Evaluation of Hi-C sequencing for the detection of gene fusions in hematologic and solid pediatric cancer samples.

HiC sequencing is a DNA-based next-generation sequencing method that preserves the 3D conformation of the genome and has shown promise in detecting genomic rearrangements in translational research studies. To evaluate HiC as a potential clinical diagnostic platform, analytical concordance with routine laboratory testing was assessed using primary pediatric leukemia and sarcoma specimens previously positive for clinically significant genomic rearrangements. Archived specimen types tested included viable and nonviable frozen leukemic cells, as well as formalin-fixed paraffin-embedded (FFPE) tumor tissues. Initially, pediatric acute myeloid leukemia (AML) and alveolar rhabdomyosarcoma (A-RMS) specimens with known genomic rearrangements were subjected to HiC analysis to assess analytical concordance. Subsequently, a discovery cohort consisting of AML and acute lymphoblastic leukemia (ALL) cases with no known genomic rearrangements based on prior clinical diagnostic testing were evaluated to determine whether HiC could detect rearrangements. Using a standard sequencing depth of 50 million raw read-pairs per sample, or approximately 5X raw genomic coverage, 100% concordance was observed between HiC and previous clinical cytogenetic and molecular testing. In the discovery cohort, a clinically relevant gene fusion was detected in 45% of leukemia cases (5/11). This study demonstrates the value of HiC sequencing to medical diagnostic testing as it identified several clinically significant rearrangements, including those that might have been missed by current clinical testing workflows. Key points: HiC sequencing is a DNA-based next-generation sequencing method that preserves the 3D conformation of the genome, facilitating detection of genomic rearrangements.HiC was 100% concordant with clinical diagnostic testing workflows for detecting clinically significant genomic rearrangements in pediatric leukemia and rhabdomyosarcoma specimens.HiC detected clinically significant genomic rearrangements not previously detected by prior clinical cytogenetic and molecular testing.HiC performed well with archived non-viable and viable frozen leukemic cell samples, as well as archived formalin-fixed paraffin-embedded tumor tissue specimens.

3D genome mapping identifies subgroup-specific chromosome conformations and tumor-dependency genes in ependymoma.

Ependymoma is a tumor of the brain or spinal cord. The two most common and aggressive molecular groups of ependymoma are the supratentorial ZFTA-fusion associated and the posterior fossa ependymoma group A. In both groups, tumors occur mainly in young children and frequently recur after treatment. Although molecular mechanisms underlying these diseases have recently been uncovered, they remain difficult to target and innovative therapeutic approaches are urgently needed. Here, we use genome-wide chromosome conformation capture (Hi-C), complemented with CTCF and H3K27ac ChIP-seq, as well as gene expression and DNA methylation analysis in primary and relapsed ependymoma tumors, to identify chromosomal conformations and regulatory mechanisms associated with aberrant gene expression. In particular, we observe the formation of new topologically associating domains ('neo-TADs') caused by structural variants, group-specific 3D chromatin loops, and the replacement of CTCF insulators by DNA hyper-methylation. Through inhibition experiments, we validate that genes implicated by these 3D genome conformations are essential for the survival of patient-derived ependymoma models in a group-specific manner. Thus, this study extends our ability to reveal tumor-dependency genes by 3D genome conformations even in tumors that lack targetable genetic alterations.

Complex pleiotropic genetic architecture of evolved heat stress and oxidative stress resistance in the nematode Caenorhabditis remanei.

