[Show abstract][Hide abstract] ABSTRACT: Although single genes underlying several evolutionary adaptations have been identified, the genetic basis of complex, polygenic adaptations has been far more challenging to pinpoint. Here we report that the budding yeast Saccharomyces paradoxus has recently evolved resistance to citrinin, a naturally occurring mycotoxin. Applying a genome-wide test for selection on cis-regulation, we identified five genes involved in the citrinin response that are constitutively up-regulated in S. paradoxus. Four of these genes are necessary for resistance, and are also sufficient to increase the resistance of a sensitive strain when over-expressed. Moreover, cis-regulatory divergence in the promoters of these genes contributes to resistance, while exacting a cost in the absence of citrinin. Our results demonstrate how the subtle effects of individual regulatory elements can be combined, via natural selection, into a complex adaptation. Our approach can be applied to dissect the genetic basis of polygenic adaptations in a wide range of species.
[Show abstract][Hide abstract] ABSTRACT: In addition to coding for proteins, exons can also impact transcription by encoding regulatory elements such as enhancers.
It has been debated whether such features confer heightened selective constraint, or evolve neutrally. We have addressed this
question by developing a new approach to disentangle the sources of selection acting on exonic enhancers, in which we model
the evolutionary rates of every possible substitution as a function of their effects on both protein sequence and enhancer
activity. In three exonic enhancers, we found no significant association between evolutionary rates and effects on enhancer
activity. This suggests that despite having biochemical activity, these exonic enhancers have no detectable selective constraint,
and thus are unlikely to play a major role in protein evolution.
No preview · Article · Oct 2015 · Molecular Biology and Evolution
[Show abstract][Hide abstract] ABSTRACT: DNA methylation is an epigenetic modification that plays a key role in gene regulation. Previous studies have investigated its genetic basis by mapping genetic variants that are associated with DNA methylation at specific sites, but these have been limited to microarrays that cover less than 2% of the genome and cannot account for allele-specific methylation (ASM). Other studies have performed whole-genome bisulfite sequencing on a few individuals, but these lack statistical power to identify variants associated with DNA methylation. We present a novel approach in which bisulfite-treated DNA from many individuals is sequenced together in a single pool, resulting in a truly genome-wide map of DNA methylation. Compared to methods that do not account for ASM, our approach increases statistical power to detect associations while sharply reducing cost, effort, and experimental variability. As a proof of concept, we generated deep sequencing data from a pool of 60 human cell lines; we evaluated almost twice as many CpGs as the largest microarray studies and identified over 2,000 genetic variants associated with DNA methylation. We found that these variants are highly enriched for associations with chromatin accessibility and CTCF binding but are less likely to be associated with traits indirectly linked to DNA, such as gene expression and disease phenotypes. In summary, our approach allows genome-wide mapping of genetic variants associated with DNA methylation in any tissue of any species, without the need for individual-level genotype or methylation data.
Published by Cold Spring Harbor Laboratory Press.
[Show abstract][Hide abstract] ABSTRACT: Genomic imprinting is an epigenetic process that restricts gene expression to either the maternally or paternally inherited allele. Many theories have been proposed to explain its evolutionary origin, but understanding has been limited by a paucity of data mapping the breadth and dynamics of imprinting within any organism. We generated an atlas of imprinting spanning 33 mouse and 45 human developmental stages and tissues. Nearly all imprinted genes were imprinted in early development and either retained their parent-of-origin expression in adults or lost it completely. Consistent with an evolutionary signature of parental conflict, imprinted genes were enriched for coexpressed pairs of maternally and paternally expressed genes, showed accelerated expression divergence between human and mouse, and were more highly expressed than their non-imprinted orthologs in other species. Our approach demonstrates a general framework for the discovery of imprinting in any species and sheds light on the causes and consequences of genomic imprinting in mammals.
