The evolution of lineage-specific clusters of single nucleotide substitutions in the human genome.

Genomic regions harboring large numbers of human-specific single nucleotide substitutions are of significant interest since they are potential genomic foci underlying the evolution of human-specific traits as well as human adaptive evolution. Previous studies aimed to identify such regions either used pre-defined genomic locations such as coding sequences and conserved genomic elements or employed sliding window methods. Such approaches may miss clusters of substitutions occurring in regions other than those pre-defined locations, or not be able to distinguish human-specific clusters of substitutions from regions of generally high substitution rates. Here, we conduct a 'maximal segment' analysis to scan the whole human genome to identify clusters of human-specific substitutions that occurred since the divergence of the human and the chimpanzee genomes. This method can identify species-specific clusters of substitutions while not relying on pre-defined regions. We thus identify thousands of clusters of human-specific single nucleotide substitutions. The evolution of such clusters is driven by a combination of several different evolutionary processes including increased regional mutation rate, recombination-associated processes, and positive selection. These newly identified regions of human-specific substitution clusters include large numbers of previously identified human accelerated regions, and exhibit significant enrichments of genes involved in several developmental processes. Our study provides a useful tool to study the evolution of the human genome.

Comparative analyses of DNA methylation and sequence evolution using Nasonia genomes.

The functional and evolutionary significance of DNA methylation in insect genomes remains to be resolved. Nasonia is well situated for comparative analyses of DNA methylation and genome evolution, since the genomes of a moderately distant outgroup species as well as closely related sibling species are available. Using direct sequencing of bisulfite-converted DNA, we uncovered a substantial level of DNA methylation in 17 of 18 Nasonia vitripennis genes and a strong correlation between methylation level and CpG depletion. Notably, in the sex-determining locus transformer, the exon that is alternatively spliced between the sexes is heavily methylated in both males and females, whereas other exons are only sparsely methylated. Orthologous genes of the honeybee and Nasonia show highly similar relative levels of CpG depletion, despite ~190 My divergence. Densely and sparsely methylated genes in these species also exhibit similar functional enrichments. We found that the degree of CpG depletion is negatively correlated with substitution rates between closely related Nasonia species for synonymous, nonsynonymous, and intron sites. This suggests that mutation rates increase with decreasing levels of germ line methylation. Thus, DNA methylation is prevalent in the Nasonia genome, may participate in regulatory processes such as sex determination and alternative splicing, and is correlated with several aspects of genome and sequence evolution.

DNA methylation is widespread and associated with differential gene expression in castes of the honeybee, Apis mellifera.

The recent, unexpected discovery of a functional DNA methylation system in the genome of the social bee Apis mellifera underscores the potential importance of DNA methylation in invertebrates. The extent of genomic DNA methylation and its role in A. mellifera remain unknown, however. Here we show that genes in A. mellifera can be divided into 2 distinct classes, one with low-CpG dinucleotide content and the other with high-CpG dinucleotide content. This dichotomy is explained by the gradual depletion of CpG dinucleotides, a well-known consequence of DNA methylation. The loss of CpG dinucleotides associated with DNA methylation also may explain the unusual mutational patterns seen in A. mellifera that lead to AT-rich regions of the genome. A detailed investigation of this dichotomy implicates DNA methylation in A. mellifera development. High-CpG genes, which are predicted to be hypomethylated in germlines, are enriched with functions associated with developmental processes, whereas low-CpG genes, predicted to be hypermethylated in germlines, are enriched with functions associated with basic biological processes. Furthermore, genes more highly expressed in one caste than another are overrepresented among high-CpG genes. Our results highlight the potential significance of epigenetic modifications, such as DNA methylation, in developmental processes in social insects. In particular, the pervasiveness of DNA methylation in the genome of A. mellifera provides fertile ground for future studies of phenotypic plasticity and genomic imprinting.

Sociality is linked to rates of protein evolution in a highly social insect.

