Identification and Gene Expression Analysis of a Taxonomically Restricted Cysteine-Rich Protein Family in Reef-Building Corals

The amount of genomic sequence information continues to grow at an exponential rate, while the identification and characterization of genes without known homologs remains a major challenge. For non-model organisms with limited resources for manipulative studies, high-throughput transcriptomic data combined with bioinformatics methods provide a powerful approach to obtain initial insights into the function of unknown genes. In this study, we report the identification and characterization of a novel family of putatively secreted, small, cysteine-rich proteins herein named Small Cysteine-Rich Proteins (SCRiPs). Their discovery in expressed sequence tag (EST) libraries from the coral Montastraea faveolata required the performance of an iterative search strategy based on BLAST and Hidden-Markov-Model algorithms. While a discernible homolog could neither be identified in the genome of the sea anemone Nematostella vectensis, nor in a large EST dataset from the symbiotic sea anemone Aiptasia pallida, we identified SCRiP sequences in multiple scleractinian coral species. Therefore, we postulate that this gene family is an example of lineage-specific gene expansion in reef-building corals. Previously published gene expression microarray data suggest that a sub-group of SCRiPs is highly responsive to thermal stress. Furthermore, data from microarray experiments investigating developmental gene expression in the coral Acropora millepora suggest that different SCRiPs may play distinct roles in the development of corals. The function of these proteins remains to be elucidated, but our results from in silico, transcriptomic, and phylogenetic analyses provide initial insights into the evolution of SCRiPs, a novel, taxonomically restricted gene family that may be responsible for a lineage-specific trait in scleractinian corals

An Atlas of the Speed of Copy Number Changes in Animal Gene Families and Its Implications

The notion that gene duplications generating new genes and functions is commonly accepted in evolutionary biology. However, this assumption is more speculative from theory rather than well proven in genome-wide studies. Here, we generated an atlas of the rate of copy number changes (CNCs) in all the gene families of ten animal genomes. We grouped the gene families with similar CNC dynamics into rate pattern groups (RPGs) and annotated their function using a novel bottom-up approach. By comparing CNC rate patterns, we showed that most of the species-specific CNC rates groups are formed by gene duplication rather than gene loss, and most of the changes in rates of CNCs may be the result of adaptive evolution. We also found that the functions of many RPGs match their biological significance well. Our work confirmed the role of gene duplication in generating novel phenotypes, and the results can serve as a guide for researchers to connect the phenotypic features to certain gene duplications

SUMOylation of DRIL1 Directs Its Transcriptional Activity Towards Leukocyte Lineage-Specific Genes

DRIL1 is an ARID family transcription factor that can immortalize primary mouse fibroblasts, bypass RASV12-induced cellular senescence and collaborate with RASV12 or MYC in mediating oncogenic transformation. It also activates immunoglobulin heavy chain transcription and engages in heterodimer formation with E2F to stimulate E2F-dependent transcription. Little, however, is known about the regulation of DRIL1 activity. Recently, DRIL1 was found to interact with the SUMO-conjugating enzyme Ubc9, but the functional relevance of this association has not been assessed. Here, we show that DRIL1 is sumoylated both in vitro and in vivo at lysine 398. Moreover, we provide evidence that PIASy functions as a specific SUMO E3-ligase for DRIL1 and promotes its sumoylation both in vitro and in vivo. Furthermore, consistent with the subnuclear localization of PIASy in the Matrix-Associated Region (MAR), SUMO-modified DRIL1 species are found exclusively in the MAR fraction. This post-translational modification interferes neither with the subcellular localization nor the DNA-binding activity of the protein. In contrast, DRIL1 sumoylation impairs its interaction with E2F1 in vitro and modifies its transcriptional activity in vivo, driving transcription of subset of genes regulating leukocyte fate. Taken together, these results identify sumoylation as a novel post-translational modification of DRIL1 that represents an important mechanism for targeting and modulating DRIL1 transcriptional activity

Identification of Fast-Evolving Genes in the Scleractinian Coral Acropora Using Comparative EST Analysis

To identify fast-evolving genes in reef-building corals, we performed direct comparative sequence analysis with expressed sequence tag (EST) datasets from two acroporid species: Acropora palmata from the Caribbean Sea and A. millepora from the Great Barrier Reef in Australia. Comparison of 589 independent sequences from 1,421 A. palmata contigs, with 10,247 A. millepora contigs resulted in the identification of 196 putative homologues. Most of the homologous pairs demonstrated high amino acid similarities (over 90%). Comparisons of putative homologues showing low amino acid similarities (under 90%) among the Acropora species to the near complete datasets from two other cnidarians (Hydra magnipapillata and Nematostella vectensis) implied that some were non-orthologous. Within 86 homologous pairs, 39 exhibited dN/dS ratios significantly less than 1, suggesting that these genes are under purifying selection associated with functional constraints. Eight independent genes showed dN/dS ratios exceeding 1, while three deviated significantly from 1, suggesting that these genes may play important roles in the adaptive evolution of Acropora. Our results also indicated that CEL-III lectin was under positive selection, consistent with a possible role in immunity or symbiont recognition. Further studies are needed to clarify the possible functions of the genes under positive selection to provide insight into the evolutionary process of corals