[Show abstract][Hide abstract] ABSTRACT: Population epigenetic studies have been seeking to identify differences in DNA methylation between specific exposures, demographic factors, or diseases in accessible tissues, but relatively little is known about how inter-individual variability differs between these tissues. This study presents an analysis of DNA methylation differences between matched peripheral blood mononuclear cells (PMBCs) and buccal epithelial cells (BECs), the two most accessible tissues for population studies, in 998 promoter-located CpG sites. Specifically we compared probe-wise DNA methylation variance, and how this variance related to demographic factors across the two tissues. PBMCs had overall higher DNA methylation than BECs, and the two tissues tended to differ most at genomic regions of low CpG density. Furthermore, although both tissues showed appreciable probe-wise variability, the specific regions and magnitude of variability differed strongly between tissues. Lastly, through exploratory association analysis, we found indication of differential association of BEC and PBMC with demographic variables. The work presented here offers insight into variability of DNA methylation between individuals and across tissues and helps guide decisions on the suitability of buccal epithelial or peripheral mononuclear cells for the biological questions explored by epigenetic studies in human populations.
Full-text · Article · Feb 2015 · Scientific Reports
[Show abstract][Hide abstract] ABSTRACT: The recent advent of ribosome profiling - sequencing of short ribosome-bound fragments of mRNA - has offered an unprecedented opportunity to interrogate the sequence features responsible for modulating translational rates. Nevertheless, numerous analyses of the first riboprofiling dataset have produced equivocal and often incompatible results. Here we analyze three independent yeast riboprofiling data sets, including two with much higher coverage than previously available, and find that all three show substantial technical sequence biases that confound interpretations of ribosomal occupancy. After accounting for these biases, we find no effect of previously implicated factors on ribosomal pausing. Rather, we find that incorporation of proline, whose unique side-chain stalls peptide synthesis in vitro, also slows the ribosome in vivo. We also reanalyze a method that implicated positively charged amino acids as the major determinant of ribosomal stalling and demonstrate that it produces false signals of stalling in low-coverage data. Our results suggest that any analysis of riboprofiling data should account for sequencing biases and sparse coverage. To this end, we establish a robust methodology that enables analysis of ribosome profiling data without prior assumptions regarding which positions spanned by the ribosome cause stalling.
[Show abstract][Hide abstract] ABSTRACT: Despite the greater functional importance of protein levels, our knowledge of gene expression evolution is based almost entirely on studies of mRNA levels. In contrast, our understanding of how translational regulation evolves has lagged far behind. Here we have applied ribosome profiling- which measures both global mRNA levels and their translation rates- to two species of Saccharomyces yeast and their interspecific hybrid in order to assess the relative contributions of changes in mRNA abundance and translation to regulatory evolution. We report that both cis and trans-acting regulatory divergence in translation are abundant, affecting at least 35% of genes. The majority of translational divergence acts to buffer changes in mRNA abundance, suggesting a widespread role for stabilizing selection acting across regulatory levels. Nevertheless, we observe evidence of lineage-specific selection acting on a number of yeast functional modules, including instances of reinforcing selection acting at both levels of regulation. Finally, we also uncover multiple instances of stop-codon readthrough that are conserved between species. Our analysis reveals the underappreciated complexity of post-transcriptional regulatory divergence and indicates that partitioning the search for the locus of selection into the binary categories of 'coding' vs. 'regulatory' may overlook a significant source of selection, acting at multiple regulatory levels along the path from genotype to phenotype.
[Show abstract][Hide abstract] ABSTRACT: Eukaryotic DNA replication follows a specific temporal program, with some genomic regions consistently replicating earlier than others, yet what determines this program is largely unknown. Highly transcribed regions have been observed to replicate in early S-phase in all plant and animal species studied to date, but this relationship is thought to be absent from both budding yeast and fission yeast. No association between cell-cycle regulated transcription and replication timing has been reported for any species.
Here I show that in budding yeast, fission yeast, and human, the genes most highly transcribed during S-phase replicate early, whereas those repressed in S-phase replicate late. Transcription during other cell-cycle phases shows either the opposite correlation with replication timing, or no relation. The relationship is strongest near late-firing origins of replication, which is not consistent with a previously proposed model---that replication timing may affect transcription---and instead suggests a potential mechanism involving the recruitment of limiting replication initiation factors during S-phase.
These results suggest that S-phase transcription may be an important determinant of DNA replication timing across eukaryotes, which may explain the well-established association between transcription and replication timing.