Eusocial insects exhibit unparalleled levels of cooperation and dominate terrestrial ecosystems. The success of eusocial insects stems from the presence of specialized castes that undertake distinct tasks. We investigated whether the evolutionary transition to societies with discrete castes was associated with changes in protein evolution. We predicted that proteins with caste-biased gene expression would evolve rapidly due to reduced antagonistic pleiotropy. We found that queen-biased proteins of the honeybee Apis mellifera did indeed evolve rapidly, as predicted. However, worker-biased proteins exhibited slower evolutionary rates than queen-biased or nonbiased proteins. We suggest that distinct selective pressures operating on caste-biased genes, rather than a general reduction in pleiotropy, explain the observed differences in evolutionary rates. Our study highlights, for the first time, the interaction between highly social behavior and dynamics of protein evolution.

Identification of early liver toxicity gene biomarkers using comparative supervised machine learning.

Screening agrochemicals and pharmaceuticals for potential liver toxicity is required for regulatory approval and is an expensive and time-consuming process. The identification and utilization of early exposure gene signatures and robust predictive models in regulatory toxicity testing has the potential to reduce time and costs substantially. In this study, comparative supervised machine learning approaches were applied to the rat liver TG-GATEs dataset to develop feature selection and predictive testing. We identified ten gene biomarkers using three different feature selection methods that predicted liver necrosis with high specificity and selectivity in an independent validation dataset from the Microarray Quality Control (MAQC)-II study. Nine of the ten genes that were selected with the supervised methods are involved in metabolism and detoxification (Car3, Crat, Cyp39a1, Dcd, Lbp, Scly, Slc23a1, and Tkfc) and transcriptional regulation (Ablim3). Several of these genes are also implicated in liver carcinogenesis, including Crat, Car3 and Slc23a1. Our biomarker gene signature provides high statistical accuracy and a manageable number of genes to study as indicators to potentially accelerate toxicity testing based on their ability to induce liver necrosis and, eventually, liver cancer.

Primate phylogenomics: developing numerous nuclear non-coding, non-repetitive markers for ecological and phylogenetic applications and analysis of evolutionary rate variation.

BACKGROUND: Genetic analyses are often limited by the availability of appropriate molecular markers. Markers from neutrally evolving genomic regions may be particularly useful for inferring evolutionary histories because they escape the constraints of natural selection. For the majority of taxa however, obtaining such markers is challenging. Advances in genomics have the potential to alleviate the shortage of neutral markers. Here we present a method to develop numerous markers from putatively neutral regions of primate genomes. RESULTS: We began with the available whole genome sequences of human, chimpanzee and macaque. Using computational methods, we identified a total of 280 potential amplicons from putatively neutral, non-coding, non-repetitive regions of these genomes. Subsequently we amplified, using experimental methods, many of these amplicons from diverse primate taxa, including a ring-tailed lemur, which is distantly related to the genomic resources. Using a subset of 10 markers, we demonstrate the utility of the developed markers in phylogenetic and evolutionary rate analyses. Particularly, we uncovered substantial evolutionary rate variation among lineages, some of which are previously not reported. CONCLUSION: We successfully developed numerous markers from putatively neutral regions of primate genomes using a strategy combining computational and experimental methods. Applying these markers to phylogenetic and evolutionary rate variation analyses exemplifies the utility of these markers. Diverse ecological and evolutionary analyses will benefit from these markers. Importantly, methods similar to those presented here can be applied to other taxa in the near future.

DNA methylation and structural and functional bimodality of vertebrate promoters.

