Comparative genomics using fugu: a tool for the identification of conserved vertebrate cis-regulatory elements

With the imminent completion of the whole genome sequence of humans, increasing attention is being focused on the annotation of cis-regulatory elements in the human genome. Comparative genomics approaches based on evolutionary conservation have proved useful in the detection of conserved cis-regulatory elements. The pufferfish, Fugu rubripes, is an attractive vertebrate model for comparative genomics, by virtue of its compact genome and maximal phylogenetic distance from mammals. Fugu has lost a large proportion of nonessential DNA, and retained single orthologs for many duplicate genes that arose in the fish lineage. Non-coding sequences conserved between fugu and mammals have been shown to be functional cis-regulatory elements. Thus, fugu is a model fish genome of choice for discovering evolutionarily conserved regulatory elements in the human genome. Such evolutionarily conserved elements are likely to be shared by all vertebrates, and related to regulatory interactions fundamental to all vertebrates. The functions of these conserved vertebrate elements can be rapidly assayed in mammalian cell lines or in transgenic systems such as zebrafish/medaka and Xenopus, followed by validation of crucial elements in transgenic rodents.     

Ancient vertebrate conserved noncoding elements have been evolving rapidly in teleost fishes

Vertebrate genomes contain thousands of conserved noncoding elements (CNEs) that often function as tissue-specific enhancers. In this study, we have identified CNEs in human, dog, chicken, Xenopus, and four teleost fishes (zebrafish, stickleback, medaka, and fugu) using elephant shark, a cartilaginous vertebrate, as the base genome and investigated the evolution of these ancient vertebrate CNEs (aCNEs) in bony vertebrate lineages. Our analysis shows that aCNEs have been evolving at different rates in different bony vertebrate lineages. Although 78-83% of CNEs have diverged beyond recognition ("lost") in different teleost fishes, only 24% and 40% have been lost in the chicken and mammalian lineages, respectively. Relative rate tests of substitution rates in CNEs revealed that the teleost fish CNEs have been evolving at a significantly higher rate than those in other bony vertebrates. In the ray-finned fish lineage, 68% of aCNEs were lost before the divergence of the four teleosts. This implicates the "fish-specific" whole-genome duplication in the accelerated evolution and the loss of a large number of both copies of duplicated CNEs in teleost fishes. The aCNEs are rich in tissue-specific enhancers and thus many of them are likely to be evolutionarily constrained cis-regulatory elements. The rapid evolution of aCNEs might have affected the expression patterns driven by them. Transgenic zebrafish assay of some human CNE enhancers that have been lost in teleosts has indicated instances of conservation or changes in trans-acting factors between mammals and fishes.     

Inverse relationship between longevity and evolutionary rate of mitochondrial proteins in mammals and birds

Recently, an unexpected, positive correlation between the rate of evolution of mitochondrial proteins and longevity was reported. Here we re-analyze this relationship in various mammalian lineages using a bayesian phylogenetic analysis of amino-acid sequences, allowing for variable evolutionary rates across sites and species. A negative relationship between protein evolutionary rate and species longevity is reported for all oxidative phosphorylation complexes. A detailed analysis of the cytochrome b in 528 mammals reinforced this result, which contradicts previous publications. Reconducting the analysis in birds yielded similar results. We explain the discrepancy between this and previous reports by our improved taxon sampling and more appropriate methodology: unlike distance-based methods, the tree-based bayesian approach can take into account the high variation of substitution rate across amino-acid sites, and the resulting multiple substitution events. We discuss how our analysis contradicts Rottenberg’s rationale, but does not dismiss his proposal of a longevity-dependent selective pressure on mitochondrial mutation rate in mammals and birds. This is because his interpretation invokes adaptation as the single evolutionary force at work, disregarding the effects of mutation, genetic drift, and purifying selection.     

