Analysing gene function after duplication

After gene duplication, mutations cause the gene copies to diverge. The classical model predicts that these mutations will generally lead to the loss of function of one gene copy; rarely, new functions will be created and both duplicate genes are conserved. In contrast, under the subfunctionalization model both duplicates are preserved due to the partition of different functions between the duplicates. A recent study provides support for the subfunctionalization model, identifying several expressed gene duplicates common to humans and mice that contain regions conserved in one duplicate but variable in the other (and vice versa). We discuss both the methodology used in this study and also how gene phylogeny may lead to additional evidence for the importance of subfunctionalization in the evolution of new genes.     

Regulatory divergence of the duplicated chromosomal loci sox11a/b by subpartitioning and sequence evolution of enhancers in zebrafish

We used the classic example of the duplicated zebrafish sox11a and -b loci to test the duplication, degeneration, complementation (DDC) model of genome evolution through whole genome duplication. While recent reports have demonstrated sub-partitioning of regulatory sequences in duplicated regions, a comparison of the regulatory capabilities of extant regulatory sequences derived from ancient ancestral elements has been scarce. Consistent with the DDC model, we find that ancestral regulatory elements distributed over several hundred kb were lost in either one or the other zebrafish duplicate, leading to subpartitioning. However, regulatory sequences kept as duplicates near both sox11 co-orthologs diverged in sequence from each other and from human elements and in the regulatory patterns they drive in transgenic zebrafish. Evolutionary analysis of the loci suggested that both zebrafish protein coding sox11 orthologs have been maintained by purifying selection, and have evolved at comparable rates, indicative of non-diverged protein functions. The duplicated regulatory elements, conversely, evolved with different divergence rates and degrees of subfunctionalization. These data show that regulatory evolution of gene expression patterns occurred both through differential loss as well as through regulatory sequence evolution in zebrafish versus human genomes.     

Genomic background predicts the fate of duplicated genes: evidence from the yeast genome

Gene duplication with subsequent divergence plays a central role in the acquisition of genes with novel function and complexity during the course of evolution. With reduced functional constraints or through positive selection, these duplicated genes may experience accelerated evolution. Under the model of subfunctionalization, loss of subfunctions leads to complementary acceleration at sites with two copies, and the difference in average rate between the sequences may not be obvious. On the other hand, the classical model of neofunctionalization predicts that the evolutionary rate in one of the two duplicates is accelerated. However, the classical model does not tell which of the duplicates experiences the acceleration in evolutionary rate. Here, we present evidence from the Saccharomyces cerevisiae genome that a duplicate located in a genomic region with a low-recombination rate is likely to evolve faster than a duplicate in an area of high recombination. This observation is consistent with population genetics theory that predicts that purifying selection is less effective in genomic regions of low recombination (Hill-Robertson effect). Together with previous studies, our results suggest the genomic background (e.g., local recombination rate) as a potential force to drive the divergence between nontandemly duplicated genes. This implies the importance of structure and complexity of genomes in the diversification of organisms via gene duplications.     

Gene duplication as a major force in evolution

Gene duplication is an important mechanism for acquiring new genes and creating genetic novelty in organisms. Many new gene functions have evolved through gene duplication and it has contributed tremendously to the evolution of developmental programmes in various organisms. Gene duplication can result from unequal crossing over, retroposition or chromosomal (or genome) duplication. Understanding the mechanisms that generate duplicate gene copies and the subsequent dynamics among gene duplicates is vital because these investigations shed light on localized and genomewide aspects of evolutionary forces shaping intra-specific and inter-specific genome contents, evolutionary relationships, and interactions. Based on whole-genome analysis of Arabidopsis thaliana, there is compelling evidence that angiosperms underwent two whole-genome duplication events early during their evolutionary history. Recent studies have shown that these events were crucial for creation of many important developmental and regulatory genes found in extant angiosperm genomes. Recent studies also provide strong indications that even yeast (Saccharomyces cerevisiae), with its compact genome, is in fact an ancient tetraploid. Gene duplication can provide new genetic material for mutation, drift and selection to act upon, the result of which is specialized or new gene functions. Without gene duplication the plasticity of a genome or species in adapting to changing environments would be severely limited. Whether a duplicate is retained depends upon its function, its mode of duplication, (i.e. whether it was duplicated during a whole-genome duplication event), the species in which it occurs, and its expression rate. The exaptation of preexisting secondary functions is an important feature in gene evolution, just as it is in morphological evolution.     

