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1.  Global similarity and local divergence in human and mouse gene co-expression networks 
Background
A genome-wide comparative analysis of human and mouse gene expression patterns was performed in order to evaluate the evolutionary divergence of mammalian gene expression. Tissue-specific expression profiles were analyzed for 9,105 human-mouse orthologous gene pairs across 28 tissues. Expression profiles were resolved into species-specific coexpression networks, and the topological properties of the networks were compared between species.
Results
At the global level, the topological properties of the human and mouse gene coexpression networks are, essentially, identical. For instance, both networks have topologies with small-world and scale-free properties as well as closely similar average node degrees, clustering coefficients, and path lengths. However, the human and mouse coexpression networks are highly divergent at the local level: only a small fraction (<10%) of coexpressed gene pair relationships are conserved between the two species. A series of controls for experimental and biological variance show that most of this divergence does not result from experimental noise. We further show that, while the expression divergence between species is genuinely rapid, expression does not evolve free from selective (functional) constraint. Indeed, the coexpression networks analyzed here are demonstrably functionally coherent as indicated by the functional similarity of coexpressed gene pairs, and this pattern is most pronounced in the conserved human-mouse intersection network. Numerous dense network clusters show evidence of dedicated functions, such as spermatogenesis and immune response, that are clearly consistent with the coherence of the expression patterns of their constituent gene members.
Conclusion
The dissonance between global versus local network divergence suggests that the interspecies similarity of the global network properties is of limited biological significance, at best, and that the biologically relevant aspects of the architectures of gene coexpression are specific and particular, rather than universal. Nevertheless, there is substantial evolutionary conservation of the local network structure which is compatible with the notion that gene coexpression networks are subject to purifying selection.
doi:10.1186/1471-2148-6-70
PMCID: PMC1601971  PMID: 16968540
2.  Functional Divergence and Evolutionary Turnover in Mammalian Phosphoproteomes 
PLoS Genetics  2014;10(1):e1004062.
Protein phosphorylation is a key mechanism to regulate protein functions. However, the contribution of this protein modification to species divergence is still largely unknown. Here, we studied the evolution of mammalian phosphoregulation by comparing the human and mouse phosphoproteomes. We found that 84% of the positions that are phosphorylated in one species or the other are conserved at the residue level. Twenty percent of these conserved sites are phosphorylated in both species. This proportion is 2.5 times more than expected by chance alone, suggesting that purifying selection is preserving phosphoregulation. However, we show that the majority of the sites that are conserved at the residue level are differentially phosphorylated between species. These sites likely result from false-negative identifications due to incomplete experimental coverage, false-positive identifications and non-functional sites. In addition, our results suggest that at least 5% of them are likely to be true differentially phosphorylated sites and may thus contribute to the divergence in phosphorylation networks between mouse and humans and this, despite residue conservation between orthologous proteins. We also showed that evolutionary turnover of phosphosites at adjacent positions (in a distance range of up to 40 amino acids) in human or mouse leads to an over estimation of the divergence in phosphoregulation between these two species. These sites tend to be phosphorylated by the same kinases, supporting the hypothesis that they are functionally redundant. Our results support the hypothesis that the evolutionary turnover of phosphorylation sites contributes to the divergence in phosphorylation profiles while preserving phosphoregulation. Overall, our study provides advanced analyses of mammalian phosphoproteomes and a framework for the study of their contribution to phenotypic evolution.
Author Summary
Understanding how differences in cellular regulation lead to phenotypic differences between species remains an open challenge in evolutionary genetics. The extensive phosphorylation data currently available allows to compare the human and mouse phosphoproteomes and to measure changes in their phosphoregulation. We found a general conservation of phosphorylation sites between these two species. However, a fraction of sites are conserved at the sequence level (the same amino acid is present in both species) but differ in their phosphorylation status. These sites represent candidate sites that have the potential to explain differences between human and mouse signalling networks that do not depend on the divergence of orthologous residues. Furthermore, we identified several sites where to a phosphorylation site in one species corresponds a non-phosphorylatable residue in the other one. These cases represent clear differences in protein regulation. Recent studies suggest that phosphorylation sites can shift position during evolution, leading to configurations in which pairs of divergent phosphorylation sites are functionally redundant. We identified more than 100 putative such cases, suggesting that divergence in amino acid does not necessarily imply functional divergence when comparing phosphoproteomes. Overall, our study provides new key concepts and data for the study of how regulatory differences may be linked to phenotypic ones at the network level.
doi:10.1371/journal.pgen.1004062
PMCID: PMC3900387  PMID: 24465218
3.  Does Positive Selection Drive Transcription Factor Binding Site Turnover? A Test with Drosophila Cis-Regulatory Modules 
PLoS Genetics  2011;7(4):e1002053.
Transcription factor binding site(s) (TFBS) gain and loss (i.e., turnover) is a well-documented feature of cis-regulatory module (CRM) evolution, yet little attention has been paid to the evolutionary force(s) driving this turnover process. The predominant view, motivated by its widespread occurrence, emphasizes the importance of compensatory mutation and genetic drift. Positive selection, in contrast, although it has been invoked in specific instances of adaptive gene expression evolution, has not been considered as a general alternative to neutral compensatory evolution. In this study we evaluate the two hypotheses by analyzing patterns of single nucleotide polymorphism in the TFBS of well-characterized CRM in two closely related Drosophila species, Drosophila melanogaster and Drosophila simulans. An important feature of the analysis is classification of TFBS mutations according to the direction of their predicted effect on binding affinity, which allows gains and losses to be evaluated independently along the two phylogenetic lineages. The observed patterns of polymorphism and divergence are not compatible with neutral evolution for either class of mutations. Instead, multiple lines of evidence are consistent with contributions of positive selection to TFBS gain and loss as well as purifying selection in its maintenance. In discussion, we propose a model to reconcile the finding of selection driving TFBS turnover with constrained CRM function over long evolutionary time.
Author Summary
Transcription factor binding sites (TFBS) turnover (i.e. lineage-specific gain and loss) is a well-documented phenomenon in eukaryote cis-regulatory modules (CRM). The wide spread of the phenomenon and the appearance of conserved expression patterns for diverged orthologous CRM led to the standing view that the observed gain and loss of TFBS were functionally and selectively neutral. To the contrary, genome-wide population genetics analyses have unequivocally identified signatures of positive selection acting in noncoding regions in general, and particularly in 5′ and 3′ untranscribed regions of genes. To specifically test the neutral versus selection hypotheses for the TFBS turnover process, we analyzed natural variation patterns within and between two closely related Drosophila species. We found the patterns of divergence and polymorphism for two types of mutations—those inferred to increase or decrease the binding affinity respectively—are not compatible with a neutral hypothesis. Instead, multiple lines of evidence suggested that positive selection has contributed to gain as well as loss of TFBS in the two lineages, with purifying selection maintaining existing TFBS in the population. Spacer sequences also showed signatures of negative and positive selection. We proposed a model of CRM evolution to reconcile the finding of frequent adaptive changes with constraints on long-term evolution.
doi:10.1371/journal.pgen.1002053
PMCID: PMC3084208  PMID: 21572512
4.  Adaptive Gene Expression Divergence Inferred from Population Genomics 
PLoS Genetics  2007;3(10):e187.
