Sea Urchin Genome Research Articles

Sea urchin immune cells as sentinels of environmental stress

Echinoderms, an ancient and very successful phylum of marine invertebrates, play a central role in the maintenance of ecosystem integrity and are constantly exposed to environmental pressure, including: predation, changes in temperature and pH, hypoxia, pathogens, UV radiation, metals, toxicants, and emerging pollutants like nanomaterials. The annotation of the sea urchin genome, so closely related to humans and other vertebrate genomes, revealed an unusually complex immune system, which may be the basis for why sea urchins can adapt to different marine environments and survive even in hazardous conditions. In this review, we give a brief overview of the morphological features and recognized functions of echinoderm immune cells with a focus on studies correlating stress and immunity in the sea urchin. Immune cells from adult Paracentrotus lividus, which have been introduced in the last fifteen years as sentinels of environmental stress, are valid tools to uncover basic molecular and regulatory mechanisms of immune responses, supporting their use in immunological research. Here we summarize laboratory and field studies that reveal the amenability of sea urchin immune cells for toxicological testing.

In silico determination of the effect of multi-target drugs on calcium dynamics signaling network underlying sea urchin spermatozoa motility.

The motility of spermatozoa of both Lytechinus pictus and Strongylocentrotus purpuratus sea urchin species is modulated by the egg-derived decapeptide speract via an oscillatory [Ca2+]-dependent signaling pathway. Comprehension of this pathway is hence directly related to the understanding of regulated sperm swimming. Niflumic acid (NFA), a nonsteroidal anti-inflammatory drug alters several ion channels. Though unspecific, NFA profoundly affects how sea urchin sperm respond to speract, increasing the [Ca2+]i oscillation period, amplitude, peak and average level values of the responses in immobilized and swimming cells. A previous logical network model we developed for the [Ca2+] dynamics of speract signaling cascade in sea urchin sperm allows integrated dissection of individual and multiple actions of NFA. Among the channels affected by NFA are: hyperpolarization-activated and cyclic nucleotide gated Na+ channels (HCN), [Ca2+]-dependent Cl− channels (CaCC) and [Ca2+]-dependent K+ channels (CaKC), all present in the sea urchin genome. Here, using our model we investigated the effect of blocking in silico HCN and CaCC channels suggested by experiments. Regarding CaKC channels, arguments can be provided for either their blockage or activation by NFA. Our study yielded two scenarios compliant with experimental observations: i) under CaKC inhibition, this [Ca2+]-dependent K+ channel should be different from the Slo1 channel and ii) under activation of the CaKC channel, another [Ca2+] channel not considered previously in the network is required, such as the pH-dependent CatSper channel. Additionally, our findings predict cause-effect relations resulting from a selective inhibition of those channels. Knowledge of these relations may be of consequence for a variety of electrophysiological studies and have an impact on drug related investigations. Our study contributes to a better grasp of the network dynamics and suggests further experimental work.

Effects of seawater acidification on gene expression: resolving broader-scale trends in sea urchins.

Sea urchins are ecologically and economically important calcifying organisms threatened by acidification of the global ocean caused by anthropogenic CO2 emissions. Propelled by the sequencing of the purple sea urchin (Strongylocentrotus purpuratus) genome, profiling changes in gene expression during exposure to high pCO2 seawater has emerged as a powerful and increasingly common method to infer the response of urchins to ocean change. However, analyses of gene expression are sensitive to experimental methodology, and comparisons between studies of genes regulated by ocean acidification are most often made in the context of major caveats. Here we perform meta-analyses as a means of minimizing experimental discrepancies and resolving broader-scale trends regarding the effects of ocean acidification on gene expression in urchins. Analyses across eight studies and four urchin species largely support prevailing hypotheses about the impact of ocean acidification on marine calcifiers. The predominant expression pattern involved the down-regulation of genes within energy-producing pathways, a clear indication of metabolic depression. Genes with functions in ion transport were significantly over-represented and are most plausibly contributing to intracellular pH regulation. Expression profiles provided extensive evidence for an impact on biomineralization, epitomized by the down-regulation of seven spicule matrix proteins. In contrast, expression profiles provided limited evidence for CO2-mediated developmental delay or induction of a cellular stress response. Congruence between studies of gene expression and the ocean acidification literature in general validates the accuracy of gene expression in predicting the consequences of ocean change and justifies its continued use in future studies.

