Evolutionary growth process of highly conserved sequences in vertebrate genomes

Genome sequence comparison between evolutionarily distant species revealed ultraconserved elements (UCEs) among mammals under strong purifying selection. Most of them were also conserved among vertebrates. Because they tend to be located in the flanking regions of developmental genes, they would have fundamental roles in creating vertebrate body plans. However, the evolutionary origin and selection mechanism of these UCEs remain unclear. Here we report that UCEs arose in primitive vertebrates, and gradually grew in vertebrate evolution. We searched for UCEs in two teleost fishes, Tetraodon nigroviridis and Oryzias latipes, and found 554 UCEs with 100% identity over 100 bps. Comparison of teleost and mammalian UCEs revealed 43 pairs of common, jawed-vertebrate UCEs (jUCE) with high sequence identities, ranging from 83.1% to 99.2%. Ten of them retain lower similarities to the Petromyzon marinus genome, and the substitution rates of four non-exonic jUCEs were reduced after the teleost-mammal divergence, suggesting that robust conservation had been acquired in the jawed vertebrate lineage. Our results indicate that prototypical UCEs originated before the divergence of jawed and jawless vertebrates and have been frozen as perfect conserved sequences in the jawed vertebrate lineage. In addition, our comparative sequence analyses of UCEs and neighboring regions resulted in a discovery of lineage-specific conserved sequences. They were added progressively to prototypical UCEs, suggesting step-wise acquisition of novel regulatory roles. Our results indicate that conserved non-coding elements (CNEs) consist of blocks with distinct evolutionary history, each having been frozen since different evolutionary era along the vertebrate lineage.     

Identification and characterization of new long conserved noncoding sequences in vertebrates

Comparative sequence analyses have identified highly conserved genomic DNA sequences, including noncoding sequences, between humans and other species. By performing whole-genome comparisons of human and mouse, we have identified 611 conserved noncoding sequences longer than 500 bp, with more than 95% identity between the species. These long conserved noncoding sequences (LCNS) include 473 new sequences that do not overlap with previously reported ultraconserved elements (UCE), which are defined as aligned sequences longer than 200 bp with 100% identity in human, mouse, and rat. The LCNS were distributed throughout the genome except for the Y chromosome and often occurred in clusters within regions with a low density of coding genes. Many of the LCNS were also highly conserved in other mammals, chickens, frogs, and fish; however, we were unable to find orthologous sequences in the genomes of invertebrate species. In order to examine whether these conserved sequences are functionally important or merely mutational cold spots, we directly measured the frequencies of ENU-induced germline mutations in the LCNS of the mouse. By screening about 40.7 Mb, we found 35 mutations, including mutations at nucleotides that were conserved between human and fish. The mutation frequencies were equivalent to those found in other genomic regions, including coding sequences and introns, suggesting that the LCNS are not mutational cold spots at all. Taken together, these results suggest that mutations occur with equal frequency in LCNS but are eliminated by natural selection during the course of evolution.

     

Comparison of Pax1/9 locus reveals 500-Myr-old syntenic block and evolutionary conserved noncoding regions

Identification of conserved genomic regions within and between different genomes is crucial when studying genome evolution. Here, we described regions of strong synteny conservation between vertebrate deuterostomes (tetrapods and teleosts) and invertebrate deuterostomes (amphioxus and sea urchin). The shared gene contents across phylogenetically distant species demonstrate that the conservation of the regions stemmed from an ancestral segment instead of a series of independent convergent events. Comparison of the syntenic regions allows us to postulate the primitive gene organization in the last common ancestor of deuterostomes and the evolutionary events that occurred to the 3 distinct lineages of sea urchin, amphioxus, and vertebrates after their separation. In addition, alignment of the syntenic regions led to the identification of 8 noncoding evolutionarily conserved regions shared between amphioxus and vertebrates. To our knowledge, this is the first report of conserved noncoding sequences shared by vertebrates and nonvertebrates. These noncoding sequences have high possibility of being elements that regulate neighboring genes. They are likely to be a factor in the maintenance of conserved synteny over long phylogenetic distance in different deuterostome lineages.     