Detailed studies of individual genes have shown that gene expression divergence often results from adaptive evolution of regulatory sequence. Genome-wide analyses, however, have yet to unite patterns of gene expression with polymorphism and divergence to infer population genetic mechanisms underlying expression evolution. Here, we combined genomic expression data—analyzed in a phylogenetic context—with whole genome light-shotgun sequence data from six Drosophila simulans lines and reference sequences from D. melanogaster and D. yakuba. These data allowed us to use molecular population genetics to test for neutral versus adaptive gene expression divergence on a genomic scale. We identified recent and recurrent adaptive evolution along the D. simulans lineage by contrasting sequence polymorphism within D. simulans to divergence from D. melanogaster and D. yakuba. Genes that evolved higher levels of expression in D. simulans have experienced adaptive evolution of the associated 3′ flanking and amino acid sequence. Concomitantly, these genes are also decelerating in their rates of protein evolution, which is in agreement with the finding that highly expressed genes evolve slowly. Interestingly, adaptive evolution in 5′ cis-regulatory regions did not correspond strongly with expression evolution. Our results provide a genomic view of the intimate link between selection acting on a phenotype and associated genic evolution.
Author Summary
Changes in patterns of gene expression likely contribute greatly to phenotypic differences among closely related organisms. However, the evolutionary mechanisms, such as Darwinian selection and random genetic drift, which are underlying differences in patterns of expression, are only now being understood on a genomic level. We combine measurements of gene expression and whole-genome sequence data to investigate the relationship between the forces driving sequence evolution and expression divergence among closely related fruit flies. We find that Darwinian selection acting on regions that may control gene expression is associated with increases in gene expression levels. Investigation of the functional consequences of adaptive evolution on regulating gene expression is clearly warranted. The genetic tools available in Drosophila make functional experiments possible and will shed light on how closely related species have responded to reproductive, pathogenic, and environmental pressures.
doi:10.1371/journal.pgen.0030187
PMCID: PMC2042001  PMID: 17967066
5.  Evolution of the Correlation between Expression Divergence and Protein Divergence in Mammals 
Genome Biology and Evolution  2013;5(7):1324-1335.
Divergence of protein sequences and gene expression patterns are two fundamental mechanisms that generate organismal diversity. Here, we have used genome and transcriptome data from eight mammals and one bird to study the positive correlation of these two processes throughout mammalian evolution. We demonstrate that the correlation is stable over time and most pronounced in neural tissues, which indicates that it is the result of strong negative selection. The correlation is not driven by genes with specific functions and may instead best be viewed as an evolutionary default state, which can nevertheless be evaded by certain gene types. In particular, genes with developmental and neural functions are skewed toward changes in gene expression, consistent with selection against pleiotropic effects associated with changes in protein sequences. Surprisingly, we find that the correlation between expression divergence and protein divergence is not explained by between-gene variation in expression level, tissue specificity, protein connectivity, or other investigated gene characteristics, suggesting that it arises independently of these gene traits. The selective constraints on protein sequences and gene expression patterns also fluctuate in a coordinate manner across phylogenetic branches: We find that gene-specific changes in the rate of protein evolution in a specific mammalian lineage tend to be accompanied by similar changes in the rate of expression evolution. Taken together, our findings highlight many new aspects of the correlation between protein divergence and expression divergence, and attest to its role as a fundamental property of mammalian genome evolution.
doi:10.1093/gbe/evt093
PMCID: PMC3730345  PMID: 23781097
gene expression evolution; protein evolution; primates; amniotes; correlation analysis
6.  Duplicate gene evolution and expression in the wake of vertebrate allopolyploidization 
Background
The mechanism by which duplicate genes originate – whether by duplication of a whole genome or of a genomic segment – influences their genetic fates. To study events that trigger duplicate gene persistence after whole genome duplication in vertebrates, we have analyzed molecular evolution and expression of hundreds of persistent duplicate gene pairs in allopolyploid clawed frogs (Xenopus and Silurana). We collected comparative data that allowed us to tease apart the molecular events that occurred soon after duplication from those that occurred later on. We also quantified expression profile divergence of hundreds of paralogs during development and in different tissues.
Results
Our analyses indicate that persistent duplicates generated by allopolyploidization are subjected to strong purifying selection soon after duplication. The level of purifying selection is relaxed compared to a singleton ortholog, but not significantly variable over a period spanning about 40 million years. Despite persistent functional constraints, however, analysis of paralogous expression profiles indicates that quantitative aspects of their expression diverged substantially during this period.
Conclusion
These results offer clues into how vertebrate transcriptomes are sculpted in the wake of whole genome duplication (WGD), such as those that occurred in our early ancestors. That functional constraints were relaxed relative to a singleton ortholog but not significantly different in the early compared to the later stage of duplicate gene evolution suggests that the timescale for a return to pre-duplication levels is drawn out over tens of millions of years – beyond the age of these tetraploid species. Quantitative expression divergence can occur soon after WGD and with a magnitude that is not correlated with the rate of protein sequence divergence. On a coarse scale, quantitative expression divergence appears to be more prevalent than spatial and temporal expression divergence, and also faster or more frequent than other processes that operate at the protein level, such as some types of neofunctionalization.
doi:10.1186/1471-2148-8-43
PMCID: PMC2275784  PMID: 18261230
7.  Next generation sequencing and analysis of a conserved transcriptome of New Zealand's kiwi 
Background
Kiwi is a highly distinctive, flightless and endangered ratite bird endemic to New Zealand. To understand the patterns of molecular evolution of the nuclear protein-coding genes in brown kiwi (Apteryx australis mantelli) and to determine the timescale of avian history we sequenced a transcriptome obtained from a kiwi embryo using next generation sequencing methods. We then assembled the conserved protein-coding regions using the chicken proteome as a scaffold.
Results
Using 1,543 conserved protein coding genes we estimated the neutral evolutionary divergence between the kiwi and chicken to be ~45%, which is approximately equal to the divergence computed for the human-mouse pair using the same set of genes. A large fraction of genes was found to be under high selective constraint, as most of the expressed genes appeared to be involved in developmental gene regulation. Our study suggests a significant relationship between gene expression levels and protein evolution. Using sequences from over 700 nuclear genes we estimated the divergence between the two basal avian groups, Palaeognathae and Neognathae to be 132 million years, which is consistent with previous studies using mitochondrial genes.
Conclusions
The results of this investigation revealed patterns of mutation and purifying selection in conserved protein coding regions in birds. Furthermore this study suggests a relatively cost-effective way of obtaining a glimpse into the fundamental molecular evolutionary attributes of a genome, particularly when no closely related genomic sequence is available.
doi:10.1186/1471-2148-10-387
PMCID: PMC3009673  PMID: 21156082
8.  Divergence of nucleosome positioning between two closely related yeast species: genetic basis and functional consequences 
Inter-species hybrids can be used to dissect the relative contribution of cis and trans effects to the evolution of nucleosome positioning. Most (∼70%) differences in nucleosome positioning between two closely related yeast species are due to cis effects.Cis effects are primarily due to divergence of AT-rich nucleosome-disfavoring sequences, but are not associated with divergence of nucleosome-favoring sequences.Differences in nucleosome positioning propagate to multiple adjacent nucleosomes, supporting the statistical positioning hypothesis.Divergence of nucleosome positioning is excluded from regulatory elements and is not correlated with gene expression divergence, suggesting a neutral mode of evolution.