Two-phase resolution of polyploidy in the Arabidopsis metabolic network gives rise to relative and absolute dosage constraints

The abundance of detected ancient polyploids in extant genomes raises questions regarding evolution after whole-genome duplication (WGD). For instance, what rules govern the preservation or loss of the duplicated genes created by WGD? We explore this question by contrasting two possible preservation forces: selection on relative and absolute gene dosages. Constraints on the relative dosages of central network genes represent an important force for maintaining duplicates (the dosage balance hypothesis). However, preservation may also result from selection on the absolute abundance of certain gene products. The metabolic network of the model plant Arabidopsis thaliana is a powerful system for comparing these hypotheses. We analyzed the surviving WGD-produced duplicate genes in this network, finding evidence that the surviving duplicates from the most recent WGD (WGD-α) are clustered in the network, as predicted by the dosage balance hypothesis. A flux balance analysis suggests an association between the survival of duplicates from a more ancient WGD (WGD-β) and reactions with high metabolic flux. We argue for an interplay of relative and absolute dosage constraints, such that the relative constraints imposed by the recent WGD are still being resolved by evolution, while they have been essentially fully resolved for the ancient event.     

Detecting correlation between sequence and expression divergences in a comparative analysis of human serpin genes

Physiological functions and characteristic structures of the serpin gene superfamily have been studied extensively, yet the evolution of the serpin genes remains unclear. Gene duplication in this superfamily may shed light on this issue. Two models are used to predict the preservation of duplicated genes: the classical model and the duplication–degeneration–complementation (DDC) model. In this study, we analyzed the phylogenetic relationships of 33 human serpin genes and the expression data of some members of the serpin superfamily from a DNA microarray of human leukemia U937 cells with stably inducible expression of the leukemia-related AML1-ETO gene. We then determined the utility of the DDC model by mapping serpin superfamily expression data to the phylogenetic tree. The correlation between sequence and expression divergences as measured by the Pearson correlation coefficient indicated that human serpin genes evolved under the DDC model. Our study provides a new strategy for comparative analysis of gene sequences and microarray data.     

Detecting correlation between sequence and expression divergences in a comparative analysis of human serpin genes

Physiological functions and characteristic structures of the serpin gene superfamily have been studied extensively, yet the evolution of the serpin genes remains unclear. Gene duplication in this superfamily may shed light on this issue. Two models are used to predict the preservation of duplicated genes: the classical model and the duplication-degeneration-complementation (DDC) model. In this study, we analyzed the phylogenetic relationships of 33 human serpin genes and the expression data of some members of the serpin superfamily from a DNA microarray of human leukemia U937 cells with stably inducible expression of the leukemia-related AML1-ETO gene. We then determined the utility of the DDC model by mapping serpin superfamily expression data to the phylogenetic tree. The correlation between sequence and expression divergences as measured by the Pearson correlation coefficient indicated that human serpin genes evolved under the DDC model. Our study provides a new strategy for comparative analysis of gene sequences and microarray data.     

Evolution of Stress-Regulated Gene Expression in Duplicate Genes of Arabidopsis thaliana

Due to the selection pressure imposed by highly variable environmental conditions, stress sensing and regulatory response mechanisms in plants are expected to evolve rapidly. One potential source of innovation in plant stress response mechanisms is gene duplication. In this study, we examined the evolution of stress-regulated gene expression among duplicated genes in the model plant Arabidopsis thaliana. Key to this analysis was reconstructing the putative ancestral stress regulation pattern. By comparing the expression patterns of duplicated genes with the patterns of their ancestors, duplicated genes likely lost and gained stress responses at a rapid rate initially, but the rate is close to zero when the synonymous substitution rate (a proxy for time) is >~0.8. When considering duplicated gene pairs, we found that partitioning of putative ancestral stress responses occurred more frequently compared to cases of parallel retention and loss. Furthermore, the pattern of stress response partitioning was extremely asymmetric. An analysis of putative cis-acting DNA regulatory elements in the promoters of the duplicated stress-regulated genes indicated that the asymmetric partitioning of ancestral stress responses are likely due, at least in part, to differential loss of DNA regulatory elements; the duplicated genes losing most of their stress responses were those that had lost more of the putative cis-acting elements. Finally, duplicate genes that lost most or all of the ancestral responses are more likely to have gained responses to other stresses. Therefore, the retention of duplicates that inherit few or no functions seems to be coupled to neofunctionalization. Taken together, our findings provide new insight into the patterns of evolutionary changes in gene stress responses after duplication and lay the foundation for testing the adaptive significance of stress regulatory changes under highly variable biotic and abiotic environments.     