Identification and characterization of new long conserved noncoding sequences in vertebrates

Comparative sequence analyses have identified highly conserved genomic DNA sequences, including noncoding sequences, between humans and other species. By performing whole-genome comparisons of human and mouse, we have identified 611 conserved noncoding sequences longer than 500 bp, with more than 95% identity between the species. These long conserved noncoding sequences (LCNS) include 473 new sequences that do not overlap with previously reported ultraconserved elements (UCE), which are defined as aligned sequences longer than 200 bp with 100% identity in human, mouse, and rat. The LCNS were distributed throughout the genome except for the Y chromosome and often occurred in clusters within regions with a low density of coding genes. Many of the LCNS were also highly conserved in other mammals, chickens, frogs, and fish; however, we were unable to find orthologous sequences in the genomes of invertebrate species. In order to examine whether these conserved sequences are functionally important or merely mutational cold spots, we directly measured the frequencies of ENU-induced germline mutations in the LCNS of the mouse. By screening about 40.7 Mb, we found 35 mutations, including mutations at nucleotides that were conserved between human and fish. The mutation frequencies were equivalent to those found in other genomic regions, including coding sequences and introns, suggesting that the LCNS are not mutational cold spots at all. Taken together, these results suggest that mutations occur with equal frequency in LCNS but are eliminated by natural selection during the course of evolution.     

A genetic map of Cottus gobio (Pisces, Teleostei) based on microsatellites can be linked to the physical map of Tetraodon nigroviridis

To initiate QTL studies in the nonmodel fish Cottus gobio we constructed a genetic map based on 171 microsatellite markers. The mapping panel consisted of F1 intercrosses between two divergent Cottus lineages from the River Rhine System. Basic local alignment search tool (BLAST) searches with the flanking sequences of the microsatellite markers yielded a significant (e < 10(-5)) hit with the Tetraodon nigroviridis genomic sequence for 45% of the Cottus loci. Remarkably, most of these hits were due to short highly conserved noncoding stretches. These have an average length of 40 bp and are on average 92% conserved. Comparison of the map locations between the two genomes revealed extensive conserved synteny, suggesting that the Tetraodon genomic sequence will serve as an excellent genomic reference for at least the Acanthopterygii, which include evolutionarily interesting fish groups such as guppies (Poecilia), cichlids (Tilapia) or Xiphophorus (Platy). The apparent high density of short conserved noncoding stretches in these fish genomes will highly facilitate the identification of genes that have been identified in QTL mapping strategies of evolutionary relevant traits.     

Evolutionary conservation of DNA methylation in CpG sites within ultraconserved noncoding elements

Ultraconserved noncoding elements (UCNEs) constitute less than 1 Mb of vertebrate genomes and are impervious to accumulating mutations. About 4000 UCNEs exist in vertebrate genomes, each at least 200 nucleotides in length, sharing greater than 95% sequence identity between human and chicken. Despite extreme sequence conservation over 400 million years of vertebrate evolution, we show both ordered interspecies and within-species interindividual variation in DNA methylation in these regions. Here, we surveyed UCNEs with high CpG density in 56 species finding half to be intermediately methylated and the remaining near 0% or 100%. Intermediately methylated UCNEs displayed a greater range of methylation between mouse tissues. In a human population, most UCNEs showed greater variation than the LINE1 transposon, a frequently used epigenetic biomarker. Global methylation was found to be inversely correlated to hydroxymethylation across 60 vertebrates. Within UCNEs, DNA methylation is flexible, conserved between related species, and relaxed from the underlying sequence selection pressure, while remaining heritable through speciation.     

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.     

Tuning in to the signals: noncoding sequence conservation in vertebrate genomes

Aligning and comparing genomic sequences enables the identification of conserved sequence signatures and can enrich for coding and noncoding functional regions. In vertebrates, the comparison of human and rodent genomes and the comparison of evolutionarily distant genomes, such as human and pufferfish, have identified specific sets of 'ultraconserved' sequence elements associated with the control of early development. However, is this just the tip of a 'conservation iceberg' or do these sequences represent a specific class of regulatory element? Studies on the zebrafish phox2b gene region and the ENCODE project suggest that many regulatory elements are not highly conserved, posing intriguing questions about the relationship between noncoding sequence conservation and function and the evolution of regulatory sequences.     

Human and mouse ABCA1 comparative sequencing and transgenesis studies revealing novel regulatory sequences

The expression of ABCA1, a major participant in apolipoprotein-mediated cholesterol efflux, is regulated by a variety of factors, including intracellular cholesterol concentration. To identify sequences involved in its regulation, we sequenced and compared approximately 200 kb of mouse and human DNA containing the ABCA1 gene. Furthermore, expression of the human gene containing different 5' ends was examined in transgenic mice. Sequence comparison revealed multiple conserved noncoding sequences. The two most highly conserved noncoding elements (CNS1, 88% identity over 498 bp; CNS2, 81% identity over 214 bp) were also highly conserved in other organisms. Mice containing the human ABCA1 gene, 70 kb of upstream DNA, and 35 kb of downstream DNA expressed the transgene similarly to endogenous Abca1. A second transgene beginning 3' to exon 1 was expressed only in liver, providing strong evidence of an unsuspected liver-specific promoter. The identified conserved noncoding sequences invite further investigation to elucidate ABCA1 regulation.     

