate that disruption of muscle cell adhesion structures might be the primary event of muscle degeneration occurring in the absence of dystrophin, in C. elegans as well as vertebrates.

The C. elegans dense body: anchoring and signaling structure of the muscle.

During evolution, both the architecture and the cellular physiology of muscles have been remarkably maintained. Striated muscles of invertebrates, although less complex, strongly resemble vertebrate skeletal muscles. In particular, the basic contractile unit called the sarcomere is almost identical between vertebrates and invertebrates. In vertebrate muscles, sarcomeric actin filaments are anchored to attachment points called Z-disks, which are linked to the extra-cellular matrix (ECM) by a muscle specific focal adhesion site called the costamere. In this review, we focus on the dense body of the animal model Caenorhabditis elegans. The C. elegans dense body is a structure that performs two in one roles at the same time, that of the Z-disk and of the costamere. The dense body is anchored in the muscle membrane and provides rigidity to the muscle by mechanically linking actin filaments to the ECM. In the last few years, it has become increasingly evident that, in addition to its structural role, the dense body also performs a signaling function in muscle cells. In this paper, we review recent advances in the understanding of the C. elegans dense body composition and function.