Comparative genomics using Fugu reveals insights into regulatory subfunctionalization

Fish-mammal genomic alignments were used to compare over 800 conserved non-coding elements that associate with genes that have undergone fish-specific duplication and retention, revealing a pattern of element retention and loss between paralogs indicative of subfunctionalization

Ancient Pbx-Hox signatures define hundreds of vertebrate developmental enhancers

Background: Gene regulation through cis-regulatory elements plays a crucial role in development and disease. A major aim of the post-genomic era is to be able to read the function of cis-regulatory elements through scrutiny of their DNA sequence. Whilst comparative genomics approaches have identified thousands of putative regulatory elements, our knowledge of their mechanism of action is poor and very little progress has been made in systematically de-coding them. Results: Here, we identify ancient functional signatures within vertebrate conserved non-coding elements (CNEs) through a combination of phylogenetic footprinting and functional assay, using genomic sequence from the sea lamprey as a reference. We uncover a striking enrichment within vertebrate CNEs for conserved binding-site motifs of the Pbx-Hox hetero-dimer. We further show that these predict reporter gene expression in a segment specific manner in the hindbrain and pharyngeal arches during zebrafish development. Conclusions: These findings evoke an evolutionary scenario in which many CNEs evolved early in the vertebrate lineage to co-ordinate Hox-dependent gene-regulatory interactions that pattern the vertebrate head. In a broader context, our evolutionary analyses reveal that CNEs are composed of tightly linked transcription-factor binding-sites (TFBSs), which can be systematically identified through phylogenetic footprinting approaches. By placing a large number of ancient vertebrate CNEs into a developmental context, our findings promise to have a significant impact on efforts toward de-coding gene-regulatory elements that underlie vertebrate development, and will facilitate building general models of regulatory element evolution

A Reporter Assay in Lamprey Embryos Reveals Both Functional Conservation and Elaboration of Vertebrate Enhancers

The sea lamprey is an important model organism for investigating the evolutionary origins of vertebrates. As more vertebrate genome sequences are obtained, evolutionary developmental biologists are becoming increasingly able to identify putative gene regulatory elements across the breadth of the vertebrate taxa. The identification of these regions makes it possible to address how changes at the genomic level have led to changes in developmental gene regulatory networks and ultimately to the evolution of morphological diversity. Comparative genomics approaches using sea lamprey have already predicted a number of such regulatory elements in the lamprey genome. Functional characterisation of these sequences and other similar elements requires efficient reporter assays in lamprey. In this report, we describe the development of a transient transgenesis method for lamprey embryos. Focusing on conserved non-coding elements (CNEs), we use this method to investigate their functional conservation across the vertebrate subphylum. We find instances of both functional conservation and lineage-specific functional evolution of CNEs across vertebrates, emphasising the utility of functionally testing homologous CNEs in their host species

Parallel evolution of conserved non-coding elements that target a common set of developmental regulatory genes from worms to humans

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Background The human genome contains thousands of non-coding sequences that are often more conserved between vertebrate species than protein-coding exons. These highly conserved non-coding elements (CNEs) are associated with genes that coordinate development, and have been proposed to act as transcriptional enhancers. Despite their extreme sequence conservation in vertebrates, sequences homologous to CNEs have not been identified in invertebrates. Results Here we report that nematode genomes contain an alternative set of CNEs that share sequence characteristics, but not identity, with their vertebrate counterparts. CNEs thus represent a very unusual class of sequences that are extremely conserved within specific animal lineages yet are highly divergent between lineages. Nematode CNEs are also associated with developmental regulatory genes, and include well-characterized enhancers and transcription factor binding sites, supporting the proposed function of CNEs as cis-regulatory elements. Most remarkably, 40 of 156 human CNE-associated genes with invertebrate orthologs are also associated with CNEs in both worms and flies. Conclusion A core set of genes that regulate development is associated with CNEs across three animal groups (worms, flies and vertebrates). We propose that these CNEs reflect the parallel evolution of alternative enhancers for a common set of developmental regulatory genes in different animal groups. This 're-wiring' of gene regulatory networks containing key developmental coordinators was probably a driving force during the evolution of animal body plans. CNEs may, therefore, represent the genomic traces of these 'hard-wired' core gene regulatory networks that specify the development of each alternative animal body plan.Published versio

Calcitonin: characterisation and expression in a teleost fish, Fugu rubripes

The present report describes the structure and expression of the calcitonin gene in Fugu rubripes. It is composed of 4 exons and 3 introns. Splicing of exons 1, 2 and 3 generates the calcitonin pre-proprotein, while splicing of exons 1, 2 and 4 generates calcitonin gene-related protein (CGRP). Exons 1 and 2 encoding the signal sequence and the N-terminal peptide are common in both the gene products and this gene organisation has been conserved in human, rat, chicken and salmon. The gene environment around calcitonin in Fugu has been poorly conserved when compared with human, apart from a small gene cluster. The calcitonin gene in Fugu has a widespread tissue distribution but it is most highly expressed in the brain. The abundance of gene expression in the ultimobranchial gland and the pituitary indicates that these are important sites of production and that the peptide is probably secreted into the circulation and/or acts as a paracrine or autocrine controlling factor. Whilst the function of calcitonin in fish is still largely unknown, the distribution described here suggests that one of the potential functions may be as a neuropeptide.info:eu-repo/semantics/publishedVersio

CONDOR: a database resource of developmentally associated conserved non-coding elements