Phenotypic diversity is often due to changes in gene regulation, and recent studies have characterized extensive differences between the gene expression programs of closely related species (Khaitovich et al, 2006; Tirosh et al, 2009). However, very little is known about the mechanisms that drive this divergence. Here, we analyze the evolution of nucleosome positioning, by comparing the patterns of nucleosomes between two yeast species, as well as generating the allele-specific nucleosome profile in their hybrid. We ask two main questions: (1) what is the genetic basis of inter-species differences in nucleosome positioning? and (2) what is the regulatory function of these differences?
Generally speaking, we can classify the genetic basis of the divergence in nucleosome positioning into two mechanisms. First, mutations in the local DNA sequence may influence the ability to bind nucleosomes at this region; we refer to these as cis effects. Second, mutations may affect the activity of various proteins that alter nucleosome positioning either actively (e.g. chromatin-remodeling enzymes) or by simply competing with nucleosomes for binding to the same DNA sequence (e.g. transcription factors); we refer to these as trans effects.
To classify the observed inter-species differences into cis versus trans effects, we measured allele-specific nucleosome positions within the inter-specific hybrid of the two species (Wittkopp et al, 2004; Tirosh et al, 2009). The hybrid contains the alleles of both species; hence, cis effects, which involve mutations that discriminate between the two alleles, will be maintained in the hybrid so that nucleosome positioning will be different between the alleles coming from the different species. Trans effects, in contrast, will not discriminate between the two hybrid alleles from the different species, as these two alleles reside together at the same trans environment (hybrid nucleus) and are thus regulated by the same set of proteins—the combination of proteins from the two species. Using this approach, we found that ∼70% of the inter-species differences in nucleosome positioning are due to cis effects, whereas the rest is due to trans effects.
The local DNA sequence is indeed known to affect nucleosome positions, and many features of DNA sequences were proposed to influence nucleosome binding, either by rejecting nucleosomes, or by being favorable for nucleosome binding (Segal et al, 2006; Lee et al, 2007; Kaplan et al, 2009). We find, however, that nucleosome positions diverged primarily through changes in AT-rich sequences, which exclude nucleosomes, whereas mutations in sequences that correlate with high-nucleosome occupancy do not influence inter-species divergence.
Nucleosomes restrict the access of proteins to the DNA and may thus affect DNA-related processes such as transcription, recombination or replication. Indeed, promoters and regulatory sequences are often depleted of nucleosomes, and highly transcribed genes are associated with low occupancy of nucleosomes at their promoters (Lee et al, 2007). Several earlier studies also suggested that evolutionary divergence of gene expression is driven by changes in chromatin structure (Lee et al, 2006; Choi and Kim, 2008; Tirosh et al, 2008; Field et al, 2009). However, we find that nucleosome positions (or occupancy) at regulatory elements are largely conserved, and furthermore, that the inter-species differences in nucleosome positions do not correlate with gene expression differences. These results suggest that nucleosome positioning is not a central mechanism for evolutionary changes in gene regulation and that most of the observed changes may be due to neutral drift.
Does the apparent low influence of nucleosome positioning on gene expression divergence implies that nucleosome positions do not have a function in gene regulation? To address this, we examined two additional modes of gene regulation: transcriptional response to changes in growth conditions (glucose versus glycerol media), and the expression differences between different cell types (haploid versus diploid cells). Consistent with earlier studies, we found that the response to growth conditions is significantly, albeit weakly, associated with changes in nucleosome positioning. Interestingly, we also found a strikingly strong association between gene expression and nucleosomal changes in the two cell types. Taken together, these results suggest that nucleosome positioning is used preferentially for biological processes in which genes are turned on and off (e.g. different cell type), but less so during divergence of closely related species in which gradual changes accumulate over time.
Gene regulation differs greatly between related species, constituting a major source of phenotypic diversity. Recent studies characterized extensive differences in the gene expression programs of closely related species. In contrast, virtually nothing is known about the evolution of chromatin structure and how it influences the divergence of gene expression. Here, we compare the genome-wide nucleosome positioning of two closely related yeast species and, by profiling their inter-specific hybrid, trace the genetic basis of the observed differences into mutations affecting the local DNA sequences (cis effects) or the upstream regulators (trans effects). The majority (∼70%) of inter-species differences is due to cis effects, leaving a significant contribution (30%) for trans factors. We show that cis effects are well explained by mutations in nucleosome-disfavoring AT-rich sequences, but are not associated with divergence of nucleosome-favoring sequences. Differences in nucleosome positioning propagate to multiple adjacent nucleosomes, supporting the statistical positioning hypothesis, and we provide evidence that nucleosome-free regions, but not the +1 nucleosome, serve as stable border elements. Surprisingly, although we find that differential nucleosome positioning among cell types is strongly correlated with differential expression, this does not seem to be the case for evolutionary changes: divergence of nucleosome positioning is excluded from regulatory elements and is not correlated with gene expression divergence, suggesting a primarily neutral mode of evolution. Our results provide evolutionary insights to the genetic determinants and regulatory function of nucleosome positioning.
doi:10.1038/msb.2010.20
PMCID: PMC2890324  PMID: 20461072
evolution; gene regulation; nucleosome positioning
9.  Inferring Evolutionary Histories of Pathway Regulation from Transcriptional Profiling Data 
PLoS Computational Biology  2013;9(10):e1003255.
One of the outstanding challenges in comparative genomics is to interpret the evolutionary importance of regulatory variation between species. Rigorous molecular evolution-based methods to infer evidence for natural selection from expression data are at a premium in the field, and to date, phylogenetic approaches have not been well-suited to address the question in the small sets of taxa profiled in standard surveys of gene expression. We have developed a strategy to infer evolutionary histories from expression profiles by analyzing suites of genes of common function. In a manner conceptually similar to molecular evolution models in which the evolutionary rates of DNA sequence at multiple loci follow a gamma distribution, we modeled expression of the genes of an a priori-defined pathway with rates drawn from an inverse gamma distribution. We then developed a fitting strategy to infer the parameters of this distribution from expression measurements, and to identify gene groups whose expression patterns were consistent with evolutionary constraint or rapid evolution in particular species. Simulations confirmed the power and accuracy of our inference method. As an experimental testbed for our approach, we generated and analyzed transcriptional profiles of four Saccharomyces yeasts. The results revealed pathways with signatures of constrained and accelerated regulatory evolution in individual yeasts and across the phylogeny, highlighting the prevalence of pathway-level expression change during the divergence of yeast species. We anticipate that our pathway-based phylogenetic approach will be of broad utility in the search to understand the evolutionary relevance of regulatory change.
Author Summary
Comparative transcriptomic studies routinely identify thousands of genes differentially expressed between species. The central question in the field is whether and how such regulatory changes have been the product of natural selection. Can the signal of evolutionarily relevant expression divergence be detected amid the noise of changes resulting from genetic drift? Our work develops a theory of gene expression variation among a suite of genes that function together. We derive a formalism that relates empirical observations of expression of pathway genes in divergent species to the underlying strength of natural selection on expression output. We show that fitting this type of model to simulated data accurately recapitulates the parameters used to generate the simulation. We then make experimental measurements of gene expression in a panel of single-celled eukaryotic yeast species. To these data we apply our inference method, and identify pathways with striking evidence for accelerated or constrained regulatory evolution, in particular species and across the phylogeny. Our method provides a key advance over previous approaches in that it maximizes the power of rigorous molecular-evolution analysis of regulatory variation even when data are relatively sparse. As such, the theory and tools we have developed will likely find broad application in the field of comparative genomics.
doi:10.1371/journal.pcbi.1003255
PMCID: PMC3794907  PMID: 24130471
10.  Ontogeny and phylogeny: molecular signatures of selection, constraint, and temporal pleiotropy in the development of Drosophila 
BMC Biology  2009;7:42.
