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Drosophila GAGA factor (GAF) is a nuclear protein, conserved along evolution, with multiple roles in gene regulation, chromatin remodelling, Polycomb-dependent silencing, and insulator functions (1). GAF recognizes and specifically binds GAGAG consensus DNA motif by its C2H2-type zinc-finger domain, and interacts with other regulatory factors by its BTB/POZ domain. We have identified plGaga, a cDNA coding for a putative GAF of the sea urchin P. lividus, which showed significant sequence similarity with Drosophila and vertebrate GAFs. Real time RT-PCR revealed that the plGaga RNA is always present during embryo development decreasing rapidly in abundance at the larval pluteus stage. We raised a specific antibody against the sea urchin GAF and used it in whole-mount immunofluorescence assays, showing that the protein was distributed predominantly to the nuclei of the whole embryo. By Chromatin Immunoprecipitation experiments, we showed that the sea urchin GAF not only binds the sns5 insulator, which is essential for the regulation of the sea urchin early-histone genes (2,3), but also to other GAGA containing sequences in the early-histone gene cluster. 1 Matharu NK et al. 2010 JMB 400 (3): 434–447 2 Melfi R et Al. 2001 JMB (1); 304:753-63 3 Cavalieri V et al. 2013 Nucleic Acids Res 37: 7407-7415

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.

The building of the skeleton in the indirect developing sea urchin embryo is a complex morphogenetic process that is executed by the Primary Mesenchyme Cells or PMCs (Ettensohn et al, 1997; Wilt 2002). It is well known that the PMCs acquire most of the positional and temporal information from the overlying ectoderm for skeletal initiation and growth (Armstrong 1993; Cavalieri et al, 2003; Röttinger et al, 2008). In this study, we analyze the function of a novel gene, encoding for a tripartite motif-containing (TRIM) protein denoted strim1, that adds up to the list of genes constituting the epithelial-mesenchymal signaling network. We show that strim1 is expressed in ectoderm regions adjacent to the bilateral clusters of PMCs. Strim1 misexpression causes the number of PMCs to double and leads to skeletal abnormalities. By micromere transplantations, we establish that skeletal defects depend upon strim1 misexpression in ectoderm cells. Reciprocally, knock-down of strim1 function abrogates PMC migration and blocks skeletogenesis. Identical phenotype is shown by chimeras in which strim1 function is blocked selectively in the ectoderm. We also show that clonal expression of strim1 into ectoderm cells from knocked-down embryos restores the correct skeletogenic program. Finally, we report that strim1 triggers the expression of the ectodermspecific gene pax2/5/8, and the PMC marker sm30 (an ectoderm signaling dependent gene). We conclude that strim1 function is able to elicit specific gene expression both in ectoderm cells and PMCs to guide the biomineralization during morphogenesis. References Armstrong, N, Hardin, J and McClay, DR (1993). Cell-cell interactions regulate skeleton formation in the sea urchin embryo. Development 119, 833-40. Cavalieri, V, Spinelli, G and Di Bernardo, M (2003). Impairing Otp homeodomain function in oral ectoderm cells affects skeletogenesis in sea urchin embryos. Dev Biol 262, 107-18. Ettensohn, CA, Guss, KA, Hodor, PG, and Malinda, KM (1997). The morphogenesis of the skeletal system of the sea urchin embryo, in: Collier JR (Ed.), Reproductive Biology of Invertebrates, vol. VII: Progress in Developmental Biology, Oxford & IBH publishing Co. Pvt. Ltd. New Delhi, Calcutta. 225-265. Röttinger, E, Saudemont, A, Duboc, V, Besnardeau, L, McClay, D and Lepage, T (2008). FGF signals guide migration of mesenchymal cells, control skeletal morphogenesis and regulate gastrulation during sea urchin development. Development 135, 353-65. Wilt, FH (2002). Biomineralization of the spicules of sea urchin embryos. Zoolog Sci 19, 253-61.

Transposons constitute a significant component of the eukaryotic genome. Among others, Helitrons represent a novel major class of eukaryotic transposons, and are fundamentally different from classical ones in terms of their structure and mechanism of transposition [1]. In particular, Helitrons constitute ~1% of the sea urchin genome [2]. By a in silico approach focused on the genome of the Mediterranean sea urchin Paracentrotus lividus, we have predicted regions of high sequence identity to a Helitron-N2 (HeN2) element in the strim1 locus. Of interest, HeN2 sequences lies within the 5’ non coding region of the strim1a and strim1b genes, spanning from -105 to -2255 (the start codon ATG of strim1 is defined as +1). As reported in other species, HeN2 is a non autonomous transposable element, lacking the sequences coding for the DNA polymerase and Helicase enzymes. A deep computational inspection of the HeN2 element revealed its structure. It consists of two terminal palindromic sequences (U5 and U3), and eleven tandemly arranged 155bp-long direct repeats (DR1-11). It is well known that the TRIMcontaining gene family, to which strim1 belongs, is widely complex and heterogeneous due to the combination of exon shuffling, duplication and/or deletion events that likely occurred during evolution [3]. Interestingly, Helitrons are a formidable evolutionary tool owing to their ability to capture host genes [4] and transposition plays important roles in the evolution of duplicated genes [5]. Therefore our finding could suggest a mechanism for the evolution of the TRIM multigenic family. References 1. Kapitonov VV and Jurka J (2007). Helitrons on a roll: eukaryotic rollingcircle transposons. TRENDS in Genetics 23(10), 521-9. 2. Kapitonov VV, Jurka J (2005). Helitron-N2_SP, a family of nonautonomous Helitrons in the sea urchin genome. Repbase Reports 5(11), 395-395. 3. Sardiello, M, Cairo, S, Fontanella, M, Ballabio, A, and Meroni, G (2008). Genomic analysis of the TRIM family reveals two groups of genes with distinct evolutionary properties. BMC Evolutionary Biology 8:225- 4. Gupta S, Gallavotti A, Stryker GA, Schmidt RJ, Lal SK (2005). A novel class of Helitron-related transposable elements in maize contain portions of multiple pseudogenes. Plant Mol Biol 57, 115-27. 5. Han, MV, Demuth, JP, McGrath, CL, Casola, C and Hahn MW (2009). Adaptive evolution of young gene duplicates in mammals. Genome Res 19(5):859-67.