Surveying phylogenetic footprints in large gene clusters: applications to Hox cluster duplications

Evolutionarily conserved non-coding genomic sequences represent a potentially rich source for the discovery of gene regulatory regions. Since these elements are subject to stabilizing selection they evolve much more slowly than adjacent non-functional DNA. These so-called phylogenetic footprints can be detected by comparison of the sequences surrounding orthologous genes in different species. Therefore the loss of phylogenetic footprints as well as the acquisition of conserved non-coding sequences in some lineages, but not in others, can provide evidence for the evolutionary modification of cis-regulatory elements. We introduce here a statistical model of footprint evolution that allows us to estimate the loss of sequence conservation that can be attributed to gene loss and other structural reasons. This approach to studying the pattern of cis-regulatory element evolution, however, requires the comparison of relatively long sequences from many species. We have therefore developed an efficient software tool for the identification of corresponding footprints in long sequences from multiple species. We apply this novel method to the published sequences of HoxA clusters of shark, human, and the duplicated zebrafish and Takifugu clusters as well as the published HoxB cluster sequences. We find that there is a massive loss of sequence conservation in the intergenic region of the HoxA clusters, consistent with the finding in [Chiu et al., PNAS 99 (2002) 5492]. The loss of conservation after cluster duplication is more extensive than expected from structural reasons. This suggests that binding site turnover and/or adaptive modification may also contribute to the loss of sequence conservation.     

Purifying selection and positive selection on the myxovirus resistance gene in mammals and chickens

The myxovirus resistance gene (Mx) expresses antiviral activity in many species, e.g. mouse, human and chicken. It is not clear if the antiviral activity of Mx has evolved in these species to inhibit a set of species-specific pathogens, nor what factors drive Mx evolution in different animal lineages. Therefore, it is important to determine the evolutionary pattern of Mx and positively selected sites which affect the antiviral activity of the Mx gene in mammals and birds. We used sequence comparisons among species to detect positively selected sites by conducting phylogenetic analysis. The two-ratio model was significantly better than the one-ratio model in four species (mouse, rat, chicken and duck, p<0.05). Although selection pressure varied among different lineages, Mx had strong purifying selection in mammals and positive selection in chicken and duck lineages. Relative rate test revealed that Mx evolved faster in chickens than in ducks (Tajima's relative rate test, chi(2)=7.17, p<0.01). In the further analysis using a branch-site model A test, 8 sites were positively selected in the chicken lineage while no positive selection signals were observed for any site in the other lineages. The branch-site model A test had a omega value of 4.374 for the chicken lineage (2Deltal=14.20, d.f.=1, p<0.001). Comparisons of all currently available Mx mRNA sequences showed that these predicted positively selected sites had been fixed in the chicken lineage, suggesting that the chicken Mx gene evolved within the species to resist newly challenging environments. There is an increased selection constraint leading to mammals, while positive selection has acted on the chicken Mx.     

Increased rate of hair keratin gene loss in the cetacean lineage

Background

Hair represents an evolutionary innovation that appeared early on mammalian evolutionary history, and presumably contributed significantly to the rapid radiation of the group. An interesting event in hair evolution has been its secondary loss in some mammalian groups, such as cetaceans, whose hairless phenotype appears to be an adaptive response to better meet the environmental conditions. To determine whether different repertoire of keratin genes among mammals can potentially explain the phenotypic hair features of different lineages, we characterized the type I and II clusters of alpha keratins from eight mammalian species, including the hairless dolphin and minke whale representing the order Cetacea.

Results

We combined the available genomic information with phylogenetic analysis to conduct a comprehensive analysis of the evolutionary patterns of keratin gene clusters. We found that both type I and II gene clusters are fairly conserved among the terrestrial mammals included in this study, with lineage specific gene duplication and gene loss. Nevertheless, there is also evidence for an increased rate of pseudogenization in the cetacean lineage when compared to their terrestrial relatives, especially among the hair type keratins.

Conclusions

Here we present a comprehensive characterization of alpha-keratin genes among mammals and elucidate the mechanisms involved in the evolution of this gene family. We identified lineage-specific gene duplications and gene loss among the Laurasiatherian and Euarchontoglires species included in the study. Interestingly, cetaceans present an increased loss of hair-type keratin genes when compared to other terrestrial mammals. As suggested by the ‘less-is-more’ hypothesis, we do not rule out the possibility that the gene loss of hair-type keratin genes in these species might be associated to the hairless phenotype and could have been adaptive in response to new selective pressures imposed by the colonization of a new habitat. Our study provides support for the idea that pseudogenes are not simply ‘genomic fossils’ but instead have adaptive roles during the evolutionary process.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-869) contains supplementary material, which is available to authorized users.     