Human promoters divide into 2 classes, the low CpG (LCG) and the high CpG (HCG), based on their CpG dinucleotide content. The LCG class of promoters is hypermethylated and is associated with tissue-specific genes, whereas the HCG class is hypomethylated and associated with broadly expressed genes. By analyzing several chordate genomes separated for hundreds of millions of years, here we show that the divide between low CpG and high CpG promoters is conserved in several distantly related vertebrate taxa (including human, chicken, frog, lizard, and fish) but not in close invertebrate outgroups (sea squirts). Furthermore, LCG and HCG promoters are distinctively associated with tissue-specific and broadly expressed genes in these distantly related vertebrate taxa. Our results indicate that the function of DNA methylation on gene expression is conserved across these vertebrate taxa and suggest that the 2 classes of promoters have evolved early in vertebrate evolution, as a consequence of the advent of global DNA methylation.

Mutations of different molecular origins exhibit contrasting patterns of regional substitution rate variation.

Transitions at CpG dinucleotides, referred to as "CpG substitutions", are a major mutational input into vertebrate genomes and a leading cause of human genetic disease. The prevalence of CpG substitutions is due to their mutational origin, which is dependent on DNA methylation. In comparison, other single nucleotide substitutions (for example those occurring at GpC dinucleotides) mainly arise from errors during DNA replication. Here we analyzed high quality BAC-based data from human, chimpanzee, and baboon to investigate regional variation of CpG substitution rates. We show that CpG substitutions occur approximately 15 times more frequently than other single nucleotide substitutions in primate genomes, and that they exhibit substantial regional variation. Patterns of CpG rate variation are consistent with differences in methylation level and susceptibility to subsequent deamination. In particular, we propose a "distance-decaying" hypothesis, positing that due to the molecular mechanism of a CpG substitution, rates are correlated with the stability of double-stranded DNA surrounding each CpG dinucleotide, and the effect of local DNA stability may decrease with distance from the CpG dinucleotide.Consistent with our "distance-decaying" hypothesis, rates of CpG substitution are strongly (negatively) correlated with regional G+C content. The influence of G+C content decays as the distance from the target CpG site increases. We estimate that the influence of local G+C content extends up to 1,500 approximately 2,000 bps centered on each CpG site. We also show that the distance-decaying relationship persisted when we controlled for the effect of long-range homogeneity of nucleotide composition. GpC sites, in contrast, do not exhibit such "distance-decaying" relationship. Our results highlight an example of the distinctive properties of methylation-dependent substitutions versus substitutions mostly arising from errors during DNA replication. Furthermore, the negative relationship between G+C content and CpG rates may provide an explanation for the observation that GC-rich SINEs show lower CpG rates than other repetitive elements.

Heterogeneous genomic molecular clocks in primates.

Using data from primates, we show that molecular clocks in sites that have been part of a CpG dinucleotide in recent past (CpG sites) and non-CpG sites are of markedly different nature, reflecting differences in their molecular origins. Notably, single nucleotide substitutions at non-CpG sites show clear generation-time dependency, indicating that most of these substitutions occur by errors during DNA replication. On the other hand, substitutions at CpG sites occur relatively constantly over time, as expected from their primary origin due to methylation. Therefore, molecular clocks are heterogeneous even within a genome. Furthermore, we propose that varying frequencies of CpG dinucleotides in different genomic regions may have contributed significantly to conflicting earlier results on rate constancy of mammalian molecular clock. Our conclusion that different regions of genomes follow different molecular clocks should be considered when inferring divergence times using molecular data and in phylogenetic analysis.

A Novel Open Access Web Portal for Integrating Mechanistic and Toxicogenomic Study Results.

