Genome-wide analysis of the repertoire of TRIM genes in sea urchins

The eukaryotic TRIM (TRIpartite Motif) super-family represents one of the largest classes of putative E3 ubiquitin ligases involved in several processes, including epigenetic control of development and disease. In the post-genomic era, new approaches allow genome-wide studies of gene family. In particular, we performed a comprehensive analysis of the TRIM repertoire in selected sea urchin species. By combining iterations of ab initio predictions and pairwise comparative methods, we first retrieved the full complement of TRIM genes in Strongylocentrotus purpuratus, whose full genome sequence was available. Interestingly, such a DNA sequence set includes not previously classified, echinoderm-specific, TRIM genes as well as multiple copies of known ones. We also retrieved a landscape of cDNA sequences from staged EST libraries, indicating that most of these genes are actively transcribed during development. Phylogenetic analysis of the deduced proteins, using set of TRIMs from various species, revealed a degree of genetic variation between species. Worth of mention, we predicted the occurrence of transposition events involving some of these genes, according with the documented rapid evolution of this family. Next, we adopted heuristic algorithms and post-processing steps to investigate the evolutionarily distant Paracentrotus lividus, Allocentrotus fragilis and Lytechinus variegatus genomes, whose sequencing projects are actually in progress. We assembled partial pools of TRIM genes and specifically associated them to EST-derived cDNA sequences. Such a collection of data should provide a framework for unravel gene regulatory networks involving TRIM genes from an evolutionary perspective. Indeed, in the Pl species, we have previously isolated and functionally characterized the cDNA sequence encoding the first echinoderm TRIM factor, Strim1. Here, we identified five strim1 genes, all sharing a intronless organization, and roughly located their cis-regulatory apparatus

EFFECT OF Γ-AMINOBUTYRRIC ACID (GABA) EXPOSURE ON EMBRYOGENESIS OF PARACENTROTUS LIVIDUS AND IDENTIFICATION OF GABA-RECEPTOR GENES IN SEA URCHINS

Developmental processes are controlled by regulatory genes encoding for transcription factors and signaling molecules. Functional relationships between these genes are described by gene regulatory networks (GRN), models which allow integration of various levels of information. The sea urchin embryo is an experimental model system which offers many advantages for the analysis of GRN. Recently, the GRN that governs the biomineralization of the sea urchin embryonic skeleton has begun to be deciphered. Preliminary evidence suggest that the γ- aminobutyric acid (GABA) signaling pathway is involved in skeletal morphogenesis during development of the sea urchin. GABA is a molecule synthesized by nearly all organism, from bacteria to humans, and it acts through ionotropic and metabotropic receptors (GABAA-Rs and GABAB-Rs, respectively). We report that Paracentrotus lividus embryos exposed to GABA at concentrations ranging from 0.01 to 1.0 mM showed aberrations in axial patterning, with a dose dependent effect. Washout experiments allowed to determine that the period of sensitivity is restricted from the blastula to the gastrula stage. In order to identify GABA-R genes we performed a comprehensive in silico analysis in selected sea urchin species (P. lividus, Strongylocentrotus purpuratus, and Lytechinus variegatus), and in phylogenetically related organisms, such as the hemichordate Saccoglossus kowalevskii, the chordate Ciona intestinalis, and the nematode Caenorhabditis elegans. By combining iteration of ab initio predictions and pairwise comparative methods, we identified the orthologous genes encoding for GABAB1 and GABAB2, the two subunits which assemble GABAB-R, and we confirmed that all of these organisms possess a unique α/β GABAA-R gene pair clustered in the genome. Furthermore, we have observed that the reciprocal disposition of GABAA-R genes is also evolutionarily conserved. Interestingly, in adjacent position to these genes, we have identified an additional gene, which shows significant sequence similarity to a invertebrate-specific GABAA-R gene. Indeed, such a gene has been only identified in C. elegans, Drosophila melanogaster, and Nematostella vectensis. We also retrieved several cDNA sequences from staged EST databases of the three sea urchin species inspected, indicating that these genes are actively transcribed during development. Some selected cDNA plasmids were also isolated from P. lividus total RNA samples and fully sequenced. Hypothetical proteins were deduced and used for phylogenetic analysis, including a selection of vertebrate and invertebrate GABAA-R subunit sequences. The resulting phylogenetic tree strongly support the hypothesis that the sea urchins contain genes encoding for both canonical and invertebrate-specific GABAA-R subunits. Such a collection of data should provide a support to better understand the involvement of GABA-signalling pathway in the skeletal GR

The Compass-like Locus, Exclusive to the Ambulacrarians, Encodes a Chromatin Insulator Binding Protein in the Sea Urchin Embryo