Knockdown of duplicated zebrafish hoxb1 genes reveals distinct roles in hindbrain patterning and a novel mechanism of duplicate gene retention

We have used a morpholino-based knockdown approach to investigate the functions of a pair of zebrafish Hox gene duplicates, hoxb1a and hoxb1b, which are expressed during development of the hindbrain. We find that the zebrafish hoxb1 duplicates have equivalent functions to mouse Hoxb1 and its paralogue Hoxa1. Thus, we have revealed a 'function shuffling' among genes of paralogue group 1 during the evolution of vertebrates. Like mouse Hoxb1, zebrafish hoxb1a is required for migration of the VIIth cranial nerve branchiomotor neurons from their point of origin in hindbrain rhombomere 4 towards the posterior. By contrast, zebrafish hoxb1b, like mouse Hoxa1, is required for proper segmental organization of rhombomere 4 and the posterior hindbrain. Double knockdown experiments demonstrate that the zebrafish hoxb1 duplicates have partially redundant functions. However, using an RNA rescue approach, we reveal that these duplicated genes do not have interchangeable biochemical functions: only hoxb1a can properly pattern the VIIth cranial nerve. Despite this difference in protein function, we provide evidence that the hoxb1 duplicate genes were initially maintained in the genome because of complementary degenerative mutations in defined cis-regulatory elements.     

How Often Do Duplicated Genes Evolve New Functions?

A recently duplicated gene can either fix a null allele (becoming a pseudogene) or fix an (advantageous) allele giving a slightly different function, starting it on the road to evolving a new function. Here we examine the relative probabilities of these two events under a simple model. Null alleles are assumed to be neutral; linkage effects are ignored, as are unequal crossing over and gene conversion. These assumptions likely make our results underestimates for the probability that an advantageous allele is fixed first. When new advantageous mutations are additive with selection coefficient s and the ratio of advantageous to null mutations is ρ, the probability an advantageous allele is fixed first is ([1 - e(-S)]/[ρS] + 1)(-1), where S = 4N(e)s with N(e) the effective population size. The probability that a duplicate locus becomes a pseudogene, as opposed to evolving a new gene function, is high unless ρS & 1. However, even if advantageous mutations are very rare relative to null mutations, for sufficiently large populations ρS & 1 and new gene function, rather than pseudogene formation, is the expected fate of most duplicated genes.     

Degree of Functional Divergence in Duplicates Is Associated with Distinct Roles in Plant Evolution

Gene duplication is a major mechanism to create new genes. After gene duplication, some duplicated genes undergo functionalization, whereas others largely maintain redundant functions. Duplicated genes comprise various degrees of functional diversification in plants. However, the evolutionary fate of high and low diversified duplicates is unclear at genomic scale. To infer high and low diversified duplicates in Arabidopsis thaliana genome, we generated a prediction method for predicting whether a pair of duplicate genes was subjected to high or low diversification based on the phenotypes of knock-out mutants. Among 4,017 pairs of recently duplicated A. thaliana genes, 1,052 and 600 are high and low diversified duplicate pairs, respectively. The predictions were validated based on the phenotypes of generated knock-down transgenic plants. We determined that the high diversified duplicates resulting from tandem duplications tend to have lineage-specific functions, whereas the low diversified duplicates produced by whole-genome duplications are related to essential signaling pathways. To assess the evolutionary impact of high and low diversified duplicates in closely related species, we compared the retention rates and selection pressures on the orthologs of A. thaliana duplicates in two closely related species. Interestingly, high diversified duplicates resulting from tandem duplications tend to be retained in multiple lineages under positive selection. Low diversified duplicates by whole-genome duplications tend to be retained in multiple lineages under purifying selection. Taken together, the functional diversities determined by different duplication mechanisms had distinct effects on plant evolution.     