Applying toxicogenomics to improving the safety profile of drug candidates and crop protection molecules is most useful when it identifies relevant biological and mechanistic information that highlights risks and informs risk mitigation strategies. Pathway-based approaches, such as gene set enrichment analysis, integrate toxicogenomic data with known biological process and pathways. Network methods help define unknown biological processes and offer data reduction advantages. Integrating the 2 approaches would improve interpretation of toxicogenomic information. Barriers to the routine application of these methods in genome-wide transcriptomic studies include a need for "hands-on" computer programming experience, the selection of 1 or more analysis methods (eg pathway analysis methods), the sensitivity of results to algorithm parameters, and challenges in linking differential gene expression to variation in safety outcomes. To facilitate adoption and reproducibility of gene expression analysis in safety studies, we have developed Collaborative Toxicogeomics, an open-access integrated web portal using the Django web framework. The software, developed with the Python programming language, is modular, extensible and implements "best-practice" methods in computational biology. New study results are compared with over 4000 rodent liver experiments from Drug Matrix and open TG-GATEs. A unique feature of the software is the ability to integrate clinical chemistry and histopathology-derived outcomes with results from gene expression studies, leading to relevant mechanistic conclusions. We describe its application by analyzing the effects of several toxicants on liver gene expression and exemplify application to predicting toxicity study outcomes upon chronic treatment from expression changes in acute-duration studies.

Functional relevance of CpG island length for regulation of gene expression.

CpG islands mark CpG-enriched regions in otherwise CpG-depleted vertebrate genomes. While the regulatory importance of CpG islands is widely accepted, it is little appreciated that CpG islands vary greatly in lengths. For example, CpG islands in the human genome vary ∼30-fold in their lengths. Here we report findings suggesting that the lengths of CpG islands have functional consequences. Specifically, we show that promoters associated with long CpG islands (long-CGI promoters) are distinct from other promoters. First, long-CGI promoters are uniquely associated with genes with an intermediate level of gene expression breadths. Notably, intermediate expression breadths require the most complex mode of gene regulation, from the standpoint of information content. Second, long-CGI promoters encode more RNA polymerase II (Polr2a) binding sites than other promoters. Third, the actual binding patterns of Polr2a occur in a more tissue-specific manner in long-CGI promoters compared to other CGI promoters. Moreover, long-CGI promoters contain the largest numbers of experimentally characterized transcription start sites compared to other promoters, and the types of transcription start sites in them are biased toward tissue-specific patterns of gene expression. Finally, long-CGI promoters are preferentially associated with genes involved in development and regulation. Together, these findings indicate that functionally relevant variations of CpG islands exist. By investigating consequences of certain CpG island traits, we can gain additional insights into the mechanism and evolution of regulatory complexity of gene expression.

Evolutionary rate variation in Old World monkeys.

We analysed over 8 million base pairs of bacterial artificial chromosome-based sequence alignments of four Old World monkeys and the human genome. Our findings are as follows. (i) Genomic divergences among several Old World monkeys mirror those between well-studied hominoids. (ii) The X-chromosome evolves slower than autosomes, in accord with 'male-driven evolution'. However, the degree of male mutation bias is lower in Old World monkeys than in hominoids. (iii) Evolutionary rates vary significantly between lineages. The baboon branch shows a particularly slow molecular evolution. Thus, lineage-specific evolutionary rate variation is a common theme of primate genome evolution. (iv) In contrast to the overall pattern, mutations originating from DNA methylation exhibit little variation between lineages. Our study illustrates the potential of primates as a model system to investigate genome evolution, in particular to elucidate molecular mechanisms of substitution rate variation.

Variable molecular clocks in hominoids.

Generation time is an important determinant of a neutral molecular clock. There are several human-specific life history traits that led to a substantially longer generation time in humans than in other hominoids. Indeed, a long generation time is considered an important trait that distinguishes humans from their closest relatives. Therefore, humans may exhibit a significantly slower molecular clock as compared to other hominoids. To investigate this hypothesis, we performed a large-scale analysis of lineage-specific rates of single-nucleotide substitutions among hominoids. We found that humans indeed exhibit a significant slowdown of molecular evolution compared to chimpanzees and other hominoids. However, the amount of fixed differences between humans and chimpanzees appears extremely small, suggesting a very recent evolution of human-specific life history traits. Notably, chimpanzees also exhibit a slower rate of molecular evolution compared to gorillas and orangutans in the regions analyzed.