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Lineage-specific expansions and contractions of the bitter taste receptor gene repertoire in vertebrates

The sense of bitter taste plays a critical role in how organisms avoid generally bitter toxic and harmful substances. Previous studies revealed that there were 25 intact bitter taste receptor (T2R) genes in humans and 34 in mice. However, because the recent chicken genome project reported only three T2R genes, it appears that extensive gene expansions occurred in the lineage leading to mammals or extensive gene contractions occurred in the lineage leading to birds. Here, I examined the T2R gene repertoire in placental mammals (dogs, Canis familiaris; and cows, Bos taurus), marsupials (opossums, Monodelphis domestica), amphibians (frogs, Xenopus tropicalis), and fishes (zebrafishes, Danio rerio; and pufferfishes, Takifugu rubripes) to investigate the birth-and-death process of T2R genes throughout vertebrate evolution. I show that (1) the first extensive gene expansions occurred before the divergence of mammals from reptiles/birds but after the divergence of amniotes (reptiles/birds/mammals) from amphibians, (2) subsequent gene expansions continuously took place in the ancestral mammalian lineage and the lineage leading to amphibians, as evidenced by the presence of 15, 18, 26, and 49 intact T2R genes in the dog, cow, opossum, and frog genome, respectively, and (3) contractions of the gene repertoire happened in the lineage leading to chickens. Thus, continuous gene expansions have shaped the T2R repertoire in mammals, but the contractions subsequent to the first round of expansions have made the chicken T2R repertoire narrow. These dramatic changes in the repertoire size might reflect the daily intake of foods from an external environment as a driving force of evolution.     

Molecular Cloning and Evolutionary Analysis of GJB6 in Mammals

GJB6 plays a crucial role in hearing. In mammals, bats use ultrasonic echolocation for orientation and locating prey. To investigate the evolution of GJB6 in mammals, we cloned the full-length coding region of GJB6 from 16 species of bats and 4 other mammal species and compared them with orthologous sequences in 11 other mammals. The results show purifying selection on GJB6 in mammals, as well as in the bat lineage, which indicates an important role for GJB6 in mammal hearing. We also found one unique amino acid substitution shared by 16 species of bats and 10 shared by two species of artiodactyls. This positioned the artiodactyls at an abnormal location in the gene tree. In addition, the cytoplasmic loop and carboxy terminus were more variable than other domains in all the mammals. These results demonstrate that GJB6 is basically conserved in mammals but has undergone relatively rapid evolution in particular lineages and domains.     

Teleost Fish Genomes Contain a Diverse Array of L1 RetrotransposonLineages That Exhibit a Low Copy Number and High Rate of Turnover

Retrotransposable elements exhibit a wide range of variation in population dynamics, abundance, and lineage diversity among host genomes across taxa. This range of diversity is illustrated by a single well-defined constituent monophyletic clade of L1 non-LTR retrotransposons that is shared between mammalian and teleost fish genomes. Despite the clear phylogenetic relationships that exist between mammalian and teleost L1 sequences, these elements exhibit markedly different dynamics within their respective taxa. While mammalian genomes typically contain a single, abundant lineage of L1 elements that traces millions of years of evolution, the zebraflsh genome was recently shown to exhibit a high diversity of ancient lineages coexisting at a very low copy number and apparently exhibiting a high rate of turnover. In the present study, a combination of degenerate PCR, lineage-specific PCR, and genomic Southern blot analysis is utilized to demonstrate high L1 lineage diversity, low copy number, and a high proportion of polymorphic inserts in the genomes of the killifish species, Fundulus heteroclitus. Additional species surveyed by degenerate PCR include Cyprinodon variegatus, Rivulus marmoratus, and Menidia beryllina. These results further support the generality of the differences that exist in host–element dynamics between teleost fish and mammalian genomes with regard to L1 retrotransposons.Reviewing Editor: Dr. Axel Meyer     

The relationship between body mass and relative investment in testes mass in amniotes and other vertebrates

In terrestrial placental mammals, there is a well‐known negative allometric relationship between body mass and relative investment in testes mass. Such a negative relationship means that males of relatively monogamous small species invest proportionately more in their reproductive tissues than males of more polyandrous larger species. The selective pressure responsible for this relationship remains unclear and is it not known if this is a general allometric relationship that is similar across all vertebrate lineages. To investigate this, we conducted the first comparison of relationships between body mass and testes mass (using percentage testes mass as the dependent variable) across a variety of vertebrate groups. In all amniote lineages examined, the allometric relationship between body mass and testes mass was relatively strong and negative. We show, for the first time, that reptiles, birds and terrestrial placental mammals followed the same allometric relationship and, contrary to previous expectations, this relationship is sigmoidal rather than linear. Within this data set, there was no significant difference between this general amniote relationship and any of the 13 orders of reptiles, birds and terrestrial placental mammals examined. As a result, we propose that a sigmoidal relationship should be considered the default assumption for the form of the body mass – testes mass relationship within the amniote lineage. However, we also identify significant differences within some additional mammal groups (marsupials, bats and cetaceans). In each of these cases, only some sub‐groupings differed significantly from the general amniote relationship. In contrast to the amniotes, the relationship is relatively weak and positive in teleost fish and frogs suggesting that a negative allometric relationship is not universal in vertebrates. We explore whether variation in the body mass – testes mass relationships can be linked to sperm competition or a variety of ecological characteristics, either for amniotes in particular or vertebrates in general.     