Background
Karl Ernst Von Baer noted that species tend to show greater morphological divergence in later stages of development when compared to earlier stages. Darwin originally interpreted these observations via a selectionist framework, suggesting that divergence should be greatest during ontogenic stages in which organisms experienced varying 'conditions of existence' and opportunity for differential selection. Modern hypotheses have focused on the notion that genes and structures involved in early development will be under stronger purifying selection due to the deleterious pleiotropic effects of mutations propagating over the course of ontogeny, also known as the developmental constraint hypothesis.
Results
Using developmental stage-specific expressed sequence tag (EST) libraries, we tested the 2 hypotheses by comparing the rates of evolution of 7,180 genes obtained from 6 species of the Drosophila melanogaster group with respect to ontogeny, and sex and reproduction-related functions in gonadal tissues. Supporting morphological observations, we found evidence of a pattern of increasing mean evolutionary rate in genes that are expressed in subsequent stages of development. Furthermore, supporting expectations that early expressed genes are constrained in divergence, we found that embryo stage genes are involved in a higher mean number of interactions as compared to later stages. We noted that the accelerated divergence of genes in the adult stage is explained by those expressed specifically in the male gonads, whose divergence is driven by positive selection. In addition, accelerated gonadal gene divergence occurs only in the adult stage, suggesting that the effects of selection are observed primarily at the stages during which they are expected occur. Finally, we also found a significant correlation between temporal specificity of gene expression and evolutionary rate, supporting expectations that genes with ubiquitous expression are under stronger constraint.
Conclusion
Taken together, these results support both the developmental constraint hypothesis limiting the divergence of early expressed developmentally important genes, leading to a gradient of divergence rates over ontogeny (embryonic < larval/pupal < adult), as well as Darwin's 'selection opportunity' hypothesis leading to increased divergence in adults, particularly in the case of reproductive tissues. We suggest that a constraint early/opportunity late model best explains divergence over ontogeny.
doi:10.1186/1741-7007-7-42
PMCID: PMC2722573  PMID: 19622136
11.  Faster-X Evolution of Gene Expression in Drosophila 
PLoS Genetics  2012;8(10):e1003013.
DNA sequences on X chromosomes often have a faster rate of evolution when compared to similar loci on the autosomes, and well articulated models provide reasons why the X-linked mode of inheritance may be responsible for the faster evolution of X-linked genes. We analyzed microarray and RNA–seq data collected from females and males of six Drosophila species and found that the expression levels of X-linked genes also diverge faster than autosomal gene expression, similar to the “faster-X” effect often observed in DNA sequence evolution. Faster-X evolution of gene expression was recently described in mammals, but it was limited to the evolutionary lineages shortly following the creation of the therian X chromosome. In contrast, we detect a faster-X effect along both deep lineages and those on the tips of the Drosophila phylogeny. In Drosophila males, the dosage compensation complex (DCC) binds the X chromosome, creating a unique chromatin environment that promotes the hyper-expression of X-linked genes. We find that DCC binding, chromatin environment, and breadth of expression are all predictive of the rate of gene expression evolution. In addition, estimates of the intraspecific genetic polymorphism underlying gene expression variation suggest that X-linked expression levels are not under relaxed selective constraints. We therefore hypothesize that the faster-X evolution of gene expression is the result of the adaptive fixation of beneficial mutations at X-linked loci that change expression level in cis. This adaptive faster-X evolution of gene expression is limited to genes that are narrowly expressed in a single tissue, suggesting that relaxed pleiotropic constraints permit a faster response to selection. Finally, we present a conceptional framework to explain faster-X expression evolution, and we use this framework to examine differences in the faster-X effect between Drosophila and mammals.
Author Summary
As species diverge over evolutionary time, they accumulate differences in the sequences of their genes and how those genes are expressed. We show that gene expression changes accumulate faster for genes on the X chromosome than for genes on the other chromosomes (autosomes) in Drosophila (the “faster-X” effect). The X chromosome is only found in a single copy in males, whereas the autosomes are found in two copies in both sexes. To compensate for the reduced dosage of X-linked genes in males, a molecular complex binds the Drosophila X chromosome to upregulate gene expression in males. We demonstrate that genes that escape this dosage compensation process have faster evolving expression levels. X-linked genes are inherited in a unique manner, and we hypothesize that this permits a faster rate of adaptive evolution, thereby driving the faster-X evolution of gene expression. We compare these observations with the recently described faster-X evolution of gene expression in mammals, and we explain how differences in dosage compensation, mutation rate, and population size could affect the extent of the faster-X effect.
doi:10.1371/journal.pgen.1003013
PMCID: PMC3469423  PMID: 23071459
12.  A Neutral Model of Transcriptome Evolution 
PLoS Biology  2004;2(5):e132.
Microarray technologies allow the identification of large numbers of expression differences within and between species. Although environmental and physiological stimuli are clearly responsible for changes in the expression levels of many genes, it is not known whether the majority of changes of gene expression fixed during evolution between species and between various tissues within a species are caused by Darwinian selection or by stochastic processes. We find the following: (1) expression differences between species accumulate approximately linearly with time; (2) gene expression variation among individuals within a species correlates positively with expression divergence between species; (3) rates of expression divergence between species do not differ significantly between intact genes and expressed pseudogenes; (4) expression differences between brain regions within a species have accumulated approximately linearly with time since these regions emerged during evolution. These results suggest that the majority of expression differences observed between species are selectively neutral or nearly neutral and likely to be of little or no functional significance. Therefore, the identification of gene expression differences between species fixed by selection should be based on null hypotheses assuming functional neutrality. Furthermore, it may be possible to apply a molecular clock based on expression differences to infer the evolutionary history of tissues.
Analysis of differences in gene expression between primate species suggests that the majority of them are selectively neutral and likely to have little or no functional consequences
doi:10.1371/journal.pbio.0020132
PMCID: PMC406393  PMID: 15138501
13.  ZIPK: A Unique Case of Murine-Specific Divergence of a Conserved Vertebrate Gene 
PLoS Genetics  2007;3(10):e180.
Zipper interacting protein kinase (ZIPK, also known as death-associated protein kinase 3 [DAPK3]) is a Ser/Thr kinase that functions in programmed cell death. Since its identification eight years ago, contradictory findings regarding its intracellular localization and molecular mode of action have been reported, which may be attributed to unpredicted differences among the human and rodent orthologs. By aligning the sequences of all available ZIPK orthologs, from fish to human, we discovered that rat and mouse sequences are more diverged from the human ortholog relative to other, more distant, vertebrates. To test experimentally the outcome of this sequence divergence, we compared rat ZIPK to human ZIPK in the same cellular settings. We found that while ectopically expressed human ZIPK localized to the cytoplasm and induced membrane blebbing, rat ZIPK localized exclusively within nuclei, mainly to promyelocytic leukemia oncogenic bodies, and induced significantly lower levels of membrane blebbing. Among the unique murine (rat and mouse) sequence features, we found that a highly conserved phosphorylation site, previously shown to have an effect on the cellular localization of human ZIPK, is absent in murines but not in earlier diverging organisms. Recreating this phosphorylation site in rat ZIPK led to a significant reduction in its promyelocytic leukemia oncogenic body localization, yet did not confer full cytoplasmic localization. Additionally, we found that while rat ZIPK interacts with PAR-4 (also known as PAWR) very efficiently, human ZIPK fails to do so. This interaction has clear functional implications, as coexpression of PAR-4 with rat ZIPK caused nuclear to cytoplasm translocation and induced strong membrane blebbing, thus providing the murine protein a possible adaptive mechanism to compensate for its sequence divergence. We have also cloned zebrafish ZIPK and found that, like the human and unlike the murine orthologs, it localizes to the cytoplasm, and fails to bind the highly conserved PAR-4 protein. This further supports the hypothesis that murine ZIPK underwent specific divergence from a conserved consensus. In conclusion, we present a case of species-specific divergence occurring in a specific branch of the evolutionary tree, accompanied by the acquisition of a unique protein–protein interaction that enables conservation of cellular function.