High opsin diversity in a non-visual infaunal brittle star.

BackgroundIn metazoans, opsins are photosensitive proteins involved in both vision and non-visual photoreception. Echinoderms have no well-defined eyes but several opsin genes were found in the purple sea urchin (Strongylocentrotus purpuratus) genome. Molecular data are lacking for other echinoderm classes although many species are known to be light sensitive.ResultsIn this study focused on the European brittle star Amphiura filiformis, we first highlighted a blue-green light sensitivity using a behavioural approach. We then identified 13 new putative opsin genes against eight bona fide opsin genes in the genome of S. purpuratus. Six opsins were included in the rhabdomeric opsin group (r-opsins). In addition, one putative ciliary opsin (c-opsin), showing high similarity with the c-opsin of S. purpuratus (Sp-opsin 1), one Go opsin similar to Sp-opsins 3.1 and 3.2, two basal-branch opsins similar to Sp-opsins 2 and 5, and two neuropsins similar to Sp-opsin 8, were identified. Finally, two sequences from one putative RGR opsin similar to Sp-opsin 7 were also detected. Adult arm transcriptome analysis pinpointed opsin mRNAs corresponding to one r-opsin, one neuropsin and the homologue of Sp-opsin 2. Opsin phylogeny was determined by maximum likelihood and Bayesian analyses. Using antibodies designed against c- and r-opsins from S. purpuratus, we detected putative photoreceptor cells mainly in spines and tube feet of A. filiformis, respectively. The r-opsin expression pattern is similar to the one reported in S. purpuratus with cells labelled at the tip and at the base of the tube feet. In addition, r-opsin positive cells were also identified in the radial nerve of the arm. C-opsins positive cells, expressed in pedicellariae, spines, tube feet and epidermis in S. purpuratus were observed at the level of the spine stroma in the brittle star.ConclusionLight perception in A. filiformis seems to be mediated by opsins (c- and r-) in, at least, spines, tube feet and in the radial nerve cord. Other non-visual opsin types could participate to the light perception process indicating a complex expression pattern of opsins in this infaunal brittle star.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1035) contains supplementary material, which is available to authorized users.

SGEBP, a giant protein from starfish oocytes able to interact with ERK

The mitogen-activated protein kinase (MAPK) pathway is a key regulator of animal meiotic divisions. It involves cascades of kinases whose specificity has been shown to depend on binding proteins acting as scaffolds. We searched for proteins interacting with starfish extracellular signal-regulated kinase (ERK) using the yeast two-hybrid system. An interacting clone was found to encode the 5' region of a giant 16.7-kb transcript encoded by an intronless gene. The corresponding 630-kDa protein could not be detected by Western blot, but the meiotic spindle was labelled by immunolocalization with an antibody against the ERK-binding domain. A related gene was found in the genome of another starfish species, and similarities were also found to a 42.9-kb open reading frame in the sea urchin genome. Yet, no conserved protein-binding domain was detected in the amino acid sequence(s) compared to all the known motifs. Structure prediction software indicated that the encoded proteins are probably disordered while a query of the disordered protein database indicated some similarity with vertebrates microtubule-associated protein 2 (MAP2). This predicts that SGEBP may function as a space-filling polymer, having a role in both cytoskeleton organization and ERK targeting.