The adaptation of complex organisms to changing environments has been a central question in evolutionary quantitative genetics since its inception. The structure of the genotype-phenotype maps is critical because pleiotropic effects can generate widespread correlated responses to selection and potentially restrict the extent of evolutionary change. In this study, we use experimental evolution to dissect the genetic architecture of natural variation for acute heat stress and oxidative stress response in the nematode Caenorhabiditis remanei. Previous work in the classic model nematode Caenorhabiditis elegans has found that abiotic stress response is controlled by a handful of genes of major effect and that mutations in any one of these genes can have widespread pleiotropic effects on multiple stress response traits. Here, we find that acute heat stress response and acute oxidative response in C. remanei are polygenic, complex traits, with hundreds of genomic regions responding to selection. In contrast to expectation from mutation studies, we find that evolved acute heat stress and acute oxidative stress response for the most part display independent genetic bases. This lack of correlation is reflected at the levels of phenotype, gene expression, and in the genomic response to selection. Thus, while these findings support the general view that rapid adaptation can be generated by changes at hundreds to thousands of sites in the genome, the architecture of segregating variation is likely to be determined by the pleiotropic structure of the underlying genetic networks.

Immunogenetic and tolerance strategies against a novel parasitoid of wild field crickets.

Among the parasites of insects, endoparasitoids impose a costly challenge to host defenses because they use their host's body for the development and maturation of their eggs or larvae, and ultimately kill the host. Tachinid flies are highly specialized acoustically orienting parasitoids, with first instar mobile larvae that burrow into the host's body to feed. We investigated the possibility that Teleogryllus oceanicus field crickets employ postinfestation strategies to maximize survival when infested with the larvae of the parasitoid fly Ormia ochracea. Using crickets from the Hawaiian Islands of Kauai, where the parasitoid is present, and crickets from the Cook Islands (Mangaia), where the parasitoid is absent, we evaluated fitness consequences of infestation by comparing feeding behavior, reproductive capacity, and survival of males experimentally infested with O. ochracea larvae. We also evaluated mechanisms underlying host responses by comparing gene expression in crickets infested with fly larvae for different lengths of time with that of uninfested control crickets. We observed weak population differences in fitness (spermatophore production) and survival (total survival time postinfestation). These responses generally did not show an interaction between population and the number of larva hosts carried or by host body condition. Gene expression patterns also revealed population differences in response to infestation, but we did not find evidence for consistent differences in genes associated with immunity or stress response. One possibility is that any postinfestation evolved resistance does not involve genes associated with these particular functional categories. More likely, these results suggest that coevolution with the fly does not strongly select for either postinfestation resistance or tolerance of parasitoid larvae in male crickets.

Traffic influences nutritional quality of roadside plants for monarch caterpillars.

Roadside habitats are increasingly being targeted for restoration and conservation. Roadside habitats often exhibit altered soil and plant chemistry due to pollution from maintenance (e.g. de-icing salt), car deterioration, and exhaust. Roadside plants may attract animals due to elevated levels of sodium or nitrogen, but high concentrations of heavy metals and sodium can be toxic, potentially setting an ecological trap. In this study, we determine how roads influence the chemistry of common milkweed (Asclepias syriaca) as it is the primary roadside host plant for the declining monarch butterfly (Danaus plexippus) in the eastern United States. Even though road salt is applied during the winter, we detect enhanced sodium along roads the following growing season. Road salts increase soil sodium, which in turn elevates host-plant foliar sodium (occasionally to toxic levels in <10% of plants) and sodium content in monarch caterpillars feeding on these plants. Sodium levels of milkweed leaves are highest close to the edge of busy roads. Some heavy metals (lead, zinc) are also elevated in roadside soils or plants. Nitrogen content was affected by adjacent agricultural use, but not traffic volume or proximity to a road. Other potential road pollutants (e.g. nickel) were not elevated in soil or plants. Despite a clear signature of road pollution in the chemistry of milkweed, most plants are likely still suitable for developing monarchs. Nonetheless, restoration investments in snowy regions should prioritize sites with lower-traffic density that are further from the road edge to minimize toxic impacts of high sodium. To extend this research to other insects of conservation concern, future work should characterize the nutritional quality of nectar, pollen, and other species of host-plants in roadside habitats.

Tolerance of Novel Toxins through Generalized Mechanisms: Simulating Gradual Host Shifts of Butterflies.