Author Summary
Mammals are a fairly young class of animals, first appearing about 70 million years ago. Such recent common descent does not allow the evolutionary process to create much diversity within the class, and indeed, the physiology among different mammals is remarkably similar. This similarity enables the use of various small mammals, especially rats and mice, as model systems for the study of biological phenomenon and disease. Experiments unfeasible or unethical to perform on humans are conducted on these model animals, with the postulation that insights gained from them are applicable to the human system. In this article, we present an exception to this rule. We bring evidence that ZIPK, a gene with important roles in programmed cell death, has undergone accelerated evolution in the rat and mouse, thus diverging considerably from a well-conserved consensus in all vertebrates, from fish to man. We also show that this sequence divergence caused changes in the protein's properties, including its localization within the cell, and the proteins with which it interacts. Still, the basic biologic function of ZIPK is conserved in both systems, and we propose an adaptive mechanism that compensates for the sequence divergence in rodents.
doi:10.1371/journal.pgen.0030180
PMCID: PMC2041995  PMID: 17953487
14.  Adaptive Evolution and the Birth of CTCF Binding Sites in the Drosophila Genome 
PLoS Biology  2012;10(11):e1001420.
Comparative ChIP-seq data reveal adaptive evolution of insulator protein CTCF binding in multiple Drosophila species.
Changes in the physical interaction between cis-regulatory DNA sequences and proteins drive the evolution of gene expression. However, it has proven difficult to accurately quantify evolutionary rates of such binding change or to estimate the relative effects of selection and drift in shaping the binding evolution. Here we examine the genome-wide binding of CTCF in four species of Drosophila separated by between ∼2.5 and 25 million years. CTCF is a highly conserved protein known to be associated with insulator sequences in the genomes of human and Drosophila. Although the binding preference for CTCF is highly conserved, we find that CTCF binding itself is highly evolutionarily dynamic and has adaptively evolved. Between species, binding divergence increased linearly with evolutionary distance, and CTCF binding profiles are diverging rapidly at the rate of 2.22% per million years (Myr). At least 89 new CTCF binding sites have originated in the Drosophila melanogaster genome since the most recent common ancestor with Drosophila simulans. Comparing these data to genome sequence data from 37 different strains of Drosophila melanogaster, we detected signatures of selection in both newly gained and evolutionarily conserved binding sites. Newly evolved CTCF binding sites show a significantly stronger signature for positive selection than older sites. Comparative gene expression profiling revealed that expression divergence of genes adjacent to CTCF binding site is significantly associated with the gain and loss of CTCF binding. Further, the birth of new genes is associated with the birth of new CTCF binding sites. Our data indicate that binding of Drosophila CTCF protein has evolved under natural selection, and CTCF binding evolution has shaped both the evolution of gene expression and genome evolution during the birth of new genes.
Author Summary
A large proportion of the diversity of living organisms results from differential regulation of gene transcription. Transcriptional regulation is thought to differ between species because of evolutionary changes in the physical interactions between regulatory DNA elements and DNA-binding proteins; these can generate variation in the spatial and temporal patterns of gene expression. The mechanisms by which these protein–DNA interactions evolve is therefore an important question in evolutionary biology. Does adaptive evolution play a role, or is the process dominated by neutral genetic drift? Insulator proteins are a special group of DNA-binding proteins—instead of directly serving to activate or repress genes, they can function to coordinate the interactions between other regulatory elements (such as enhancers and promoters). Additionally, insulator proteins can limit the spreading of chromatin condensation and help to demarcate the boundaries of regulatory domains in the genome. In spite of their critical role in genome regulation, little is known about the evolution of interactions between insulator proteins and DNA. Here, we use ChIP-seq to examine the distribution of binding sites for CTCF, a highly conserved insulator protein, in four closely related Drosophila species. We find that genome-wide binding profiles of CTCF are highly dynamic across evolutionary time, with frequent births of new CTCF-DNA interactions, and we demonstrate that this evolutionary process is driven by natural selection. By comparing these with RNA-seq data, we find that gain or loss of CTCF binding impacts the expression levels of nearby genes and correlates with structural evolution of the genome. Together these results suggest a potential mechanism of regulatory re-wiring through adaptive evolution of CTCF binding.
doi:10.1371/journal.pbio.1001420
PMCID: PMC3491045  PMID: 23139640
15.  Evolution of Regulatory Sequences in 12 Drosophila Species 
PLoS Genetics  2009;5(1):e1000330.
Characterization of the evolutionary constraints acting on cis-regulatory sequences is crucial to comparative genomics and provides key insights on the evolution of organismal diversity. We study the relationships among orthologous cis-regulatory modules (CRMs) in 12 Drosophila species, especially with respect to the evolution of transcription factor binding sites, and report statistical evidence in favor of key evolutionary hypotheses. Binding sites are found to have position-specific substitution rates. However, the selective forces at different positions of a site do not act independently, and the evidence suggests that constraints on sites are often based on their exact binding affinities. Binding site loss is seen to conform to a molecular clock hypothesis. The rate of site loss is transcription factor–specific and depends on the strength of binding and, in some cases, the presence of other binding sites in close proximity. Our analysis is based on a novel computational method for aligning orthologous CRMs on a tree, which rigorously accounts for alignment uncertainties and exploits binding site predictions through a unified probabilistic framework. Finally, we report weak purifying selection on short deletions, providing important clues about overall spatial constraints on CRMs. Our results present a complex picture of regulatory sequence evolution, with substantial plasticity that depends on a number of factors. The insights gained in this study will help us to understand the combinatorial control of gene regulation and how it evolves. They will pave the way for theoretical models that are cognizant of the important determinants of regulatory sequence evolution and will be critical in genome-wide identification of non-coding sequences under purifying or positive selection.
Author Summary
The spatial–temporal expression pattern of a gene, which is crucial to its function, is controlled by cis-regulatory DNA sequences. Forming the basic units of regulatory sequences are transcription factor binding sites, often organized into larger modules that determine gene expression in response to combinatorial environmental signals. Understanding the conservation and change of regulatory sequences is critical to our knowledge of the unity as well as diversity of animal development and phenotypes. In this paper, we study the evolution of sequences involved in the regulation of body patterning in the Drosophila embryo. We find that mutations of nucleotides within a binding site are constrained by evolutionary forces to preserve the site's binding affinity to the cognate transcription factor. Functional binding sites are frequently destroyed during evolution and the rate of loss across evolutionary spans is roughly constant. We also find that the evolutionary fate of a site strongly depends on its context; a pair of interacting sites are more likely to survive mutational forces than isolated sites. Together, these findings provide new insights and pose new challenges to our understanding of cis-regulatory sequences and their evolution.
doi:10.1371/journal.pgen.1000330
PMCID: PMC2607023  PMID: 19132088
16.  Conservation of Regional Gene Expression in Mouse and Human Brain 
PLoS Genetics  2007;3(4):e59.