Evolution of yolk protein genes in the Echinodermata

Vitellogenin genes (vtg) encode large lipid transfer proteins (LLTPs) that are typically female-specific, functioning as precursors to major yolk proteins (MYPs). Within the phylum Echinodermata, however, the MYP of the Echinozoa (Echinoidea + Holothuroidea) is expressed by an unrelated transferrin-like gene that has a reproductive function in both sexes. We investigated egg proteins in the Asterozoa (Asteroidea + Ophiuroidea), a sister clade to the Echinozoa, showing that eggs of the asteroid Parvulastra exigua contain a vitellogenin protein (Vtg). vtg is expressed by P. exigua, a species with large eggs and nonfeeding larvae, and by the related asterinid Patiriella regularis which has small eggs and feeding larvae. In the Asteroidea, therefore, the reproductive function of vtg is conserved despite significant life history evolution. Like the echinozoan MYP gene, asteroid vtg is expressed in both sexes and may play a role in the development of both ovaries and testes. Phylogenetic analysis indicated that a putative Vtg from the sea urchin genome, a likely pseudogene, does not clade with asteroid Vtg. We propose the following sequence as a potential pathway for the evolution of YP genes in the Echinodermata: (1) the ancestral echinoderm produced YPs derived from Vtg, (2) bisexual vtg expression subsequently evolved in the echinoderm lineage, (3) the reproductive function of vtg was assumed by a transferrin-like gene in the ancestral echinozoan, and (4) redundant echinozoan vtg was released from stabilizing selection.

Dynamic Evolution of Toll-Like Receptor Multigene Families in Echinoderms

The genome sequence of the purple sea urchin, Strongylocentrotus purpuratus, a large and long-lived invertebrate, provides a new perspective on animal immunity. Analysis of this genome uncovered a highly complex immune system in which the gene families that encode homologs of the pattern recognition receptors that form the core of vertebrate innate immunity are encoded in large multigene families. The sea urchin genome contains 253 Toll-like receptor (TLR) sequences, more than 200 Nod-like receptors and 1095 scavenger receptor cysteine-rich domains, a 10-fold expansion relative to vertebrates. Given their stereotypic protein structure and simple intron-exon architecture, the TLRs are the most tractable of these families for more detailed analysis. A role for these receptors in immune defense is suggested by their similarity to TLRs in other organisms, sequence diversity, and expression in immunologically active tissues, including phagocytes. The complexity of the sea urchin TLR multigene families is largely derived from expansions independent of those in vertebrates and protostomes, although a small family of TLRs with structure similar to that of Drosophila Toll can be traced to an ancient eumetazoan ancestor. Several other echinoderm sequences are now available, including Lytechinus variegatus, as well as partial sequences from two other sea urchin species. Here, we present an analysis of the invertebrate deuterostome TLRs with emphasis on the echinoderms. Representatives of most of the S. purpuratus TLR subfamilies and homologs of the mccTLR sequences are found in L. variegatus, although the L. variegatus TLR gene family is notably smaller (68 TLR sequences). The phylogeny of these genes within sea urchins highlights lineage-specific expansions at higher resolution than is evident at the phylum level. These analyses identify quickly evolving TLR subfamilies that are likely to have novel immune recognition functions and other, more stable, subfamilies that may function more similarly to those of vertebrates.

Expression pattern of polyketide synthase-2 during sea urchin development

Polyketide synthases (PKSs) are a large group of proteins responsible for the biosynthesis of polyketide compounds, which are mainly found in bacteria, fungi, and plants. Polyketides have a wide array of biological functions, including antibiotic, antifungal, predator defense, and light responses. In this study, we describe the developmental expression pattern of pks2, one of two pks found in the sea urchin genome. Throughout development, pks2 expression was restricted to skeletogenic cells and their precursors. Pks2 was first detected during the blastula stage. The transcript level peaked at hatched blastula, when all skeletogenic cell precursors expressed pks2. This was followed by a steady decline in expression in the skeletogenic cells on the aboral side of the embryo. By the prism stage, pks2 expression was limited to only 3–4 skeletogenic cells localized on the oral side.

