Detecting Functional Divergence after Gene Duplication through Evolutionary Changes in Posttranslational Regulatory Sequences

Gene duplication is an important evolutionary mechanism that can result in functional divergence in paralogs due to neo-functionalization or sub-functionalization. Consistent with functional divergence after gene duplication, recent studies have shown accelerated evolution in retained paralogs. However, little is known in general about the impact of this accelerated evolution on the molecular functions of retained paralogs. For example, do new functions typically involve changes in enzymatic activities, or changes in protein regulation? Here we study the evolution of posttranslational regulation by examining the evolution of important regulatory sequences (short linear motifs) in retained duplicates created by the whole-genome duplication in budding yeast. To do so, we identified short linear motifs whose evolutionary constraint has relaxed after gene duplication with a likelihood-ratio test that can account for heterogeneity in the evolutionary process by using a non-central chi-squared null distribution. We find that short linear motifs are more likely to show changes in evolutionary constraints in retained duplicates compared to single-copy genes. We examine changes in constraints on known regulatory sequences and show that for the Rck1/Rck2, Fkh1/Fkh2, Ace2/Swi5 paralogs, they are associated with previously characterized differences in posttranslational regulation. Finally, we experimentally confirm our prediction that for the Ace2/Swi5 paralogs, Cbk1 regulated localization was lost along the lineage leading to SWI5 after gene duplication. Our analysis suggests that changes in posttranslational regulation mediated by short regulatory motifs systematically contribute to functional divergence after gene duplication.     

Gene expression divergence is coupled to evolution of DNA structure in coding regions

Sequence changes in coding region and regulatory region of the gene itself (cis) determine most of gene expression divergence between closely related species. But gene expression divergence between yeast species is not correlated with evolution of primary nucleotide sequence. This indicates that other factors in cis direct gene expression divergence. Here, we studied the contribution of DNA three-dimensional structural evolution as cis to gene expression divergence. We found that the evolution of DNA structure in coding regions and gene expression divergence are correlated in yeast. Similar result was also observed between Drosophila species. DNA structure is associated with the binding of chromatin remodelers and histone modifiers to DNA sequences in coding regions, which influence RNA polymerase II occupancy that controls gene expression level. We also found that genes with similar DNA structures are involved in the same biological process and function. These results reveal the previously unappreciated roles of DNA structure as cis-effects in gene expression.     

Evolution of proteins and gene expression levels are coupled in Drosophila and are independently associated with mRNA abundance, protein length, and number of protein-protein interactions

Organismic evolution requires that variation at distinct hierarchical levels and attributes be coherently integrated, often in the face of disparate environmental and genetic pressures. A central part of the evolutionary analysis of biological systems remains to decipher the causal connections between organism-wide (or genome-wide) attributes (e.g., mRNA abundance, protein length, codon bias, recombination rate, genomic position, mutation rate, etc) as well as their role-together with mutation, selection, and genetic drift-in shaping patterns of evolutionary variation in any of the attributes themselves. Here we combine genome-wide evolutionary analysis of protein and gene expression data to highlight fundamental relationships among genomic attributes and their associations with the evolution of both protein sequences and gene expression levels. Our results show that protein divergence is positively coupled with both gene expression polymorphism and divergence. We show moreover that although the number of protein-protein interactions in Drosophila is negatively associated with protein divergence as well as gene expression polymorphism and divergence, protein-protein interactions cannot account for the observed coupling between regulatory and structural evolution. Furthermore, we show that proteins with higher rates of amino acid substitutions tend to have larger sizes and tend to be expressed at lower mRNA abundances, whereas genes with higher levels of gene expression divergence and polymorphism tend to have shorter sizes and tend to be expressed at higher mRNA abundances. Finally, we show that protein length is negatively associated with both number of protein-protein interactions and mRNA abundance and that interacting proteins in Drosophila show similar amounts of divergence. We suggest that protein sequences and gene expression are subjected to similar evolutionary dynamics, possibly because of similarity in the fitness effect (i.e., strength of stabilizing selection) of disruptions in a gene's protein sequence or its mRNA expression. We conclude that, as more and better data accumulate, understanding the causal connections among biological traits and how they are integrated over time to constrain or promote structural and regulatory evolution may finally become possible.     