Many neurodegenerative diseases have a hallmark regional and cellular pathology. Gene expression analysis of healthy tissues may provide clues to the differences that distinguish resistant and sensitive tissues and cell types. Comparative analysis of gene expression in healthy mouse and human brain provides a framework to explore the ability of mice to model diseases of the human brain. It may also aid in understanding brain evolution and the basis for higher order cognitive abilities. Here we compare gene expression profiles of human motor cortex, caudate nucleus, and cerebellum to one another and identify genes that are more highly expressed in one region relative to another. We separately perform identical analysis on corresponding brain regions from mice. Within each species, we find that the different brain regions have distinctly different expression profiles. Contrasting between the two species shows that regionally enriched genes in one species are generally regionally enriched genes in the other species. Thus, even when considering thousands of genes, the expression ratios in two regions from one species are significantly correlated with expression ratios in the other species. Finally, genes whose expression is higher in one area of the brain relative to the other areas, in other words genes with patterned expression, tend to have greater conservation of nucleotide sequence than more widely expressed genes. Together these observations suggest that region-specific genes have been conserved in the mammalian brain at both the sequence and gene expression levels. Given the general similarity between patterns of gene expression in healthy human and mouse brains, we believe it is reasonable to expect a high degree of concordance between microarray phenotypes of human neurodegenerative diseases and their mouse models. Finally, these data on very divergent species provide context for studies in more closely related species that address questions such as the origins of cognitive differences.
Author Summary
Animal models of human neurodegenerative and psychiatric disorders, particularly mouse models, have assumed a central role in biomedical research aimed at discovering the causes of disease and generating novel, mechanism-based treatments. But to what degree can a mouse brain serve as a model for a human brain? Here we begin to address this question by looking at patterns of gene expression across three corresponding regions of mouse and human brains. We find that within each species, the different regions (motor cortex, striatum, and cerebellum) have very distinct gene expression profiles. It is likely that these differences reflect distinctions in regional neurochemistry and function. We then show that genes that are enriched in one of the three areas relative to the other two in mice have the same pattern of expression in humans. Looking at the relationship between conservation of expression and amino acid sequence, we find that genes showing patterned expression generally have been more conserved than more uniformly expressed genes. This suggests that in the brain, constraints on the evolution of DNA sequence and gene expression can also be particularly high for genes with regional or tissue-specific expression.
doi:10.1371/journal.pgen.0030059
PMCID: PMC1853119  PMID: 17447843
17.  FoxO gene family evolution in vertebrates 
Background
Forkhead box, class O (FoxO) belongs to the large family of forkhead transcription factors that are characterized by a conserved forkhead box DNA-binding domain. To date, the FoxO group has four mammalian members: FoxO1, FoxO3a, FoxO4 and FoxO6, which are orthologs of DAF16, an insulin-responsive transcription factor involved in regulating longevity of worms and flies. The degree of homology between these four members is high, especially in the forkhead domain, which contains the DNA-binding interface. Yet, mouse FoxO knockouts have revealed that each FoxO gene has its unique role in the physiological process. Whether the functional divergences are primarily due to adaptive selection pressure or relaxed selective constraint remains an open question. As such, this study aims to address the evolutionary mode of FoxO, which may lead to the functional divergence.
Results
Sequence similarity searches have performed in genome and scaffold data to identify homologues of FoxO in vertebrates. Phylogenetic analysis was used to characterize the family evolutionary history by identifying two duplications early in vertebrate evolution. To determine the mode of evolution in vertebrates, we performed a rigorous statistical analysis with FoxO gene sequences, including relative rate ratio tests, branch-specific dN/dS ratio tests, site-specific dN/dS ratio tests, branch-site dN/dS ratio tests and clade level amino acid conservation/variation patterns analysis. Our results suggest that FoxO is constrained by strong purifying selection except four sites in FoxO6, which have undergone positive Darwinian selection. The functional divergence in this family is best explained by either relaxed purifying selection or positive selection.
Conclusion
We present a phylogeny describing the evolutionary history of the FoxO gene family and show that the genes have evolved through duplications followed by purifying selection except for four sites in FoxO6 fixed by positive selection lie mostly within the non-conserved optimal PKB motif in the C-terminal part. Relaxed selection may play important roles in the process of functional differentiation evolved through gene duplications as well.
doi:10.1186/1471-2148-9-222
PMCID: PMC2746812  PMID: 19732467
18.  Evolutionary Processes Acting on Candidate cis-Regulatory Regions in Humans Inferred from Patterns of Polymorphism and Divergence 
PLoS Genetics  2009;5(8):e1000592.
Analysis of polymorphism and divergence in the non-coding portion of the human genome yields crucial information about factors driving the evolution of gene regulation. Candidate cis-regulatory regions spanning more than 15,000 genes in 15 African Americans and 20 European Americans were re-sequenced and aligned to the chimpanzee genome in order to identify potentially functional polymorphism and to characterize and quantify departures from neutral evolution. Distortions of the site frequency spectra suggest a general pattern of selective constraint on conserved non-coding sites in the flanking regions of genes (CNCs). Moreover, there is an excess of fixed differences that cannot be explained by a Gamma model of deleterious fitness effects, suggesting the presence of positive selection on CNCs. Extensions of the McDonald-Kreitman test identified candidate cis-regulatory regions with high probabilities of positive and negative selection near many known human genes, the biological characteristics of which exhibit genome-wide trends that differ from patterns observed in protein-coding regions. Notably, there is a higher probability of positive selection in candidate cis-regulatory regions near genes expressed in the fetal brain, suggesting that a larger portion of adaptive regulatory changes has occurred in genes expressed during brain development. Overall we find that natural selection has played an important role in the evolution of candidate cis-regulatory regions throughout hominid evolution.
Author Summary
It has been suggested that changes in gene expression may have played a more important role in the evolution of modern humans than changes in protein-coding sequences. In order to identify signatures of natural selection on candidate cis-regulatory regions, we examined single nucleotide polymorphisms obtained from the complete re-sequencing of conserved non-coding sites (CNCs) in the flanking regions of over 15,000 genes in 35 humans. Patterns of allele frequencies in CNCs indicate the presence of both positive and negative selection acting on standing variation within these candidate cis-regulatory regions, particularly for the 5′ and 3′ UTRs of genes. Gene-specific tests comparing levels of polymorphism and divergence identify several genes with strong signatures of selection on candidate cis-regulatory regions and suggest that the biological characteristics of genes subject to selection are different between coding and candidate cis-regulatory regions with respect to gene expression and function. For example, we find stronger signatures of positive selection in candidate cis-regulatory regions near genes expressed in the fetal brain, which we do not observe in a concurrent analysis on protein-coding regions. Our results suggest that both positive and negative selection have acted on candidate cis-regulatory regions and that the evolution of non-coding DNA has played an important role throughout hominid evolution.
doi:10.1371/journal.pgen.1000592
PMCID: PMC2714078  PMID: 19662163
19.  Evolution and Survival on Eutherian Sex Chromosomes 
PLoS Genetics  2009;5(7):e1000568.