[Show abstract][Hide abstract] ABSTRACT: Despite recent advances in our ability to detect adaptive evolution involving the cis-regulation of gene expression, our knowledge of the molecular mechanisms underlying these adaptations has lagged far behind. Across all model organisms, the causal mutations have been discovered for only a handful of gene expression adaptations, and even for these, mechanistic details (e.g. the trans-regulatory factors involved) have not been determined. We previously reported a polygenic gene expression adaptation involving down-regulation of the ergosterol biosynthesis pathway in the budding yeast Saccharomyces cerevisiae. Here we investigate the molecular mechanism of a cis-acting mutation affecting a member of this pathway, ERG28. We show that the causal mutation is a two-base deletion in the promoter of ERG28 that strongly reduces the binding of two transcription factors, Sok2 and Mot3, thus abolishing their regulation of ERG28. This down-regulation increases resistance to a widely used antifungal drug targeting ergosterol, similar to mutations disrupting this pathway in clinical yeast isolates. The identification of the causal genetic variant revealed that the selection likely occurred after the deletion was already present at high frequency in the population, rather than when it was a new mutation. These results provide a detailed view of the molecular mechanism of a cis-regulatory adaptation, and underscore the importance of this view to our understanding of evolution at the molecular level.
[Show abstract][Hide abstract] ABSTRACT: Measuring natural selection on genomic elements involved in the cis-regulation of gene expression-such as transcriptional enhancers and promoters-is critical for understanding the evolution of genomes, yet it remains a major challenge. Many studies have attempted to detect positive or negative selection in these noncoding elements by searching for those with the fastest or slowest rates of evolution, but this can be problematic. Here we introduce a new approach to this issue, and demonstrate its utility on three mammalian transcriptional enhancers. Using results from saturation mutagenesis studies of these enhancers, we classified all possible point mutations as up-regulating, down-regulating, or silent, and determined which of these mutations have occurred on each branch of a phylogeny. Applying a framework analogous to Ka/Ks in protein-coding genes, we measured the strength of selection on up-regulating and down-regulating mutations, in specific branches as well as entire phylogenies. We discovered distinct modes of selection acting on different enhancers: while all three have experienced negative selection against down-regulating mutations, the selection pressures on up-regulating mutations vary. In one case we detected positive selection for up-regulation, while the other two had no detectable selection on up-regulating mutations. Our methodology is applicable to the growing number of saturation mutagenesis data sets, and provides a detailed picture of the mode and strength of natural selection acting on cis-regulatory elements.
[Show abstract][Hide abstract] ABSTRACT: The order of genes along metazoan chromosomes has generally been thought to be largely random, with few implications for organismal function. However, two recent studies, reporting hundreds of pairs of genes that have remained linked in diverse metazoan species over hundreds of millions of years of evolution, suggest widespread functional implications for gene order. These associations appear to largely reflect cis-regulatory constraints, with either (i) multiple genes sharing transcriptional regulatory elements, or (ii) regulatory elements for a developmental gene being found within a neighboring 'bystander' gene (known as a genomic regulatory block). We discuss implications, questions raised, and new research directions arising from these studies, as well as evidence for similar phenomena in other eukaryotic groups.
No preview · Article · Jun 2013 · Trends in Genetics
[Show abstract][Hide abstract] ABSTRACT: The molecular basis of adaptation, and in particular the relative roles of protein-coding vs. gene expression changes, has long been the subject of speculation and debate. Recently, the genotyping of diverse human populations has led to the identification of many putative "local adaptations" that differ between populations. Here I show that these local adaptations are over 10-fold more likely to affect gene expression than amino acid sequence. In addition, a novel framework for identifying polygenic local adaptations detects recent positive selection on the expression levels of genes involved in UV radiation response, immune cell proliferation, and diabetes-related pathways. These results provide the first examples of polygenic gene expression adaptation in humans, as well as the first genome-scale support for the hypothesis that changes in gene expression have driven human adaptation.
[Show abstract][Hide abstract] ABSTRACT: The time required to transcribe genes with long primary transcripts may limit their ability to be expressed in cells with
short mitotic cycles, a phenomenon termed intron delay. As such short cycles are a hallmark of the earliest stages of insect
development, we tested the impact of intron delay on the Drosophila developmental transcriptome. We find that long zygotically expressed genes show substantial delay in expression relative
to their shorter counterparts, which is not observed for maternally deposited transcripts. Patterns of RNA-seq coverage along
transcripts show that this delay is consistent with their inability to completely transcribe long transcripts, but not with
transcriptional initiation-based regulatory control. We further show that highly expressed zygotic genes maintain compact
transcribed regions across the Drosophila phylogeny, allowing conservation of embryonic expression patterns. We propose that the physical constraints of intron delay
affect patterns of expression and the evolution of gene structure of a substantial portion of the Drosophila transcriptome.