Predominant gain of promoter TATA box after gene duplication associated with stress responses

TATA box, the core promoter element, exists in a broad range of eukaryotes, and the expression of TATA-containing genes usually responds to various environmental stresses. Hence, the evolution of TATA-box in duplicate genes may provide some clues for the interrelationship among environmental stress, expression differentiation, and duplicate gene preservation. In the present study, we observed that the TATA box is significantly overrepresented in duplicate genes compared with singletons in human, worm, Arabidopsis, and yeast genomes. We then conducted an extensive functional genomic analysis to investigate the evolution of TATA box along over 700 yeast gene family phylogenies. After reconstructing the ancestral TATA-box states (presence or absence), we found that significantly higher numbers of TATA box gain events than loss events had occurred after yeast gene duplications-the overall gain-loss ratio is about 3-4 to 1. Interestingly, these TATA-gain duplicate genes on average have experienced greater expression divergence from the ancestral expression states than their most closely related TATA-less duplicate partners, but only under environmental stress conditions (asymmetric evolution); indeed, under normal physiological conditions, they have similar expression divergence (symmetric evolution). Moreover, we showed that TATA-gain duplicates are enriched in stress-associated functional categories but that is not the case for TATA-ancestral duplicates (those inherited from their ancestors prior to duplication). Together, we conclude that after the gene duplication, gain of the TATA box in duplicate promoters may have played an important role in yeast duplicate preservation by accelerating expression divergence that may facilitate the adaptive evolution of the organism in response to environmental changes.     

Evolutionary analysis of three gibberellin oxidase genes in rice, Arabidopsis, and soybean

GAs are plant hormones that play fundamental roles in plant growth and development. GA2ox, GA3ox, and GA20ox are three key enzymes in GA biosynthesis. These enzymes belong to the 2OG-Fe (II) oxygenase superfamily and are independently encoded by different gene families. To date, genome-wide comparative analyses of GA oxidases in plant species have not been thoroughly carried out. In the present work, 61 GA oxidase family genes from rice (Oryza sativa), Arabidopsis, and soybean (Glycine max) were identified and a full study of these genes including phylogenetic tree construction, gene structure, gene family expansion and analysis of functional motifs was performed. Based on phylogeny, most of the GA oxidases were divided into four subgroups that reflected functional classifications. Intron/intron average length of GA oxidase genes in rice analysis revealed that GA oxidase genes in rice experienced substantial evolutionary divergence. Segmental duplication events were mainly found in soybean genome. However, in rice and Arabidopsis, no single expansion pattern exhibited dominance, indicating that GA oxidase genes from these species might have been subjected to a more complex evolutionary mechanism. In addition, special functional motifs were discovered in GA20ox, GA3ox, and GA2ox, which suggested that different functional motifs are associated with differences in protein function. Taken together our results suggest that GA oxidase family genes have undergone divergent evolutionary routes, especially at the monocot-dicot split, with dynamic evolution occurring in Arabidopsis thaliana and soybean.     