Since the two eutherian sex chromosomes diverged from an ancestral autosomal pair, the X has remained relatively gene-rich, while the Y has lost most of its genes through the accumulation of deleterious mutations in nonrecombining regions. Presently, it is unclear what is distinctive about genes that remain on the Y chromosome, when the sex chromosomes acquired their unique evolutionary rates, and whether X-Y gene divergence paralleled that of paralogs located on autosomes. To tackle these questions, here we juxtaposed the evolution of X and Y homologous genes (gametologs) in eutherian mammals with their autosomal orthologs in marsupial and monotreme mammals. We discovered that genes on the X and Y acquired distinct evolutionary rates immediately following the suppression of recombination between the two sex chromosomes. The Y-linked genes evolved at higher rates, while the X-linked genes maintained the lower evolutionary rates of the ancestral autosomal genes. These distinct rates have been maintained throughout the evolution of X and Y. Specifically, in humans, most X gametologs and, curiously, also most Y gametologs evolved under stronger purifying selection than similarly aged autosomal paralogs. Finally, after evaluating the current experimental data from the literature, we concluded that unique mRNA/protein expression patterns and functions acquired by Y (versus X) gametologs likely contributed to their retention. Our results also suggest that either the boundary between sex chromosome strata 3 and 4 should be shifted or that stratum 3 should be divided into two strata.
Author Summary
Using recently available marsupial and monotreme genomes, we investigated nascent sex chromosome evolution in mammals. We show that, in eutherian mammals, X and Y genes acquired distinct evolutionary rates and functional constraints immediately after recombination suppression; X-linked genes maintained lower, ancestral (autosomal), rates, whereas the evolutionary rates of Y-linked genes increased. Most X and, unexpectedly, Y genes evolved under stronger purifying selection than similarly aged autosomal paralogs. However, we also observed that the divergence of gametologs and paralogs shared similar features. In addition, many Y-linked copies evolved unique functions and expression patterns compared to their counterparts on the X chromosome. Therefore, our results suggest that to be retained on the Y chromosome, genes need to acquire separately valuable expression and/or functions to be safeguarded by purifying selection.
doi:10.1371/journal.pgen.1000568
PMCID: PMC2704370  PMID: 19609352
20.  Heterotachy in Mammalian Promoter Evolution 
PLoS Genetics  2006;2(4):e30.
We have surveyed the evolutionary trends of mammalian promoters and upstream sequences, utilising large sets of experimentally supported transcription start sites (TSSs). With 30,969 well-defined TSSs from mouse and 26,341 from human, there are sufficient numbers to draw statistically meaningful conclusions and to consider differences between promoter types. Unlike previous smaller studies, we have considered the effects of insertions, deletions, and transposable elements as well as nucleotide substitutions. The rate of promoter evolution relative to that of control sequences has not been consistent between lineages nor within lineages over time. The most pronounced manifestation of this heterotachy is the increased rate of evolution in primate promoters. This increase is seen across different classes of mutation, including substitutions and micro-indel events. We investigated the relationship between promoter and coding sequence selective constraint and suggest that they are generally uncorrelated. This analysis also identified a small number of mouse promoters associated with the immune response that are under positive selection in rodents. We demonstrate significant differences in divergence between functional promoter categories and identify a category of promoters, not associated with conventional protein-coding genes, that has the highest rates of divergence across mammals. We find that evolutionary rates vary both on a fine scale within mammalian promoters and also between different functional classes of promoters. The discovery of heterotachy in promoter evolution, in particular the accelerated evolution of primate promoters, has important implications for our understanding of human evolution and for strategies to detect primate-specific regulatory elements.
Synopsis
Promoters are crucial to the regulation of gene expression. They are of considerable interest to molecular biologists from a functional perspective and to a much wider audience, as sequence changes within promoters are likely to be a substantial contributor to disease predisposition and the divergence of species. In mammals, promoters have been extensively studied in a case-by-case manner, but the more general mechanisms of promoter evolution are little understood. The authors have undertaken an extensive study of evolutionary trends across experimentally defined promoters. They have discovered that the relative rate of promoter evolution varies between lineages and is substantially accelerated in primates. The authors conclude that the predominant cause is variation in the mutation rate specifically within promoter regions. This finding has important implications for comparative genomics, in particular the identification of functional sites within promoters. The large datasets in this study have also allowed the pattern of evolution to be considered between different types of promoter, giving new insight into their distinct biology.
doi:10.1371/journal.pgen.0020030
PMCID: PMC1449885  PMID: 16683025
21.  MicroRNA-Driven Developmental Remodeling in the Brain Distinguishes Humans from Other Primates 
PLoS Biology  2011;9(12):e1001214.
Comparison of human, chimpanzee, and macaque brain transcriptomes reveals a significant developmental remodeling in the human prefrontal cortex, potentially shaped by microRNA.
While multiple studies have reported the accelerated evolution of brain gene expression in the human lineage, the mechanisms underlying such changes are unknown. Here, we address this issue from a developmental perspective, by analyzing mRNA and microRNA (miRNA) expression in two brain regions within macaques, chimpanzees, and humans throughout their lifespan. We find that constitutive gene expression divergence (species differences independent of age) is comparable between humans and chimpanzees. However, humans display a 3–5 times faster evolutionary rate in divergence of developmental patterns, compared to chimpanzees. Such accelerated evolution of human brain developmental patterns (i) cannot be explained by life-history changes among species, (ii) is twice as pronounced in the prefrontal cortex than the cerebellum, (iii) preferentially affects neuron-related genes, and (iv) unlike constitutive divergence does not depend on cis-regulatory changes, but might be driven by human-specific changes in expression of trans-acting regulators. We show that developmental profiles of miRNAs, as well as their target genes, show the fastest rates of human-specific evolutionary change, and using a combination of computational and experimental methods, we identify miR-92a, miR-454, and miR-320b as possible regulators of human-specific neural development. Our results suggest that different mechanisms underlie adaptive and neutral transcriptome divergence, and that changes in the expression of a few key regulators may have been a major driving force behind rapid evolution of the human brain.
Author Summary
Species evolution is often depicted as a slow and continuous process punctuated by rapid changes. One example of the latter is the evolution of human cognition–emergence of an exceedingly complex phenotype within a few million years. What genetic mechanisms might have driven this process? Nearly 40 years ago, it was proposed that human-specific gene expression changes, rather than changes in protein sequence, might underlie human cognitive evolution. Here we compare gene expression throughout postnatal brain development in humans, chimpanzees, and macaques. We find that simple changes in gene expression levels, plausibly driven by mutations in cis-regulatory elements, accumulate at similar rates in all three evolutionary lineages. What sharply distinguishes humans from other species is change in the timing and shape of developmental expression patterns. This is particularly pronounced in the prefrontal cortex, where 4-fold more genes show more human-specific developmental changes than chimpanzee-specific ones. Notably, our results indicate that this massive developmental remodeling of the human cortex, which affects hundreds of genes, might be driven by expression changes of only a few key regulators, such as microRNAs. Genes affected by this remodeling are preferentially associated with neural activity, thereby suggesting a link to the evolution of human cognition.
doi:10.1371/journal.pbio.1001214
PMCID: PMC3232219  PMID: 22162950
22.  Divergence of Mammalian Higher Order Chromatin Structure Is Associated with Developmental Loci 
PLoS Computational Biology  2013;9(4):e1003017.