Organisms encounter a wide range of toxic compounds in their environments, from chemicals that serve anticonsumption or anticompetition functions to pollutants and pesticides. Although we understand many detoxification mechanisms that allow organisms to consume toxins typical of their diet, we know little about why organisms vary in their ability to tolerate entirely novel toxins. We tested whether variation in generalized stress responses, such as antioxidant pathways, may underlie variation in reactions to novel toxins and, if so, their associated costs. We used an artificial diet to present cabbage white butterfly caterpillars (Pieris rapae) with plant material containing toxins not experienced in their evolutionary history. Families that maintained high performance (e.g., high survival, fast development time, large body size) on diets containing one novel toxic plant also performed well when exposed to two other novel toxic plants, consistent with a generalized response. Variation in constitutive (but not induced) expression of genes involved in oxidative stress responses was positively related to performance on the novel diets. While we did not detect reproductive trade-offs of this generalized response, there was a tendency to have less melanin investment in the wings, consistent with the role of melanin in oxidative stress responses. Taken together, our results support the hypothesis that variation in generalized stress responses, such as genes involved in oxidative stress responses, may explain the variation in tolerance to entirely novel toxins and may facilitate colonization of novel hosts and environments.

Unprecedented reorganization of holocentric chromosomes provides insights into the enigma of lepidopteran chromosome evolution.

Chromosome evolution presents an enigma in the mega-diverse Lepidoptera. Most species exhibit constrained chromosome evolution with nearly identical haploid chromosome counts and chromosome-level gene collinearity among species more than 140 million years divergent. However, a few species possess radically inflated chromosomal counts due to extensive fission and fusion events. To address this enigma of constraint in the face of an exceptional ability to change, we investigated an unprecedented reorganization of the standard lepidopteran chromosome structure in the green-veined white butterfly (Pieris napi). We find that gene content in P. napi has been extensively rearranged in large collinear blocks, which until now have been masked by a haploid chromosome number close to the lepidopteran average. We observe that ancient chromosome ends have been maintained and collinear blocks are enriched for functionally related genes suggesting both a mechanism and a possible role for selection in determining the boundaries of these genome-wide rearrangements.

Environmental and Evolutionary Drivers of the Modular Gene Regulatory Network Underlying Phenotypic Plasticity for Stress Resistance in the Nematode Caenorhabditis remanei.

Organisms can cope with stressful environments via a combination of phenotypic plasticity at the individual level and adaptation at the population level. Changes in gene expression can play an important role in both. Significant advances in our understanding of gene regulatory plasticity and evolution have come from comparative studies in the field and laboratory. Experimental evolution provides another powerful path by which to learn about how differential regulation of genes and pathways contributes to both acclimation and adaptation. Here we present results from one such study using the nematode Caenorhabditis remanei We selected one set of lines to withstand heat stress and another oxidative stress. We then compared transcriptional responses to acute heat stress of both and an unselected control to the ancestral population using a weighted gene coexpression network analysis, finding that the transcriptional response is primarily dominated by a plastic response that is shared in the ancestor and all evolved populations. In addition, we identified several modules that respond to artificial selection by (1) changing the baseline level of expression, (2) altering the magnitude of the plastic response, or (3) a combination of the two. Our findings therefore reveal that while patterns of transcriptional response can be perturbed with short bouts of intense selection, the overall ancestral structure of transcriptional plasticity is largely maintained over time.

Genomic adaptation to agricultural environments: cabbage white butterflies (Pieris rapae) as a case study.