Allelic imbalance in Drosophila hybrid heads: exons, isoforms, and evolution

Unraveling how regulatory divergence contributes to species differences and adaptation requires identifying functional variants from among millions of genetic differences. Analysis of allelic imbalance (AI) reveals functional genetic differences in cis regulation and has demonstrated differences in cis regulation within and between species. Regulatory mechanisms are often highly conserved, yet differences between species in gene expression are extensive. What evolutionary forces explain widespread divergence in cis regulation? AI was assessed in Drosophila melanogaster-Drosophila simulans hybrid female heads using RNA-seq technology. Mapping bias was virtually eliminated by using genotype-specific references. Allele representation in DNA sequencing was used as a prior in a novel Bayesian model for the estimation of AI in RNA. Cis regulatory divergence was common in the organs and tissues of the head with 41% of genes analyzed showing significant AI. Using existing population genomic data, the relationship between AI and patterns of sequence evolution was examined. Evidence of positive selection was found in 30% of cis regulatory divergent genes. Genes involved in defense, RNAi/RISC complex genes, and those that are sex regulated are enriched among adaptively evolving cis regulatory divergent genes. For genes in these groups, adaptive evolution may play a role in regulatory divergence between species. However, there is no evidence that adaptive evolution drives most of the cis regulatory divergence that is observed. The majority of genes showed patterns consistent with stabilizing selection and neutral evolutionary processes.     

Evolution of yellow gene regulation and pigmentation in Drosophila

BACKGROUND: Changes in developmental gene expression are central to phenotypic evolution, but the genetic mechanisms underlying these changes are not well understood. Interspecific differences in gene expression can arise from evolutionary changes in cis-regulatory DNA and/or in the expression of trans-acting regulatory proteins, but few case studies have distinguished between these mechanisms. Here, we compare the regulation of the yellow gene, which is required for melanization, among distantly related Drosophila species with different pigment patterns and determine the phenotypic effects of divergent Yellow expression. RESULTS: Yellow expression has diverged among D. melanogaster, D. subobscura, and D. virilis and, in all cases, correlates with the distribution of black melanin. Species-specific Yellow expression patterns were retained in D. melanogaster transformants carrying the D. subobscura and D. virilis yellow genes, indicating that sequence evolution within the yellow gene underlies the divergence of Yellow expression. Evolutionary changes in the activity of orthologous cis-regulatory elements are responsible for differences in abdominal Yellow expression; however, cis-regulatory element evolution is not the sole cause of divergent Yellow expression patterns. Transformation of the D. melanogaster yellow gene into D. virilis altered its expression pattern, indicating that trans-acting factors that regulate the D. melanogaster yellow gene have also diverged between these two species. Finally, we found that the phenotypic effects of evolutionary changes in Yellow expression depend on epistatic interactions with other genes. CONCLUSIONS: Evolutionary changes in Yellow expression correlate with divergent melanin patterns and are a result of evolution in both cis- and trans-regulation. These changes were likely necessary for the divergence of pigmentation, but evolutionary changes in other genes were also required.     

Evolutionary significance of gene expression divergence

Recent large-scale studies of evolutionary changes in gene expression among mammalian species have led to the proposal that gene expression divergence may be neutral with respect to organismic fitness. Here, we employ a comparative analysis of mammalian gene sequence divergence and gene expression divergence to test the hypothesis that the evolution of gene expression is predominantly neutral. Two models of neutral gene expression evolution are considered: 1-purely neutral evolution (i.e., no selective constraint) of gene expression levels and patterns and 2-neutral evolution accompanied by selective constraint. With respect to purely neutral evolution, levels of change in gene expression between human-mouse orthologs are correlated with levels of gene sequence divergence that are determined largely by purifying selection. In contrast, evolutionary changes of tissue-specific gene expression profiles do not show such a correlation with sequence divergence. However, divergence of both gene expression levels and profiles are significantly lower for orthologous human-mouse gene pairs than for pairs of randomly chosen human and mouse genes. These data clearly point to the action of selective constraint on gene expression divergence and are inconsistent with the purely neutral model; however, there is likely to be a neutral component in evolution of gene expression, particularly, in tissues where the expression of a given gene is low and functionally irrelevant. The model of neutral evolution with selective constraint predicts a regular, clock-like accumulation of gene expression divergence. However, relative rate tests of the divergence among human-mouse-rat orthologous gene sets reveal clock-like evolution for gene sequence divergence, and to a lesser extent for gene expression level divergence, but not for the divergence of tissue-specific gene expression profiles. Taken together, these results indicate that gene expression divergence is subject to the effects of purifying selective constraint and suggest that it might also be substantially influenced by positive Darwinian selection.