Several recent studies have examined different aspects of mammalian higher order chromatin structure – replication timing, lamina association and Hi-C inter-locus interactions — and have suggested that most of these features of genome organisation are conserved over evolution. However, the extent of evolutionary divergence in higher order structure has not been rigorously measured across the mammalian genome, and until now little has been known about the characteristics of any divergent loci present. Here, we generate a dataset combining multiple measurements of chromatin structure and organisation over many embryonic cell types for both human and mouse that, for the first time, allows a comprehensive assessment of the extent of structural divergence between mammalian genomes. Comparison of orthologous regions confirms that all measurable facets of higher order structure are conserved between human and mouse, across the vast majority of the detectably orthologous genome. This broad similarity is observed in spite of many loci possessing cell type specific structures. However, we also identify hundreds of regions (from 100 Kb to 2.7 Mb in size) showing consistent evidence of divergence between these species, constituting at least 10% of the orthologous mammalian genome and encompassing many hundreds of human and mouse genes. These regions show unusual shifts in human GC content, are unevenly distributed across both genomes, and are enriched in human subtelomeric regions. Divergent regions are also relatively enriched for genes showing divergent expression patterns between human and mouse ES cells, implying these regions cause divergent regulation. Particular divergent loci are strikingly enriched in genes implicated in vertebrate development, suggesting important roles for structural divergence in the evolution of mammalian developmental programmes. These data suggest that, though relatively rare in the mammalian genome, divergence in higher order chromatin structure has played important roles during evolution.
Author Summary
The mammalian genome is organised into large multi-megabase domains defined by their physical structure, or higher order chromatin structure. Although these structures are believed to be well conserved between species, there have been few studies attempting to quantify such conservation, or identify divergent structures. We find that regions showing clear evidence of divergence in higher order chromatin structure encompass at least 10% of the mammalian genome, and include many hundreds of genes whose regulation may have been affected. At least some of these genes have been directly implicated in evolutionary innovations to vertebrate developmental programmes, so divergent regions may have been disproportionately important during evolution. In addition, we show that divergent regions occur in large stretches of more than 2 Mb in the human genome and are enriched towards telomeres at the ends of human chromosomes. This may reflect shifts in the nuclear organisation and regulatory functions of chromatin domains between human and mouse.
doi:10.1371/journal.pcbi.1003017
PMCID: PMC3617018  PMID: 23592965
23.  Evolution of a Membrane Protein Regulon in Saccharomyces 
Molecular Biology and Evolution  2012;29(7):1747-1756.
Expression variation is widespread between species. The ability to distinguish regulatory change driven by natural selection from the consequences of neutral drift remains a major challenge in comparative genomics. In this work, we used observations of mRNA expression and promoter sequence to analyze signatures of selection on groups of functionally related genes in Saccharomycete yeasts. In a survey of gene regulons with expression divergence between Saccharomyces cerevisiae and S. paradoxus, we found that most were subject to variation in trans-regulatory factors that provided no evidence against a neutral model. However, we identified one regulon of membrane protein genes controlled by unlinked cis- and trans-acting determinants with coherent effects on gene expression, consistent with a history of directional, nonneutral evolution. For this membrane protein group, S. paradoxus alleles at regulatory loci were associated with elevated expression and altered stress responsiveness relative to other yeasts. In a phylogenetic comparison of promoter sequences of the membrane protein genes between species, the S. paradoxus lineage was distinguished by a short branch length, indicative of strong selective constraint. Likewise, sequence variants within the S. paradoxus population, but not across strains of other yeasts, were skewed toward low frequencies in promoters of genes in the membrane protein regulon, again reflecting strong purifying selection. Our results support a model in which a distinct expression program for the membrane protein genes in S. paradoxus has been preferentially maintained by negative selection as the result of an increased importance to organismal fitness. These findings illustrate the power of integrating expression- and sequence-based tests of natural selection in the study of evolutionary forces that underlie regulatory change.
doi:10.1093/molbev/mss017
PMCID: PMC3375471  PMID: 22319167
regulatory variation; natural selection; Saccharomyces
24.  MicroRNA Expression and Regulation in Human, Chimpanzee, and Macaque Brains 
PLoS Genetics  2011;7(10):e1002327.
Among other factors, changes in gene expression on the human evolutionary lineage have been suggested to play an important role in the establishment of human-specific phenotypes. However, the molecular mechanisms underlying these expression changes are largely unknown. Here, we have explored the role of microRNA (miRNA) in the regulation of gene expression divergence among adult humans, chimpanzees, and rhesus macaques, in two brain regions: prefrontal cortex and cerebellum. Using a combination of high-throughput sequencing, miRNA microarrays, and Q-PCR, we have shown that up to 11% of the 325 expressed miRNA diverged significantly between humans and chimpanzees and up to 31% between humans and macaques. Measuring mRNA and protein expression in human and chimpanzee brains, we found a significant inverse relationship between the miRNA and the target genes expression divergence, explaining 2%–4% of mRNA and 4%–6% of protein expression differences. Notably, miRNA showing human-specific expression localize in neurons and target genes that are involved in neural functions. Enrichment in neural functions, as well as miRNA–driven regulation on the human evolutionary lineage, was further confirmed by experimental validation of predicted miRNA targets in two neuroblastoma cell lines. Finally, we identified a signature of positive selection in the upstream region of one of the five miRNA with human-specific expression, miR-34c-5p. This suggests that miR-34c-5p expression change took place after the split of the human and the Neanderthal lineages and had adaptive significance. Taken together these results indicate that changes in miRNA expression might have contributed to evolution of human cognitive functions.
Author Summary
Humans are remarkably similar to apes and monkeys on the genome sequence level but remain remarkably distinct with respect to cognitive abilities. How could human cognition evolve within such a short evolutionary time? Among many hypotheses, evolution in expression of a few key regulators affecting hundreds of their target genes was proposed as one possible solution. Here, we tested this notion by studying expression divergence of a specific type of regulatory RNA, microRNA (miRNA), and its effect on gene expression profiles in brains of humans, chimpanzees, and rhesus macaques. Our results indicate that changes in miRNA expression have played a considerable role in the establishment of gene expression divergence between human brains and brains of non-human primates at both mRNA and protein expression levels. Furthermore, we find indications that some of the human-specific gene expression profiles caused by miRNA expression divergence might be associated with evolution of human-specific functions.
doi:10.1371/journal.pgen.1002327
PMCID: PMC3192836  PMID: 22022286
25.  Evidence for Widespread Degradation of Gene Control Regions in Hominid Genomes 
PLoS Biology  2005;3(2):e42.
Although sequences containing regulatory elements located close to protein-coding genes are often only weakly conserved during evolution, comparisons of rodent genomes have implied that these sequences are subject to some selective constraints. Evolutionary conservation is particularly apparent upstream of coding sequences and in first introns, regions that are enriched for regulatory elements. By comparing the human and chimpanzee genomes, we show here that there is almost no evidence for conservation in these regions in hominids. Furthermore, we show that gene expression is diverging more rapidly in hominids than in murids per unit of neutral sequence divergence. By combining data on polymorphism levels in human noncoding DNA and the corresponding human–chimpanzee divergence, we show that the proportion of adaptive substitutions in these regions in hominids is very low. It therefore seems likely that the lack of conservation and increased rate of gene expression divergence are caused by a reduction in the effectiveness of natural selection against deleterious mutations because of the low effective population sizes of hominids. This has resulted in the accumulation of a large number of deleterious mutations in sequences containing gene control elements and hence a widespread degradation of the genome during the evolution of humans and chimpanzees.
A comparison of hominid and rodent lineages reveals that the gene control regions of hominids are not conserved and are accumulating mutations, suggesting widespread degradation of the hominid genome
doi:10.1371/journal.pbio.0030042
PMCID: PMC544929  PMID: 15678168

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