Preview · Article · Jan 2013 · Molecular Biology and Evolution
Ana Ariza-Cosano · Axel Visel · Len A Pennacchio · Hunter B Fraser · José Luis Gómez-Skarmeta · Manuel Irimia · José Bessa
[Show abstract][Hide abstract] ABSTRACT: In situ hybridization performed in 24hpf zebrafish embryos for gsx2 and znf423 genes. A) gsx2 expression is detected in hindbrain and forebrain at 24hpf and 48hpf (B). C) At 22hpf znf423 gene is expressed in the forebrain, hindbrain, eye and spinal cord. D) At 48hpf znf423 gene is detected in the forebrain, midbrain, hindbrain and eye.
[Show abstract][Hide abstract] ABSTRACT: Enhancer activity of CNEs absent from the lineage of teleost fishes in mice and in zebrafish. A and B) Expression of Hs240 is shared by zebrafish and mice in the forebrain, being singularly expressed in the zebrafish hindbrain. C and D) The Hs426 enhancer shows similar expression in mice and zebrafish (otic vesicle, forebrain and hindbrain). E and F) A species specific expression of the Hs312 enhancer is observed in the hindbrain and midbrain of mice (E) being shared by zebrafish (F) in the spinalcord, limbs and forebrain. G and H) The expression of the H752 enhancer is shared by mice and zebrafish in muscle being mice specific for the dorsal root ganglia, trigeminal ganglion and spinal cord.
[Show abstract][Hide abstract] ABSTRACT: Background
Phenotypic evolution in animals is thought to be driven in large part by differences in gene expression patterns, which can result from sequence changes in cis-regulatory elements (cis-changes) or from changes in the expression pattern or function of transcription factors (trans-changes). While isolated examples of trans-changes have been identified, the scale of their overall contribution to regulatory and phenotypic evolution remains unclear.
Here, we attempt to examine the prevalence of trans-effects and their potential impact on gene expression patterns in vertebrate evolution by comparing the function of identical human tissue-specific enhancer sequences in two highly divergent vertebrate model systems, mouse and zebrafish. Among 47 human conserved non-coding elements (CNEs) tested in transgenic mouse embryos and in stable zebrafish lines, at least one species-specific expression domain was observed in the majority (83%) of cases, and 36% presented dramatically different expression patterns between the two species. Although some of these discrepancies may be due to the use of different transgenesis systems in mouse and zebrafish, in some instances we found an association between differences in enhancer activity and changes in the endogenous gene expression patterns between mouse and zebrafish, suggesting a potential role for trans-changes in the evolution of gene expression.
In total, our results: (i) serve as a cautionary tale for studies investigating the role of human enhancers in different model organisms, and (ii) suggest that changes in the trans environment may play a significant role in the evolution of gene expression in vertebrates.
[Show abstract][Hide abstract] ABSTRACT: Epigenetics is emerging as an attractive mechanism to explain the persistent genomic embedding of early-life experiences. Tightly linked to chromatin, which packages DNA into chromosomes, epigenetic marks primarily serve to regulate the activity of genes. DNA methylation is the most accessible and characterized component of the many chromatin marks that constitute the epigenome, making it an ideal target for epigenetic studies in human populations. Here, using peripheral blood mononuclear cells collected from a community-based cohort stratified for early-life socioeconomic status, we measured DNA methylation in the promoter regions of more than 14,000 human genes. Using this approach, we broadly assessed and characterized epigenetic variation, identified some of the factors that sculpt the epigenome, and determined its functional relation to gene expression. We found that the leukocyte composition of peripheral blood covaried with patterns of DNA methylation at many sites, as did demographic factors, such as sex, age, and ethnicity. Furthermore, psychosocial factors, such as perceived stress, and cortisol output were associated with DNA methylation, as was early-life socioeconomic status. Interestingly, we determined that DNA methylation was strongly correlated to the ex vivo inflammatory response of peripheral blood mononuclear cells to stimulation with microbial products that engage Toll-like receptors. In contrast, our work found limited effects of DNA methylation marks on the expression of associated genes across individuals, suggesting a more complex relationship than anticipated.
Preview · Article · Oct 2012 · Proceedings of the National Academy of Sciences