Rho-kinase in sea urchin eggs and embryos

The activation of sea urchin eggs at fertilization provides an ideal system for studying the molecular events involved in cellular activation. Rho GTPases, which are key signaling enzymes in eukaryotes, are involved in sustaining the activation of sea urchin eggs; however, their downstream effectors have not yet been characterized. In somatic cells, RhoA regulates a serine/threonine kinase known as Rho-kinase (ROCK). The activity of ROCK in early sea urchin development has been inferred, but not tested directly. A ROCK gene was identified in the sea urchin (Strongylocentrotus purpuratus) genome and the sequence of its cDNA determined. The sea urchin ROCK (SpROCK) sequence predicts a protein of 158 kDa with >72% and 45% identities with different protein orthologues of the kinase catalytic domain and the complete protein sequence, respectively. SpROCK mRNA levels are high in unfertilized eggs and decrease to 35% after 15 min postfertilization and remain low up to the 4 cell stage. Antibodies to the human ROCK-I kinase domain revealed SpROCK to be concentrated in the cortex of eggs and early embryos. Co-immunoprecipitation assays indicate that RhoA and SpROCK are physically associated. This association is destroyed by treatment with the C3 exoenzyme and with the ROCK antagonist H-1152. H-1152 also inhibited DNA synthesis in embryos. We conclude that the Rho-dependent signaling pathway, via SpROCK, is essential for early embryonic development.

Comparative proteomic analysis of a sea urchin ( Heliocidaris erythrogramma) antibacterial response revealed the involvement of apextrin and calreticulin

Echinoderms evolved early in the deuterostome lineage, and as such constitute model organisms for comparative physiology and immunology. The sea urchin genome sequence ( Strongylocentrotus purpuratus) revealed a complex repertoire of genes with similarities to the immune response genes of other species. To complement these genomic data, we investigated the responses of sea urchins to the injection of bacteria using a comparative proteomics approach on a closely related species. In the sea urchin, Heliocidaris erythrogramma, the relative abundance of many proteins was altered in response to the injection of both bacteria and saline, suggesting their involvement in wounding responses, while others were differentially altered in response to bacteria only. The identities of 15 proteins that differed in relative abundance were determined by mass spectrometry. These proteins revealed a significant modification in energy metabolism in coelomocytes towards the consumption of glutamate and the production of NADPH after injection, as well as an increased concentration of cell signalling molecules, such as heterotrimeric guanine nucleotide-binding protein. The injection of bacteria specifically increased the abundance of apextrin and calreticulin, suggesting that these two proteins are involved in the sequestration or inactivation of bacteria.

Origin and diversification of steroids: Co-evolution of enzymes and nuclear receptors

AbstractRecent sequencing of amphioxus and sea urchin genomes has provided important data for understanding the origins of enzymes that synthesize adrenal and sex steroids and the receptors that mediate physiological response to these vertebrate steroids. Phylogenetic analyses reveal that CYP11A and CYP19, which are involved in the synthesis of adrenal and sex steroids, first appear in the common ancestor of amphioxus and vertebrates. This correlates with recent evidence for the first appearance in amphioxus of receptors with close similarity to vertebrate steroid receptors. Other CYP450 enzymes involved in steroid synthesis can be traced back to invertebrates, in which they have at least two functions: detoxifying xenobiotics and catalyzing the synthesis of sterols that activate nuclear receptors. CYP450 metabolism of hydrophobic xenobiotics may have been a key event in the origin of ligand-activated steroid receptors from constitutively active nuclear receptors.

SpTie1/ 2 is expressed in coelomocytes, axial organ and embryos of the sea urchin Strongylocentrotus purpuratus, and is an orthologue of vertebrate Tie1 and Tie2

A full length cDNA sequence expressed in coelomocytes shows significant sequence match to vertebrate Tie1 and Tie2/TEK. Vertebrate Tie2/TEK is the receptor for the angiopoietins and plays an important role in angiogenesis and hematopoiesis, whereas Tie1 regulates the activity of Tie2. The deduced sequence of the SpTie1/2 protein has a similar order and organization of domains to the homologous vertebrate proteins including a highly conserved receptor tyrosine kinase domain in the cytoplasmic tail. The N terminus of the ectodomain has one immunoglobulin (Ig)-Tie2_1 domain, followed by an Ig domain, four epidermal growth factor domains, a second Ig domain, and three fibronectin type III domains. The SpTie1/ 2 gene is expressed in coelomocytes and the axial organ, whereas other organs do not show significant expression. The timing of embryonic expression corresponds with the differentiation of blastocoelar cells, the embryonic and larval immune cells. Searches of the sea urchin genome show several gene models encoding putative ligands and signaling proteins that might interact with SpTie1/2. We speculate that SpTie1/2 may be involved in the proliferation of sea urchin immune cells in both adults and embryos.