Essential Functions of the Histone Demethylase Lid

Drosophila Little imaginal discs (Lid) is a recently described member of the JmjC domain class of histone demethylases that specifically targets trimethylated histone H3 lysine 4 (H3K4me3). To understand its biological function, we have utilized a series of Lid deletions and point mutations to assess the role that each domain plays in histone demethylation, in animal viability, and in cell growth mediated by the transcription factor dMyc. Strikingly, we find that lid mutants are rescued to adulthood by either wildtype or enzymatically inactive Lid expressed under the control of its endogenous promoter, demonstrating that Lid's demethylase activity is not essential for development. In contrast, ubiquitous expression of UAS-Lid transgenes lacking its JmjN, C-terminal PHD domain, and C5HC2 zinc finger were unable to rescue lid homozygous mutants, indicating that these domains carry out Lid's essential developmental functions. Although Lid-dependent demethylase activity is not essential, dynamic removal of H3K4me3 may still be an important component of development, as we have observed a genetic interaction between lid and another H3K4me3 demethylase, dKDM2. We also show that Lid's essential C-terminal PHD finger binds specifically to di- and trimethylated H3K4 and that this activity is required for Lid to function in dMyc-induced cell growth. Taken together, our findings highlight the importance of Lid function in the regulated removal and recognition of H3K4me3 during development

Physiological effects of KDM5C on neural crest migration and eye formation during vertebrate development

Background: Lysine-specific histone demethylase 5C (KDM5C) belongs to the jumonji family of demethylases and is specific for the di- and tri-demethylation of lysine 4 residues on histone 3 (H3K4 me2/3). KDM5C is expressed in the brain and skeletal muscles of humans and is associated with various biologically significant processes. KDM5C is known to be associated with X-linked mental retardation and is also involved in the development of cancer. However, the developmental significance of KDM5C has not been explored yet. In the present study, we investigated the physiological roles of KDM5C during Xenopus laevis embryonic development. Results: Loss-of-function analysis using kdm5c antisense morpholino oligonucleotides indicated that kdm5c knockdown led to small-sized heads, reduced cartilage size, and malformed eyes (i.e., small-sized and deformed eyes). Molecular analyses of KDM5C functional roles using whole-mount in situ hybridization, -galactosidase staining, and reverse transcription-polymerase chain reaction revealed that loss of kdm5c resulted in reduced expression levels of neural crest specifiers and genes involved in eye development. Furthermore, transcriptome analysis indicated the significance of KDM5C in morphogenesis and organogenesis. Conclusion: Our findings indicated that KDM5C is associated with embryonic development and provided additional information regarding the complex and dynamic gene network that regulates neural crest formation and eye development. This study emphasizes the functional significance of KDM5C in Xenopus embryogenesis; however, further analysis is needed to explore the interactions of KDM5C with specific developmental genes

Hormonal signaling in cnidarians : do we understand the pathways well enough to know whether they are being disrupted?

Author Posting. © The Author, 2006. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Ecotoxicology 16 (2007): 5-13, doi:10.1007/s10646-006-0121-1.Cnidarians occupy a key evolutionary position as basal metazoans and are ecologically important as predators, prey and structure-builders. Bioregulatory molecules (e.g., amines, peptides and steroids) have been identified in cnidarians, but cnidarian signaling pathways remain poorly characterized. Cnidarians, especially hydras, are regularly used in toxicity testing, but few studies have used cnidarians in explicit testing for signal disruption. Sublethal endpoints developed in cnidarians include budding, regeneration, gametogenesis, mucus production and larval metamorphosis. Cnidarian genomic databases, microarrays and other molecular tools are increasingly facilitating mechanistic investigation of signaling pathways and signal disruption. Elucidation of cnidarian signaling processes in a comparative context can provide insight into the evolution and diversification of metazoan bioregulation. Characterizing signaling and signal disruption in cnidarians may also provide unique opportunities for evaluating risk to valuable marine resources, such as coral reefs

Gene Discovery in the Threatened Elkhorn Coral: 454 Sequencing of the Acropora palmata Transcriptome

