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.     

High intron sequence conservation across three mammalian orders suggests functional constraints

Several studies have demonstrated high levels of sequence conservation in noncoding DNA compared between two species (e.g., human and mouse), and interpreted this conservation as evidence for functional constraints. If this interpretation is correct, it suggests the existence of a hidden class of abundant regulatory elements. However, much of the noncoding sequence conserved between two species may result from chance or from small-scale heterogeneity in mutation rates. Stronger inferences are expected from sequence comparisons using more than two taxa, and by testing for spatial patterns of conservation in addition to primary sequence similarity. We used a Bayesian local alignment method to compare approximately 10 kb of intron sequence from nine genes in a pairwise manner between human, whale, and seal to test whether the degree and pattern of conservation is consistent with neutral divergence. Comparison of the three sets of conserved gapless pairwise blocks revealed the following patterns: The proportion of identical intron nucleotides averaged 47% in pairwise comparisons and 28% across the three taxa. Proportions of conserved sequence were similar in unique sequence and general mammalian repetitive elements. We simulated sequence evolution under a neutral model using published estimates of substitution rate heterogeneity for noncoding DNA and found pairwise identity at 33% and three-taxon identity at 16% of nucleotide sites. Spatial patterns of primary sequence conservation were also nonrandomly distributed within introns. Overall, segments of intron sequence closer to flanking exons were significantly more conserved than interior intron sequence. This level of intron sequence conservation is above that expected by chance and strongly suggests that intron sequences are playing a larger functional role in gene regulation than previously realized.     

Conserved noncoding sequences among cultivated cereal genomes identify candidate regulatory sequence elements and patterns of promoter evolution

Surveys for conserved noncoding sequences (CNS) among genes from monocot cereal species were conducted to assess the general properties of CNS in grass genomes and their correlation with known promoter regulatory elements. Initial comparisons of 11 orthologous maize-rice gene pairs found that previously defined regulatory motifs could be identified within short CNS but could not be distinguished reliably from random sequence matches. Among the different phylogenetic footprinting algorithms tested, the VISTA tool yielded the most informative alignments of noncoding sequence. VISTA was used to survey for CNS among all publicly available genomic sequences from maize, rice, wheat, barley, and sorghum, representing >300 gene comparisons. Comparisons of orthologous maize-rice and maize-sorghum gene pairs identified 20 bp as a minimal length criterion for a significant CNS among grass genes, with few such CNS found to be conserved across rice, maize, sorghum, and barley. The frequency and length of cereal CNS as well as nucleotide substitution rates within CNS were consistent with the known phylogenetic distances among the species compared. The implications of these findings for the evolution of cereal gene promoter sequences and the utility of using the nearly completed rice genome sequence to predict candidate regulatory elements in other cereal genes by phylogenetic footprinting are discussed.     

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.     

A comparative genomic analysis of the cow,pig, and human CFTR genes identifies potential intronic regulatory elements

The identification of sequences within noncoding regions of genes that are conserved between several species may indicate potential regulatory elements. This is important for genes with complex control mechanisms such as the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR demonstrates similar patterns of temporal and spatial expression in human and sheep, but these differ significantly in mouse cftr. The complete sheep CFTR sequence is unavailable so we annotated BAC clones encompassing the CFTR gene from two other artiodactyl species (cow and pig) for comparative sequence analysis. Regions of introns 2, 3, 10, 17a, 18, and 21 and 3' flanking sequence corresponding to human CFTR DNase I hypersensitive sites (DHS) showed high homology in the cow and pig. Cross-species sequence conservation also enabled finer mapping of other human DHS, including those in introns 1, 16, and 20. Additional potential regulatory elements not associated with human DHS were also identified.     

Analysis of sequence conservation at nucleotide resolution

One of the major goals of comparative genomics is to understand the evolutionary history of each nucleotide in the human genome sequence, and the degree to which it is under selective pressure. Ascertainment of selective constraint at nucleotide resolution is particularly important for predicting the functional significance of human genetic variation and for analyzing the sequence substructure of cis-regulatory sequences and other functional elements. Current methods for analysis of sequence conservation are focused on delineation of conserved regions comprising tens or even hundreds of consecutive nucleotides. We therefore developed a novel computational approach designed specifically for scoring evolutionary conservation at individual base-pair resolution. Our approach estimates the rate at which each nucleotide position is evolving, computes the probability of neutrality given this rate estimate, and summarizes the result in a Sequence CONservation Evaluation (SCONE) score. We computed SCONE scores in a continuous fashion across 1% of the human genome for which high-quality sequence information from up to 23 genomes are available. We show that SCONE scores are clearly correlated with the allele frequency of human polymorphisms in both coding and noncoding regions. We find that the majority of noncoding conserved nucleotides lie outside of longer conserved elements predicted by other conservation analyses, and are experiencing ongoing selection in modern humans as evident from the allele frequency spectrum of human polymorphism. We also applied SCONE to analyze the distribution of conserved nucleotides within functional regions. These regions are markedly enriched in individually conserved positions and short (<15 bp) conserved “chunks.” Our results collectively suggest that the majority of functionally important noncoding conserved positions are highly fragmented and reside outside of canonically defined long conserved noncoding sequences. A small subset of these fragmented positions may be identified with high confidence.     