Ultraconserved elements between the genomes of the plants Arabidopsis thaliana and rice

Ultraconserved elements, sequences with 100% identity with no insertions or deletions between genomes, have been found in both vertebrate and invertebrate genomes; whether plant genomes contain ultraconserved elements, however, is unknown. We consequently compared the genomes of Arabidopsis thaliana and rice, which diverged about 200 million years ago, and identified 25 ultraconserved elements that are longer than 100 bp. Similar to those previously found, ultraconserved elements in plants tend to occur in clusters and locate at noncoding regions; nevertheless, they have many distinct features. For instance, the longest ultraconserved element between the 2 plant genomes is 1491 bp, much longer than the longest one (779 bp) between the human and rodent genomes. Some biological implications are discussed, but the functions of these plant ultraconserved elements and the reasons why they are practically frozen during the evolution of millions of years remain a mystery.     

Detection of potential GDF6 regulatory elements by multispecies sequence comparisons and identification of a skeletal joint enhancer

The identification of noncoding functional elements within vertebrate genomes, such as those that regulate gene expression, is a major challenge. Comparisons of orthologous sequences from multiple species are effective at detecting highly conserved regions and can reveal potential regulatory sequences. The GDF6 gene controls developmental patterning of skeletal joints and is associated with numerous, distant cis-acting regulatory elements. Using sequence data from 14 vertebrate species, we performed novel multispecies comparative analyses to detect highly conserved sequences flanking GDF6. The complementary tools WebMCS and ExactPlus identified a series of multispecies conserved sequences (MCSs). Of particular interest are MCSs within noncoding regions previously shown to contain GDF6 regulatory elements. A previously reported conserved sequence at -64 kb was also detected by both WebMCS and ExactPlus. Analysis of LacZ-reporter transgenic mice revealed that a 440-bp segment from this region contains an enhancer for Gdf6 expression in developing proximal limb joints. Several other MCSs represent candidate GDF6 regulatory elements; many of these are not conserved in fish or frog, but are strongly conserved in mammals.     

Efficient identification of regulatory sequences in the chicken genome by a powerful combination of embryo electroporation and genome comparison

Recently expanded knowledge of gene regulation clearly indicates that the regulatory sequences of a gene, usually identified as enhancers, are widely distributed in the gene locus, revising the classical view that they are clustered in the vicinity of genes. To identify regulatory sequences for Sox2 expression governing early neurogenesis, we scanned the 50-kb region of the chicken Sox2 locus for enhancer activity utilizing embryo electroporation, resulting in identification of a number of enhancers scattered throughout the analyzed genomic span. The 'pan-neural' Sox2 expression in early embryos is actually brought about by the composite activities of five separate enhancers with distinct spatio-temporal specificities. These and other functionally defined enhancers exactly correspond to extragenic sequence blocks that are conspicuously conserved between the chicken and mammalian genomes and that are embedded in sequences with a wide range of sequence conservation between humans and mice. The sequences conserved between amniotes and teleosts correspond to subregions of the enhancer subsets which presumably represent core motifs of the enhancers, and the limited conservation partly reflects divergent expression patterns of the gene. The phylogenic distance between the chicken and mammals appears optimal for identifying a battery of genetic regulatory elements as conserved sequence blocks, and chicken embryo electroporation facilitates functional characterization of these elements.     

Characterization of the chicken histone H1 gene complement. Generation of a complete set of vertebrate H1 protein sequences

Sequence analysis of four chicken H1 histone genes described here completes the characterization of the full complement of six H1 genes in the chicken genome. Each of the six genes codes for a different H1 protein sequence, and these range in size from 217 to 224 amino acids. The proteins are distinct in sequence from the H1-related chicken H5 protein and appear to be analogous to the standard somatic mammalian H1 subtypes. The protein sequence data deduced from the genes represent the first complete set of vertebrate H1 protein sequences. Comparison of the chicken H1 gene noncoding sequences with each other and with H1 gene sequences from other organisms reveals conservation of an H1 gene-specific element, a G-rich element, and histone gene-specific 3' elements. Additional sequences are conserved between H1 genes of the chicken and other vertebrates. Comparisons also reveal variation in promoter and 3' elements between chicken genes that could play a role in the differential expression of H1 gene protein products.     