The recent wealth of genomic data has confirmed that bilaterians as simple as nematodes and as complex as humans use the same basic toolkit of transcription factors and signaling molecules to process spatial and temporal information during development (1,2). Among these regulators, the TRIM/RBCC (Tripartite motif/RING-Bbox-Coiled Coil) family represents one of the largest classes of putative E3 ubiquitin ligases that play essential roles in cell cycle regulation and development (3). It consists of rapidly evolving genes, identified in eukaryotic organisms (4,5). In the sea urchin Paracentrotus lividus we have identified a locus containing two copies, probably originated from a duplication event, of a gene coding for a TRIM-containing factor that we named strim (sea urchin TRIM). Both genes consist of a single 1839bp-long exon and share extensive sequence similarity. As embryogenesis occurs, zygotic transcription of strim starts at the very early cleavage and reaches its maximum at the morula stage. From the gastrula stage onward, strim expression fades out and very low amounts of mRNA are detectable, by RT-PCR, at prism stage. Very interestingly, whole mount in situ hybridization showed that strim transcripts are asymmetrically localized in the embryo, being restricted exclusively to the oral ectoderm. These results could suggest a role for strim in cell specification of the oral domain.

Enhancers are DNA elements which increase the transcription of associated gene in a position and distance independent manner relative to the transcription initiation site. Molecular mechanisms must operate to direct enhancers to specific promoters in complex genetic loci. The sea urchin a-histone genes are organized in several hundred tandem repeated units, each containing one copy of the five histone genes in the order 5’-H4-H2B-H3-H2A-H1-3’. Transcription is limited to the early cleavage and reaches its maximum at morula stage. After hatching these genes are repressed and maintained in the silenced state for whole life cycle of the animal. In Paracentrotus lividus, the MBF-1 activator binds to a 30 bp sequence of the H2A modulator, the only cis-acting element of the histone unit for which an enhancer activity has unambiguously demonstrated. Here we assessed the specificity of the MBF-1 factor and the molecular mechanisms that eventually limit its interaction with the adjacent promoters. We find by Chromatin ImmunePrecipitation and in vivo functional studies that the MBF-1 activator binds both the H3 and H2A regulatory sequences, suggesting that this activator is needed for the maximal expression of both genes. In addition, we find that the enhancer blocking activity of the sns5 insulator buffers the downstream H1 promoter from the H2A enhancer bound MBF-1 factor. These results suggest that the block of the H2A enhancer by the sns5 insulator might be the mechanism that regulate the ratio of 2 to 1 of core to linker histones during early development of the sea urchin embryo.

In the sea urchin embryo, a number of genes encoding transcription factors display territorial restricted expression. Among these, the zygotic Hbox12 homeobox gene is transiently transcribed in a limited number of cells of the animal-lateral half of the early Paracentrotus lividus embryo, whose descendants will constitute part of the ectoderm territory. We have explored the cis-regulatory apparatus of the gene and found that the intergenic region of the tandem Hbox12 repeats drives GFP expression in the presumptive aboral ectoderm and that a 234 bp fragment, defined Aboral Ectoderm Module, accounts for the restricted expression of the transgene. Within this module, a consensus sequence for a Sox factor and the binding of the Otx activator are both required for correct Hbox12 gene expression. Site-directed mutagenesis of the cis-regulatory elements, along with loss of function assays and ChIP analysis, strongly suggested that spatial restriction of the Hbox12 gene to the aboral ectoderm is achieved by a combination of different repressive sequence elements. Among them, we identified a Myb-like consensus, which prevents ectopic expression in the oral ectoderm cells. Further negative sequence elements necessary for repression in the endomesoderm map within the most upstream 60 bp region, while down-regulation at the gastrula stage depends on a multiple GA repeat-containing region. Altogether, these results suggest a role for Hbox12 in aboral ectoderm specification and represent our first attempt in the identification of the gene regulatory circuits involved in this process.

Core promoters and chromatin insulators (ins) may direct a transcriptional enhancer (enh) to prefer a specific promoter in complex genetic loci. Enh and ins flank the sea urchin Paracentrotus lividus α-histone H2A transcription unit in a tandem repeated cluster containing the five histone genes. In vivo competition assays of enh and ins functions reveal that the H2A enh-bound MBF-1 activator participates also in the expression of the H3 gene and that the sns5 ins buffers the downstream H1 promoter from the H2A enh. These results suggest that both the H2A enh and the sns5 ins may account for the diverse accumulation of the linker vs core nucleosomal histones during early development of the sea urchin embryo. Finally, preliminary results indicate that non-coding RNAs originate from the insulator region.