Epigenetic silencing may aid evolution by gene duplication

Gene duplication is commonly regarded as the main evolutionary path toward the gain of a new function. However, even with gene duplication, there is a loss-versus-gain dilemma: most newly born duplicates degrade to pseudogenes, since degenerative mutations are much more frequent than advantageous ones. Thus, something additional seems to be needed to shift the loss versus gain equilibrium toward functional divergence. We suggest that epigenetic silencing of duplicates might play this role in evolution. This study began when we noticed in a previous publication (Lynch M, Conery JS [2000] Science 291:1151–1155) that the frequency of functional young gene duplicates is higher in organisms that have cytosine methylation (H. sapiens, M. musculus, and A. thaliana) than in organisms that do not have methylated genomes (S. cerevisiae, D. melanogaster, and C. elegans). We find that genome data analysis confirms the likelihood of much more efficient functional divergence of gene duplicates in mammals and plants than in yeast, nematode, and fly. We have also extended the classic model of gene duplication, in which newly duplicated genes have exactly the same expression pattern, to the case when they are epigenetically silenced in a tissue- and/or developmental stage-complementary manner. This exposes each of the duplicates to negative selection, thus protecting from pseudogenization. Our analysis indicates that this kind of silencing (i) enhances evolution of duplicated genes to new functions, particularly in small populations, (ii) is quite consistent with the subfunctionalization model when degenerative but complementary mutations affect different subfunctions of the gene, and (iii) furthermore, may actually cooperate with the DDC (duplication– degeneration–complementation) process. Dedicated to the memory of Susumu Ohno     

The probability of duplicate gene preservation by subfunctionalization

It has often been argued that gene-duplication events are most commonly followed by a mutational event that silences one member of the pair, while on rare occasions both members of the pair are preserved as one acquires a mutation with a beneficial function and the other retains the original function. However, empirical evidence from genome duplication events suggests that gene duplicates are preserved in genomes far more commonly and for periods far in excess of the expectations under this model, and whereas some gene duplicates clearly evolve new functions, there is little evidence that this is the most common mechanism of duplicate-gene preservation. An alternative hypothesis is that gene duplicates are frequently preserved by subfunctionalization, whereby both members of a pair experience degenerative mutations that reduce their joint levels and patterns of activity to that of the single ancestral gene. We consider the ways in which the probability of duplicate-gene preservation by such complementary mutations is modified by aspects of gene structure, degree of linkage, mutation rates and effects, and population size. Even if most mutations cause complete loss-of-subfunction, the probability of duplicate-gene preservation can be appreciable if the long-term effective population size is on the order of 10(5) or smaller, especially if there are more than two independently mutable subfunctions per locus. Even a moderate incidence of partial loss-of-function mutations greatly elevates the probability of preservation. The model proposed herein leads to quantitative predictions that are consistent with observations on the frequency of long-term duplicate gene preservation and with observations that indicate that a common fate of the members of duplicate-gene pairs is the partitioning of tissue-specific patterns of expression of the ancestral gene.     

The probability of preservation of a newly arisen gene duplicate.

Newly emerging data from genome sequencing projects suggest that gene duplication, often accompanied by genetic map changes, is a common and ongoing feature of all genomes. This raises the possibility that differential expansion/contraction of various genomic sequences may be just as important a mechanism of phenotypic evolution as changes at the nucleotide level. However, the population-genetic mechanisms responsible for the success vs. failure of newly arisen gene duplicates are poorly understood. We examine the influence of various aspects of gene structure, mutation rates, degree of linkage, and population size (N) on the joint fate of a newly arisen duplicate gene and its ancestral locus. Unless there is active selection against duplicate genes, the probability of permanent establishment of such genes is usually no less than 1/(4N) (half of the neutral expectation), and it can be orders of magnitude greater if neofunctionalizing mutations are common. The probability of a map change (reassignment of a key function of an ancestral locus to a new chromosomal location) induced by a newly arisen duplicate is also generally >1/(4N) for unlinked duplicates, suggesting that recurrent gene duplication and alternative silencing may be a common mechanism for generating microchromosomal rearrangements responsible for postreproductive isolating barriers among species. Relative to subfunctionalization, neofunctionalization is expected to become a progressively more important mechanism of duplicate-gene preservation in populations with increasing size. However, even in large populations, the probability of neofunctionalization scales only with the square of the selective advantage. Tight linkage also influences the probability of duplicate-gene preservation, increasing the probability of subfunctionalization but decreasing the probability of neofunctionalization.     

Evolution of cis-regulatory elements in duplicated genes of yeast

An increasing number of studies report that functional divergence in duplicated genes is accompanied by gene expression changes, although the evolutionary mechanism behind this process remains unclear. Our genomic analysis on the yeast Saccharomyces cerevisiae shows that the number of shared regulatory motifs in the duplicates decreases with evolutionary time, whereas the total number of regulatory motifs remains unchanged. Moreover, genes with numerous paralogs in the yeast genome do not have especially low number of regulatory motifs. These findings indicate that degenerative complementation is not the sole mechanism behind expression divergence in yeast. Moreover, we found some evidence for the action of positive selection on cis-regulatory motifs after gene duplication. These results suggest that the evolution of functional novelty has a substantial role in yeast duplicate gene evolution.