Methods for Karyotyping and for Localization of Developmentally Relevant Genes on the Chromosomes of the Purple Sea Urchin, Strongylocentrotus purpuratus

The purple sea urchin, Strongylocentrotus purpuratus, is the only non-chordate deuterostome model with a fully sequenced genome. Chromosomal localization of individual genes and resulting gene maps are unavailable for this or for any sea urchin. As a result, the purple sea urchin genome has not been mapped onto specific chromosomes and remains inaccessible to genome-wide approaches addressing questions that require positional information for particular genes. Here we describe the first successful methods for karyotyping and localizing specific gene loci on chromosomes of Strongylocentrotus purpuratus and those of the phylogenetically related Strongylocentrotus droebachiensis. Both species have 42 chromosomes in their diploid genomes (n = 21). There are 2 large, 8 medium, and 10 small pairs, plus one putative sex pair. In both species, bindin genes were localized to 2 pair of homologous chromosomes by fluorescent in situ hybridization. Fluorescently labeled bacterial artificial chromosome clones generated from S. purpuratus for the functionally related genes brachyury, foxa, and foxb were localized to different chromosomes. Our protocols provide previously unavailable tools for developing a gene map for the purple sea urchin genome.

FGFRL1 is a neglected putative actor of the FGF signalling pathway present in all major metazoan phyla

BackgroundFibroblast Growth Factors (FGF) and their receptors are well known for having major implications in cell signalling controlling embryonic development. Recently, a gene coding for a protein closely related to FGFRs (Fibroblast Growth Factor Receptors) called FGFR5 or FGFR-like 1 (FGFRL1), has been described in vertebrates. An orthologous gene was also found in the cephalochordate amphioxus, but no orthologous genes were found by the authors in other non-vertebrate species, even if a FGFRL1 gene was identified in the sea urchin genome, as well as a closely related gene, named nou-darake, in the planarian Dugesia japonica. These intriguing data of a deuterostome-specific gene that might be implicated in FGF signalling prompted us to search for putative FGFRL1 orthologues in the completely sequenced genomes of metazoans.ResultsWe found FGFRL1 genes in the cnidarian Nematostella vectensis as well as in many bilaterian species. Our analysis also shows that FGFRL1 orthologous genes are linked in the genome with other members of the FGF signalling pathway from cnidarians to bilaterians (distance < 10 Mb). To better understand the implication of FGFRL1 genes in chordate embryonic development, we have analyzed expression patterns of the amphioxus and the mouse genes by whole mount in situ hybridization. We show that some homologous expression territories can be defined, and we propose that FGFRL1 and FGF8/17/18 were already co-expressed in the pharyngeal endoderm in the ancestor of chordates.ConclusionOur work sheds light on the existence of a putative FGF signalling pathway actor present in the ancestor of probably all metazoans, the function of which has received little attention until now.

Echinoderm Phosphorylated Matrix Proteins UTMP16 and UTMP19 Have Different Functions in Sea Urchin Tooth Mineralization