Divergence of conserved non-coding sequences: rate estimates and relative rate tests

In many eukaryotic genomes only a small fraction of the DNA codes for proteins, but the non-protein coding DNA harbors important genetic elements directing the development and the physiology of the organisms, like promoters, enhancers, insulators, and micro-RNA genes. The molecular evolution of these genetic elements is difficult to study because their functional significance is hard to deduce from sequence information alone. Here we propose an approach to the study of the rate of evolution of functional non-coding sequences at a macro-evolutionary scale. We identify functionally important non-coding sequences as Conserved Non-Coding Nucleotide (CNCN) sequences from the comparison of two outgroup species. The CNCN sequences so identified are then compared to their homologous sequences in a pair of ingroup species, and we monitor the degree of modification these sequences suffered in the two ingroup lineages. We propose a method to test for rate differences in the modification of CNCN sequences among the two ingroup lineages, as well as a method to estimate their rate of modification. We apply this method to the full sequences of the HoxA clusters from six gnathostome species: a shark, Heterodontus francisci; a basal ray finned fish, Polypterus senegalus; the amphibian, Xenopus tropicalis; as well as three mammalian species, human, rat and mouse. The results show that the evolutionary rate of CNCN sequences is not distinguishable among the three mammalian lineages, while the Xenopus lineage has a significantly increased rate of evolution. Furthermore the estimates of the rate parameters suggest that in the stem lineage of mammals the rate of CNCN sequence evolution was more than twice the rate observed within the placental amniotes clade, suggesting a high rate of evolution of cis-regulatory elements during the origin of amniotes and mammals. We conclude that the proposed methods can be used for testing hypotheses about the rate and pattern of evolution of putative cis-regulatory elements.     

Sequencing and comparative analysis of a conserved syntenic segment in the Solanaceae

Comparative genomics is a powerful tool for gaining insight into genomic function and evolution. However, in plants, sequence data that would enable detailed comparisons of both coding and noncoding regions have been limited in availability. Here we report the generation and analysis of sequences for an unduplicated conserved syntenic segment (CSS) in the genomes of five members of the agriculturally important plant family Solanaceae. This CSS includes a 105-kb region of tomato chromosome 2 and orthologous regions of the potato, eggplant, pepper, and petunia genomes. With a total neutral divergence of 0.73-0.78 substitutions/site, these sequences are similar enough that most noncoding regions can be aligned, yet divergent enough to be informative about evolutionary dynamics and selective pressures. The CSS contains 17 distinct genes with generally conserved order and orientation, but with numerous small-scale differences between species. Our analysis indicates that the last common ancestor of these species lived approximately 27-36 million years ago, that more than one-third of short genomic segments (5-15 bp) are under selection, and that more than two-thirds of selected bases fall in noncoding regions. In addition, we identify genes under positive selection and analyze hundreds of conserved noncoding elements. This analysis provides a window into 30 million years of plant evolution in the absence of polyploidization.     

Ultraconserved elements anchor thousands of genetic markers spanning multiple evolutionary timescales

Although massively parallel sequencing has facilitated large-scale DNA sequencing, comparisons among distantly related species rely upon small portions of the genome that are easily aligned. Methods are needed to efficiently obtain comparable DNA fragments prior to massively parallel sequencing, particularly for biologists working with non-model organisms. We introduce a new class of molecular marker, anchored by ultraconserved genomic elements (UCEs), that universally enable target enrichment and sequencing of thousands of orthologous loci across species separated by hundreds of millions of years of evolution. Our analyses here focus on use of UCE markers in Amniota because UCEs and phylogenetic relationships are well-known in some amniotes. We perform an in silico experiment to demonstrate that sequence flanking 2030 UCEs contains information sufficient to enable unambiguous recovery of the established primate phylogeny. We extend this experiment by performing an in vitro enrichment of 2386 UCE-anchored loci from nine, non-model avian species. We then use alignments of 854 of these loci to unambiguously recover the established evolutionary relationships within and among three ancient bird lineages. Because many organismal lineages have UCEs, this type of genetic marker and the analytical framework we outline can be applied across the tree of life, potentially reshaping our understanding of phylogeny at many taxonomic levels.