Comparison of diverged Hoxc8 early enhancer activities reveals modification of regulatory interactions at conserved cis-acting elements

The Hoxc8 early enhancer that controls the initiation and establishment of Hoxc8 expression in the developing mouse embryo is found in different vertebrate lineages including mammals, birds and fish. Mouse and Fugu Hoxc8 early enhancers (200 bp) have diverged in the composition of elements located towards the 3' region. However, they share cis-acting elements A-E located in the 5' region. Mutations at these elements in the context of the mouse Hoxc8 early enhancer affect reporter gene expression in the posterior neural tube, somites and lateral plate mesoderm of day 9.5 mouse embryos. Here, we demonstrate that mutations introduced at the same elements but in the context of the Fugu Hoxc8 early enhancer had different consequences on the reporter gene expression in transgenic mouse embryos. Furthermore, in contrast to the mouse enhancer the Fugu enhancer does not utilize elements D and E in achieving posterior neural tube and somite expression. These results suggest that the diverged sequences prevent regulatory interactions at conserved cis-acting elements. We propose that divergent sequences modify regulatory interactions at conserved elements by providing a "contextual change". Our finding that the enhancer elements do not act in a unitary fashion but function in the context of the surrounding sequence brings a new dimension to the study of cis-regulatory evolution.     

Selective Pressure on the Allantoicase Gene During Vertebrate Evolution

During vertebrate evolution, the uric acid degradation pathway has been modified and several enzymes have been lost. Consequently, the end product of purine catabolism varies from species to species. In the past few years, we have focused our attention on vertebrate allantoicase (an uricolytic pathway enzyme), whose activity is present in certain fish and amphibians only, but whose mRNA we detected also in mammals. As allantoicase activity disappeared in amniotes, we wonder why these sequences not only remain present in the mammalian genome, but are still transcribed. To elucidate this issue, we have cloned and analyzed comparable cDNA sequences of different organisms from ascidians to mammals. The analysis of the nonsynonymous–synonymous substitution rate that we performed on the coding region comprising exons 3 to 8 by means of maximum likelihood suggested that a certain amount of purifying selection is acting on the allantoicase sequences. Some implications of the preservation of an apparently unnecessary gene in higher vertebrates are discussed.     

Exploring the correlations between sequence evolution rate and phenotypic divergence across the Mammalian tree provides insights into adaptive evolution

Sequence evolution behaves in a relatively consistent manner, leading to one of the fundamental paradigms in biology, the existence of a ??molecular clock??. The molecular clock can be distilled to the concept of accumulation of substitutions, through time yielding a stable rate from which we can estimate lineage divergence. Over the last 50?years, evolutionary biologists have obtained an in-depth understanding of this clock??s nuances. It has been fine-tuned by taking into account the vast heterogeneity in rates across lineages and genes, leading to ??relaxed?? molecular clock methods for timetree reconstruction. Sequence rate varies with life history traits including body size, generation time and metabolic rate, and we review recent studies on this topic. However, few studies have explicitly examined correlates between molecular evolution and morphological evolution. The patterns observed across diverse lineages suggest that rates of molecular and morphological evolution are largely decoupled. We discuss how identifying the molecular mechanisms behind rapid functional radiations are central to understanding evolution. The vast functional divergence within mammalian lineages that have relatively ??slow?? sequence evolution refutes the hypotheses that pulses in diversification yielding major phenotypic change are the result of steady accumulation of substitutions. Patterns rather suggest phenotypic divergence is likely caused by regulatory alterations mediated through mechanisms such as insertions/deletions in functional regions. These can rapidly arise and sweep to fixation faster than predicted from a lineage??s sequence neutral substitution rate, enabling species to leapfrog between phenotypic ??islands??. We suggest research directions that could illuminate mechanisms behind the functional diversity we see today.