Studies of mineralization of embryonic spicules and of the sea urchin genome have identified several putative mineralization-related proteins. These predicted proteins have not been isolated or confirmed in mature mineralized tissues. Mature Lytechinus variegatus teeth were demineralized with 0.6 n HCl after prior removal of non-mineralized constituents with 4.0 m guanidinium HCl. The HCl-extracted proteins were fractionated on ceramic hydroxyapatite and separated into bound and unbound pools. Gel electrophoresis compared the protein distributions. The differentially present bands were purified and digested with trypsin, and the tryptic peptides were separated by high pressure liquid chromatography. NH2-terminal sequences were determined by Edman degradation and compared with the genomic sequence bank data. Two of the putative mineralization-related proteins were found. Their complete amino acid sequences were cloned from our L. variegatus cDNA library. Apatite-binding UTMP16 was found to be present in two isoforms; both isoforms had a signal sequence, a Ser-Asp-rich extracellular matrix domain, and a transmembrane and cytosolic insertion sequence. UTMP19, although rich in Glu and Thr did not bind to apatite. It had neither signal peptide nor transmembrane domain but did have typical nuclear localization and nuclear exit signal sequences. Both proteins were phosphorylated and good substrates for phosphatase. Immunolocalization studies with anti-UTMP16 show it to concentrate at the syncytial membranes in contact with the mineral. On the basis of our TOF-SIMS analyses of magnesium ion and Asp mapping of the mineral phase composition, we speculate that UTMP16 may be important in establishing the high magnesium columns that fuse the calcite plates together to enhance the mechanical strength of the mineralized tooth.

A Variant Mimicking Hyperphosphorylated 4E-BP Inhibits Protein Synthesis in a Sea Urchin Cell-Free, Cap-Dependent Translation System

Background4E-BP is a translational inhibitor that binds to eIF4E to repress cap-dependent translation initiation. This critical protein:protein interaction is regulated by the phosphorylation of 4E-BP. Hypophosphorylated 4E-BP binds to eIF4E and inhibits cap-dependent translation, whereas hyperphosphorylated forms do not. While three 4E-BP proteins exist in mammals, only one gene encoding for 4E-BP is present in the sea urchin genome. The protein product has a highly conserved core domain containing the eIF4E-binding domain motif (YxxxxLΦ) and four of the regulatory phosphorylation sites.Methodology/Principal FindingsUsing a sea urchin cell-free cap-dependent translation system prepared from fertilized eggs, we provide the first direct evidence that the sea urchin 4E-BP inhibits cap-dependent translation. We show here that a sea urchin 4E-BP variant, mimicking phosphorylation on four core residues required to abrogate binding to eIF4E, surprisingly maintains physical association to eIF4E and inhibits protein synthesis.Conclusions/SignificanceHere, we examine the involvement of the evolutionarily conserved core domain and phosphorylation sites of sea urchin 4E-BP in the regulation of eIF4E-binding. These studies primarily demonstrate the conserved activity of the 4E-BP translational repressor and the importance of the eIF4E-binding domain in sea urchin. We also show that a variant mimicking hyperphosphorylation of the four regulatory phosphorylation sites common to sea urchin and human 4E-BP is not sufficient for release from eIF4E and translation promotion. Therefore, our results suggest that there are additional mechanisms to that of phosphorylation at the four critical sites of 4E-BP that are required to disrupt binding to eIF4E.

A perturbation model of the gene regulatory network for oral and aboral ectoderm specification in the sea urchin embryo

The current gene regulatory network (GRN) for the sea urchin embryo pertains to pregastrular specification functions in the endomesodermal territories. Here we extend gene regulatory network analysis to the adjacent oral and aboral ectoderm territories over the same period. A large fraction of the regulatory genes predicted by the sea urchin genome project and shown in ancillary studies to be expressed in either oral or aboral ectoderm by 24 h are included, though universally expressed and pan-ectodermal regulatory genes are in general not. The loci of expression of these genes have been determined by whole mount in situ hybridization. We have carried out a global perturbation analysis in which expression of each gene was interrupted by introduction of morpholino antisense oligonucleotide, and the effects on all other genes were measured quantitatively, both by QPCR and by a new instrumental technology (NanoString Technologies nCounter Analysis System). At its current stage the network model, built in BioTapestry, includes 22 genes encoding transcription factors, 4 genes encoding known signaling ligands, and 3 genes that are yet unknown but are predicted to perform specific roles. Evidence emerged from the analysis pointing to distinctive subcircuit features observed earlier in other parts of the GRN, including a double negative transcriptional regulatory gate, and dynamic state lockdowns by feedback interactions. While much of the regulatory apparatus is downstream of Nodal signaling, as expected from previous observations, there are also cohorts of independently activated oral and aboral ectoderm regulatory genes, and we predict yet unidentified signaling interactions between oral and aboral territories.