<p>Abstract</p> <p>Background</p> <p>Comparative genomics is currently one of the most popular approaches to study the regulatory architecture of vertebrate genomes. Fish-mammal genomic comparisons have proved powerful in identifying conserved non-coding elements likely to be distal <it>cis-</it>regulatory modules such as enhancers, silencers or insulators that control the expression of genes involved in the regulation of early development. The scientific community is showing increasing interest in characterizing the function, evolution and language of these sequences. Despite this, there remains little in the way of user-friendly access to a large dataset of such elements in conjunction with the analysis and the visualization tools needed to study them.</p> <p>Description</p> <p>Here we present CONDOR (COnserved Non-coDing Orthologous Regions) available at: <url>http://condor.fugu.biology.qmul.ac.uk</url>. In an interactive and intuitive way the website displays data on > 6800 non-coding elements associated with over 120 early developmental genes and conserved across vertebrates. The database regularly incorporates results of ongoing <it>in vivo </it>zebrafish enhancer assays of the CNEs carried out in-house, which currently number ~100. Included and highlighted within this set are elements derived from duplication events both at the origin of vertebrates and more recently in the teleost lineage, thus providing valuable data for studying the divergence of regulatory roles between paralogs. CONDOR therefore provides a number of tools and facilities to allow scientists to progress in their own studies on the function and evolution of developmental <it>cis</it>-regulation.</p> <p>Conclusion</p> <p>By providing access to data with an approachable graphics interface, the CONDOR database presents a rich resource for further studies into the regulation and evolution of genes involved in early development.</p

Dissection of a Ciona regulatory element reveals complexity of cross-species enhancer activity

Vertebrate genomes share numerous conserved non-coding elements, many of which function as enhancer elements and are hypothesised to be under evolutionary constraint due to a need to be bound by combinations of sequence-specific transcription factors. In contrast, few such conserved elements can be detected between vertebrates and their closest invertebrate relatives. Despite this lack of sequence identity, cross-species transgenesis has identified some cases where non-coding DNA from invertebrates drives reporter gene expression in transgenic vertebrates in patterns reminiscent of the expression of vertebrate orthologues. Such instances are presumed to reflect the presence of conserved suites of binding sites in the regulatory regions of invertebrate and vertebrate orthologues, such that both regulatory elements can correctly interpret the trans-activating environment. Shuffling of binding sites has been suggested to lie behind loss of sequence conservation; however this has not been experimentally tested. Here we examine the underlying basis of enhancer activity for the Ciona intestinalis βγ-crystallin gene, which drives expression in the lens of transgenic vertebrates despite the Ciona lineage predating the evolution of the lens. We construct an interactive gene regulatory network (GRN) for vertebrate lens development, allowing network interactions to be robustly catalogued and conserved network components and features to be identified. We show that a small number of binding motifs are necessary for Ciona βγ-crystallin expression, and narrow down the likely factors that bind to these motifs. Several of these overlap with the conserved core of the vertebrate lens GRN, implicating these sites in cross species function. However when we test these motifs in a transgenic vertebrate they prove to be dispensable for reporter expression in the lens. These results show that current models depicting cross species enhancer function as dependent on conserved binding sites can be overly simplistic, with sound evolutionary inference requiring detailed dissection of underlying mechanisms

Stops making sense: translational trade-offs and stop codon reassignment

Background Efficient gene expression involves a trade-off between (i) premature termination of protein synthesis; and (ii) readthrough, where the ribosome fails to dissociate at the terminal stop. Sense codons that are similar in sequence to stop codons are more susceptible to nonsense mutation, and are also likely to be more susceptible to transcriptional or translational errors causing premature termination. We therefore expect this trade-off to be influenced by the number of stop codons in the genetic code. Although genetic codes are highly constrained, stop codon number appears to be their most volatile feature. Results In the human genome, codons readily mutable to stops are underrepresented in coding sequences. We construct a simple mathematical model based on the relative likelihoods of premature termination and readthrough. When readthrough occurs, the resultant protein has a tail of amino acid residues incorrectly added to the C-terminus. Our results depend strongly on the number of stop codons in the genetic code. When the code has more stop codons, premature termination is relatively more likely, particularly for longer genes. When the code has fewer stop codons, the length of the tail added by readthrough will, on average, be longer, and thus more deleterious. Comparative analysis of taxa with a range of stop codon numbers suggests that genomes whose code includes more stop codons have shorter coding sequences. Conclusions We suggest that the differing trade-offs presented by alternative genetic codes may result in differences in genome structure. More speculatively, multiple stop codons may mitigate readthrough, counteracting the disadvantage of a higher rate of nonsense mutation. This could help explain the puzzling overrepresentation of stop codons in the canonical genetic code and most variants

Purifying Selection in Deeply Conserved Human Enhancers Is More Consistent than in Coding Sequences

(c) 2014 De Silva et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

SAND, a New Protein Family: From Nucleic Acid to Protein Structure and Function Prediction

As a result of genome, EST and cDNA sequencing projects, there are huge numbers of predicted and/or partially characterised protein sequences compared with a relatively small number of proteins with experimentally determined function and structure. Thus, there is a considerable attention focused on the accurate prediction of gene function and structure from sequence by using bioinformatics. In the course of our analysis of genomic sequence from Fugu rubripes, we identified a novel gene, SAND, with significant sequence identity to hypothetical proteins predicted in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, a Drosophila melanogaster gene, and mouse and human cDNAs. Here we identify a further SAND homologue in human and Arabidopsis thaliana by use of standard computational tools. We describe the genomic organisation of SAND in these evolutionarily divergent species and identify sequence homologues from EST database searches confirming the expression of SAND in over 20 different eukaryotes. We confirm the expression of two different SAND paralogues in mammals and determine expression of one SAND in other vertebrates and eukaryotes. Furthermore, we predict structural properties of SAND, and characterise conserved sequence motifs in this protein family