BACKGROUND: Cnidarians, including corals and anemones, offer unique insights into metazoan evolution because they harbor genetic similarities with vertebrates beyond that found in model invertebrates and retain genes known only from non-metazoans. Cataloging genes expressed in Acropora palmata, a foundation-species of reefs in the Caribbean and western Atlantic, will advance our understanding of the genetic basis of ecologically important traits in corals and comes at a time when sequencing efforts in other cnidarians allow for multi-species comparisons. RESULTS: A cDNA library from a sample enriched for symbiont free larval tissue was sequenced on the 454 GS-FLX platform. Over 960,000 reads were obtained and assembled into 42,630 contigs. Annotation data was acquired for 57% of the assembled sequences. Analysis of the assembled sequences indicated that 83-100% of all A. palmata transcripts were tagged, and provided a rough estimate of the total number genes expressed in our samples (~18,000-20,000). The coral annotation data contained many of the same molecular components as in the Bilateria, particularly in pathways associated with oxidative stress and DNA damage repair, and provided evidence that homologs of p53, a key player in DNA repair pathways, has experienced selection along the branch separating Cnidaria and Bilateria. Transcriptome wide screens of paralog groups and transition/transversion ratios highlighted genes including: green fluorescent proteins, carbonic anhydrase, and oxidative stress proteins; and functional groups involved in protein and nucleic acid metabolism, and the formation of structural molecules. These results provide a starting point for study of adaptive evolution in corals. CONCLUSIONS: Currently available transcriptome data now make comparative studies of the mechanisms underlying coral's evolutionary success possible. Here we identified candidate genes that enable corals to maintain genomic integrity despite considerable exposure to genotoxic stress over long life spans, and showed conservation of important physiological pathways between corals and bilaterians

Massively Parallel RNA Sequencing Identifies a Complex Immune Gene Repertoire in the lophotrochozoan Mytilus edulis

The marine mussel Mytilus edulis and its closely related sister species are distributed world-wide and play an important role in coastal ecology and economy. The diversification in different species and their hybrids, broad ecological distribution, as well as the filter feeding mode of life has made this genus an attractive model to investigate physiological and molecular adaptations and responses to various biotic and abiotic environmental factors. In the present study we investigated the immune system of Mytilus, which may contribute to the ecological plasticity of this species. We generated a large Mytilus transcriptome database from different tissues of immune challenged and stress treated individuals from the Baltic Sea using 454 pyrosequencing. Phylogenetic comparison of orthologous groups of 23 species demonstrated the basal position of lophotrochozoans within protostomes. The investigation of immune related transcripts revealed a complex repertoire of innate recognition receptors and downstream pathway members including transcripts for 27 toll-like receptors and 524 C1q domain containing transcripts. NOD-like receptors on the other hand were absent. We also found evidence for sophisticated TNF, autophagy and apoptosis systems as well as for cytokines. Gill tissue and hemocytes showed highest expression of putative immune related contigs and are promising tissues for further functional studies. Our results partly contrast with findings of a less complex immune repertoire in ecdysozoan and other lophotrochozoan protostomes. We show that bivalves are interesting candidates to investigate the evolution of the immune system from basal metazoans to deuterostomes and protostomes and provide a basis for future molecular work directed to immune system functioning in Mytilus

Pre-Bilaterian Origins of the Hox Cluster and the Hox Code: Evidence from the Sea Anemone, Nematostella vectensis

BACKGROUND: Hox genes were critical to many morphological innovations of bilaterian animals. However, early Hox evolution remains obscure. Phylogenetic, developmental, and genomic analyses on the cnidarian sea anemone Nematostella vectensis challenge recent claims that the Hox code is a bilaterian invention and that no “true” Hox genes exist in the phylum Cnidaria. METHODOLOGY/PRINCIPAL FINDINGS: Phylogenetic analyses of 18 Hox-related genes from Nematostella identify putative Hox1, Hox2, and Hox9+ genes. Statistical comparisons among competing hypotheses bolster these findings, including an explicit consideration of the gene losses implied by alternate topologies. In situ hybridization studies of 20 Hox-related genes reveal that multiple Hox genes are expressed in distinct regions along the primary body axis, supporting the existence of a pre-bilaterian Hox code. Additionally, several Hox genes are expressed in nested domains along the secondary body axis, suggesting a role in “dorsoventral” patterning. CONCLUSIONS/SIGNIFICANCE: A cluster of anterior and posterior Hox genes, as well as ParaHox cluster of genes evolved prior to the cnidarian-bilaterian split. There is evidence to suggest that these clusters were formed from a series of tandem gene duplication events and played a role in patterning both the primary and secondary body axes in a bilaterally symmetrical common ancestor. Cnidarians and bilaterians shared a common ancestor some 570 to 700 million years ago, and as such, are derived from a common body plan. Our work reveals several conserved genetic components that are found in both of these diverse lineages. This finding is consistent with the hypothesis that a set of developmental rules established in the common ancestor of cnidarians and bilaterians is still at work today