BACKGROUND: Agricultural environments have long presented an opportunity to study evolution in action, and genomic approaches are opening doors for testing hypotheses about adaptation to crops, pesticides, and fertilizers. Here, we begin to develop the cabbage white butterfly (Pieris rapae) as a system to test questions about adaptation to novel, agricultural environments. We focus on a population in the north central United States as a unique case study: here, canola, a host plant, has been grown during the entire flight period of the butterfly over the last three decades. RESULTS: First, we show that the agricultural population has diverged phenotypically relative to a nonagricultural population: when reared on a host plant distantly related to canola, the agricultural population is smaller and more likely to go into diapause than the nonagricultural population. Second, drawing from deep sequencing runs from six individuals from the agricultural population, we assembled the gut transcriptome of this population. Then, we sequenced RNA transcripts from the midguts of 96 individuals from this canola agricultural population and the nonagricultural population in order to describe patterns of genomic divergence between the two. While population divergence is low, 235 genes show evidence of significant differentiation between populations. These genes are significantly enriched for cofactor and small molecule metabolic processes, and many genes also have transporter or catalytic activity. Analyses of population structure suggest the agricultural population contains a subset of the genetic variation in the nonagricultural population. CONCLUSIONS: Taken together, our results suggest that adaptation of cabbage whites to an agricultural environment occurred at least in part through selection on standing genetic variation. Both the phenotypic and genetic data are consistent with the idea that this pest has adapted to an abundant and predictable agricultural resource through a narrowing of niche breadth and loss of genetic variants rather than de novo gain of adaptive alleles. The present research develops genomic resources to pave the way for future studies using cabbage whites as a model contributing to our understanding of adaptation to agricultural environments.

Plasticity paves the way in an adaptive radiation.

Phenotypic plasticity has been hypothesized to play a central role in the evolution of phenotypic diversity across species (West-Eberhard ). Through 'genetic assimilation', phenotypes that are initially environmentally induced within species become genetically fixed over evolutionary time. While genetic assimilation has been shown to occur in both the laboratory and the field (Waddington ; Aubret & Shine ), it remains to be shown whether it can account for broad patterns of phenotypic diversity across entire adaptive radiations. Furthermore, our ignorance of the underlying molecular mechanisms has hampered our ability to incorporate phenotypic plasticity into models of evolutionary processes. In this issue of Molecular Ecology, Parsons et al. () take a significant step in filling these conceptual gaps making use of cichlid fishes as a powerful study system. Cichlid jaw and skull morphology show adaptive, plastic changes in response to early dietary experiences (Fig. 1). In this research, Parsons et al. () first show that the direction of phenotypic plasticity aligns with the major axis of phenotypic divergence across species. They then dissect the underlying genetic architecture of this plasticity, showing that it is specific to the developmental environment and implicating the patched locus in genetic assimilation (i.e. a reduction in the environmental sensitivity of that locus in the derived species).

High-specificity detection of rare alleles with Paired-End Low Error Sequencing (PELE-Seq).

BACKGROUND: Polymorphic loci exist throughout the genomes of a population and provide the raw genetic material needed for a species to adapt to changes in the environment. The minor allele frequencies of rare Single Nucleotide Polymorphisms (SNPs) within a population have been difficult to track with Next-Generation Sequencing (NGS), due to the high error rate of standard methods such as Illumina sequencing. RESULTS: We have developed a wet-lab protocol and variant-calling method that identifies both sequencing and PCR errors, called Paired-End Low Error Sequencing (PELE-Seq). To test the specificity and sensitivity of the PELE-Seq method, we sequenced control E. coli DNA libraries containing known rare alleles present at frequencies ranging from 0.2-0.4 % of the total reads. PELE-Seq had higher specificity and sensitivity than standard libraries. We then used PELE-Seq to characterize rare alleles in a Caenorhabditis remanei nematode worm population before and after laboratory adaptation, and found that minor and rare alleles can undergo large changes in frequency during lab-adaptation. CONCLUSION: We have developed a method of rare allele detection that mitigates both sequencing and PCR errors, called PELE-Seq. PELE-Seq was evaluated using control E. coli populations and was then used to compare a wild C. remanei population to a lab-adapted population. The PELE-Seq method is ideal for investigating the dynamics of rare alleles in a broad range of reduced-representation sequencing methods, including targeted amplicon sequencing, RAD-Seq, ddRAD, and GBS. PELE-Seq is also well-suited for whole genome sequencing of mitochondria and viruses, and for high-throughput rare mutation screens.