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.     

Comparative sequence analysis of 634 kb of the mouse chromosome 16 region of conserved synteny with the human velocardiofacial syndrome region on chromosome 22q11.2

Mouse genomic DNA sequence extending 634 kb on proximal mouse chromosome 16 was compared to the corresponding human sequence from chromosome 22q11.2. Haploinsufficiency for this region results in velocardiofacial syndrome (VCFS) in humans. The mouse region is rearranged into three conserved blocks relative to human, but gene content and position are highly conserved within these blocks. Examination of the boundaries of one of these blocks suggested that the evolutionary chromosomal rearrangement occurred in the mouse lineage, resulting in inactivation of the mouse orthologue of ZNF74. Sequence analysis identified 21 genes and 15 ESTs. These include 2 novel genes, Srec2 and Cals2, and previously undescribed splice variants of several other genes. Exon discovery was carried out using GRAIL2, MZEF, or comparative analysis across 491 kb of conserved mouse and human sequence. Sequence comparison was highly effective, identifying every gene and nearly every exon without the high frequency of false-positive predictions seen when algorithmic methods were used alone. In combination, these procedures identified every gene with no false-positive predictions. Comparative sequence analysis also revealed regions of extensive conservation among noncoding sequences, accounting for 6% of the sequence. A library of such sequences has been established to form a resource for generalized studies of regulatory and structural elements.     

Novel vertebrate genes and putative regulatory elements identified at kidney disease and NR2E1/fierce loci

Fierce (frc) mice are deleted for nuclear receptor 2e1 (Nr2e1), and exhibit cerebral hypoplasia, blindness, and extreme aggression. To characterize the Nr2e1 locus, which may also contain the mouse kidney disease (kd) allele, we compared sequence from human, mouse, and the puffer fish Fugu rubripes. We identified a novel gene, c222389, containing conserved elements in noncoding regions. We also discovered a novel vertebrate gene conserved across its length in prokaryotes and invertebrates. Based on a dramatic upregulation in lactating breast, we named this gene lactation elevated-1 (LACE1). Two separate 100-bp elements within the first NR2E1 intron were virtually identical between the three species, despite an estimated 450 million years of divergent evolution. These elements represent strong candidates for functional NR2E1 regulatory elements in vertebrates. A high degree of conservation across NR2E1 combined with a lack of interspersed repeats suggests that an array of regulatory elements embedded within the gene is required for proper gene expression.     

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.

     

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.     

Inferring natural selection on fine-scale chromatin organization in yeast

Despite its potential role in the evolution of complex phenotypes, the detection of negative (purifying) and positive selection on noncoding regulatory sequence has been elusive because of the inherent difficulty in predicting the functional consequences of mutations on noncoding sequence. Because the functioning of regulatory sequence depends upon both chromatin configuration and cis-regulatory factor binding, we investigate the idea that the functional conservation of regulatory regions should be associated with the conservation of sequence-dependent bending properties of DNA that determine its affinity for the nucleosome. Recent advances in the computational prediction of sequence-dependent affinity to nucleosomes provide an opportunity to distinguish between neutral and nonneutral evolution of fine-scale chromatin organization. Here, a statistical test is presented for detecting evolutionary conservation and/or adaptive evolution of nucleosome affinity from interspecies comparisons of DNA sequences. Local nucleosome affinities of homologous sequences were calculated using 2 recently published methods. A randomization test was applied to sites of mutation to evaluate the similarity of DNA-nucleosome affinity between several closely related species of Saccharomyces yeast. For most of the genes we analyzed, the conservation of local nucleosome affinity was detected at a few distinct locations in the upstream noncoding region. Our results also demonstrate that different patterns of chromatin evolution have shaped DNA-nucleosome interaction at the core promoters of TATA-containing and TATA-less genes and that elevated purifying selection has maintained low affinity for nucleosome in the core promoters of the latter group. Across the entire yeast genome, DNA-nucleosome interaction was also discovered to be significantly more conserved in TATA-less genes compared with TATA-containing genes.     

Evolution of Conserved Noncoding Sequences in Arabidopsis thaliana

Recent pangenome studies have revealed a large fraction of the gene content within a species exhibits presence–absence variation (PAV). However, coding regions alone provide an incomplete assessment of functional genomic sequence variation at the species level. Little to no attention has been paid to noncoding regulatory regions in pangenome studies, though these sequences directly modulate gene expression and phenotype. To uncover regulatory genetic variation, we generated chromosome-scale genome assemblies for thirty Arabidopsis thaliana accessions from multiple distinct habitats and characterized species level variation in Conserved Noncoding Sequences (CNS). Our analyses uncovered not only PAV and positional variation (PosV) but that diversity in CNS is nonrandom, with variants shared across different accessions. Using evolutionary analyses and chromatin accessibility data, we provide further evidence supporting roles for conserved and variable CNS in gene regulation. Additionally, our data suggests that transposable elements contribute to CNS variation. Characterizing species-level diversity in all functional genomic sequences may later uncover previously unknown mechanistic links between genotype and phenotype.