SpBase: the sea urchin genome database and web site.

SpBase is a system of databases focused on the genomic information from sea urchins and related echinoderms. It is exposed to the public through a web site served with open source software (http://spbase.org/). The enterprise was undertaken to provide an easily used collection of information to directly support experimental work on these useful research models in cell and developmental biology. The information served from the databases emerges from the draft genomic sequence of the purple sea urchin, Strongylocentrotus purpuratus and includes sequence data and genomic resource descriptions for other members of the echinoderm clade which in total span 540 million years of evolutionary time. This version of the system contains two assemblies of the purple sea urchin genome, associated expressed sequences, gene annotations and accessory resources. Search mechanisms for the sequences and the gene annotations are provided. Because the system is maintained along with the Sea Urchin Genome resource, a database of sequenced clones is also provided.

The amphioxus genome enlightens the evolution of the thyroid hormone signaling pathway

Thyroid hormones (THs) have pleiotropic effects on vertebrate development, with amphibian metamorphosis as the most spectacular example. However, developmental functions of THs in non-vertebrate chordates are largely hypothetical and even TH endogenous production has been poorly investigated. In order to get better insight into the evolution of the thyroid hormone signaling pathway in chordates, we have taken advantage of the recent release of the amphioxus genome. We found amphioxus homologous sequences to most of the genes encoding proteins involved in thyroid hormone signaling in vertebrates, except the fast-evolving thyroglobulin: sodium iodide symporter, thyroid peroxidase, deiodinases, thyroid hormone receptor, TBG, and CTHBP. As only some genes encoding proteins involved in TH synthesis regulation were retrieved (TRH, TSH receptor, and CRH receptor but not their corresponding receptors and ligands), there may be another mode of upstream regulation of TH synthesis in amphioxus. In accord with the notion that two whole genome duplications took place at the base of the vertebrate tree, one amphioxus gene often corresponded to several vertebrate homologs. However, some amphioxus specific duplications occurred, suggesting that several steps of the TH pathway were independently elaborated in the cephalochordate and vertebrate lineages. The present results therefore indicate that amphioxus is capable of producing THs. As several genes of the TH signaling pathway were also found in the sea urchin genome, we propose that the thyroid hormone signaling pathway is of ancestral origin in chordates, if not in deuterostomes, with specific elaborations in each lineage, including amphioxus.

Specificity of the Chromodomain Y Chromosome Family of Chromodomains for Lysine-methylated ARK(S/T) Motifs

Previous studies have shown two homologous chromodomain modules in the HP1 and Polycomb proteins exhibit discriminatory binding to related methyllysine residues (embedded in ARKS motifs) of the histone H3 tail. Methylated ARK(S/T) motifs have recently been identified in other chromatin factors (e.g. linker histone H1.4 and lysine methyltransferase G9a). These are thought to function as peripheral docking sites for the HP1 chromodomain. In vertebrates, HP1-like chromodomains are also present in the chromodomain Y chromosome (CDY) family of proteins adjacent to a putative catalytic motif. The human genome encodes three CDY family proteins, CDY, CDYL, and CDYL2. These have putative functions ranging from establishment of histone H4 acetylation during spermiogenesis to regulation of transcription co-repressor complexes. To delineate the biochemical functions of the CDY family chromodomains, we analyzed their specificity of methyllysine recognition. We detected substantial differences among these factors. The CDY chromodomain exhibits discriminatory binding to lysine-methylated ARK(S/T) motifs, whereas the CDYL2 chromodomain binds with comparable strength to multiple ARK(S/T) motifs. Interestingly, subtle amino acid changes in the CDYL chromodomain prohibit such binding interactions in vitro and in vivo. However, point mutations can rescue binding. In support of the in vitro binding properties of the chromodomains, the full-length CDY family proteins exhibit substantial variability in chromatin localization. Our studies underscore the significance of subtle sequence differences in a conserved signaling module for diverse epigenetic regulatory pathways.