Chromatin insulators are eukaryotic genome elements that upon binding of specific proteins display barrier and/or enhancer-blocking activity. Although several insulators have been described throughout various metazoans, much less is known about proteins that mediate their functions. This article deals with the identification and functional characterization in Paracentrotus lividus of COMPASS-like (CMPl), a novel echinoderm insulator binding protein. Phylogenetic analysis shows that the CMPl factor, encoded by the alternative spliced Cmp/Cmpl transcript, is the founder of a novel ambulacrarian-specific family of Homeodomain proteins containing the Compass domain. Specific association of CMPl with the boxB cis-element of the sns5 chromatin insulator is demonstrated by using a yeast one-hybrid system, and further corroborated by ChIP-qPCR and trans-activation assays in developing sea urchin embryos. The sns5 insulator lies within the early histone gene cluster, basically between the H2A enhancer and H1 promoter. To assess the functional role of CMPl within this locus, we challenged the activity of CMPl by two distinct experimental strategies. First we expressed in the developing embryo a chimeric protein, containing the DNA-binding domain of CMPl, which efficiently compete with the endogenous CMPl for the binding to the boxB sequence. Second, to titrate the embryonic CMPl protein, we microinjected an affinity-purified CMPl antibody. In both the experimental assays we congruently observed the loss of the enhancer-blocking function of sns5, as indicated by the specific increase of the H1 expression level. Furthermore, microinjection of the CMPl antiserum in combination with a synthetic mRNA encoding a forced repressor of the H2A enhancer-bound MBF1 factor restores the normal H1 mRNA abundance. Altogether, these results strongly support the conclusion that the recruitment of CMPl on sns5 is required for buffering the H1 promoter from the H2A enhancer activity, and this, in turn, accounts for the different level of accumulation of early linker and nucleosomal transcripts

Constitutive Promoter Occupancy by the MBF-1 Activator and Chromatin Modification of the Developmental Regulated Sea Urchin alpha-H2A Histone Gene

The tandemly repeated sea urchin α-histone genes are developmentally regulated. These genes are transcribed up to the early blastula stage and permanently silenced as the embryos approach gastrulation. As previously described, expression of the α-H2A gene depends on the binding of the MBF-1 activator to the 5′ enhancer, while down-regulation relies on the functional interaction between the 3′ sns 5 insulator and the GA repeats located upstream of the enhancer. As persistent MBF-1 binding and enhancer activity are detected in gastrula embryos, we have studied the molecular mechanisms that prevent the bound MBF-1 from trans-activating the H2A promoter at this stage of development. Here we used chromatin immunoprecipitation to demonstrate that MBF-1 occupies its site regardless of the transcriptional state of the H2A gene. In addition, we have mapped two nucleosomes specifically positioned on the enhancer and promoter regions of the repressed H2A gene. Interestingly, insertion of a 26 bp oligonucleotide between the enhancer and the TATA box, led to upregulation of the H2A gene at gastrula stage, possibly by changing the position of the TATA nucleosome. Finally, we found association of histone de-acetylase and de-acetylation and methylation of K9 of histone H3 on the promoter and insulator of the repressed H2A chromatin. These data argue for a role of a defined positioned nucleosome in the promoter and histone tail post-translational modifications, in the 3′ insulator and 5′ regulatory regions, in the repression of the α-H2A gene despite the presence of the MBF-1 activator bound to the enhance

The sea urchin embryo: A model to study Alzheimer’s beta amyloid induced toxicity

Alzheimer’s disease (AD) is the most common form of dementia. The cause of AD is closely related to the accumulation of amyloid beta peptide in the neuritic plaques. The use of animal model systems represents a good strategy to elucidate the molecular mechanism behind the development of this pathology. Here we use the Paracentrotus lividus embryo to identify molecules and pathways that can be involved in the degenerative process. As a first step, we identified the presence of an antigen related to the human APP, called PlAPP. This antigen, after gastrula stage, is processed producing a polypeptide of about 10 kDa. By immunohistochemistry we localized the PlAPP antigen in some serotonin expressing cells. Similarly, after 48 or 96 h incubation, a recombinant b-amyloid peptide, rAb42, accumulates around the intestinal tube and oesophagus. In addition, incubation of sea urchin embryos with two different solutions rich in oligomers and fibrillar aggregates of rAb42 induce activation of apoptosis as detected by TUNEL assay. Moreover, we demonstrate that aggregates induce apoptosis by extrinsic pathway activation, whereas oligomers induce apoptosis both by extrinsic and intrinsic pathway activation. Utilizing an apoptotic inhibitor, caspases activation was offset and morphological damage rescued. Taken together all these observations suggest that the sea urchin may be a simple and suitable model to characterize the mechanism underlining the cytotoxicity of Ab42