Environmentally induced changes in correlated responses to selection reveal variable pleiotropy across a complex genetic network.

Selection in novel environments can lead to a coordinated evolutionary response across a suite of characters. Environmental conditions can also potentially induce changes in the genetic architecture of complex traits, which in turn could alter the pattern of the multivariate response to selection. We describe a factorial selection experiment using the nematode Caenorhabditis remanei in which two different stress-related phenotypes (heat and oxidative stress resistance) were selected under three different environmental conditions. The pattern of covariation in the evolutionary response between phenotypes or across environments differed depending on the environment in which selection occurred, including asymmetrical responses to selection in some cases. These results indicate that variation in pleiotropy across the stress response network is highly sensitive to the external environment. Our findings highlight the complexity of the interaction between genes and environment that influences the ability of organisms to acclimate to novel environments. They also make clear the need to identify the underlying genetic basis of genetic correlations in order understand how patterns of pleiotropy are distributed across complex genetic networks.

The transgenerational effects of heat stress in the nematode Caenorhabditis remanei are negative and rapidly eliminated under direct selection for increased stress resistance in larvae.

Parents encountering stress environments can influence the phenotype of their offspring in a form of transgenerational phenotypic plasticity that has the potential to be adaptive if offspring are thereby better able to deal with future stressors. Here, we test for the existence of anticipatory parental effects in the heat stress response in the highly polymorphic nematode Caenorhabditis remanei. Rather providing an anticipatory response, parents subject to a prior heat stress actually produce offspring that are less able to survive a severe heat shock. Selection on heat shock resistance within the larvae via experimental evolution leads to a loss of sensitivity (robustness) to environmental variation during both the parental and larval periods. Whole genome transcriptional analysis of both ancestor and selected lines shows that there is weak correspondence between genetic pathways induced via temperature shifts during parental and larval periods. Parental effects can evolve very rapidly via selection acting directly on offspring.

Rapid evolution of phenotypic plasticity and shifting thresholds of genetic assimilation in the nematode Caenorhabditis remanei.

Many organisms can acclimate to new environments through phenotypic plasticity, a complex trait that can be heritable, subject to selection, and evolve. However, the rate and genetic basis of plasticity evolution remain largely unknown. We experimentally evolved outbred populations of the nematode Caenorhabditis remanei under an acute heat shock during early larval development. When raised in a nonstressful environment, ancestral populations were highly sensitive to a 36.8° heat shock and exhibited high mortality. However, initial exposure to a nonlethal high temperature environment resulted in significantly reduced mortality during heat shock (hormesis). Lines selected for heat shock resistance rapidly evolved the capacity to withstand heat shock in the native environment without any initial exposure to high temperatures, and early exposure to high temperatures did not lead to further increases in heat resistance. This loss of plasticity would appear to have resulted from the genetic assimilation of the heat induction response in the noninducing environment. However, analyses of transcriptional variation via RNA-sequencing from the selected populations revealed no global changes in gene regulation correlated with the observed changes in heat stress resistance. Instead, assays of the phenotypic response across a broader range of temperatures revealed that the induced plasticity was not fixed across environments, but rather the threshold for the response was shifted to higher temperatures over evolutionary time. These results demonstrate that apparent genetic assimilation can result from shifting thresholds of induction across environments and that analysis of the broader environmental context is critically important for understanding the evolution of phenotypic plasticity.

An RNA-dependent RNA polymerase is required for paramutation in maize.

Paramutation is an allele-dependent transfer of epigenetic information, which results in the heritable silencing of one allele by another. Paramutation at the b1 locus in maize is mediated by unique tandem repeats that communicate in trans to establish and maintain meiotically heritable transcriptional silencing. The mop1 (mediator of paramutation1) gene is required for paramutation, and mop1 mutations reactivate silenced Mutator elements. Plants carrying mutations in the mop1 gene also stochastically exhibit pleiotropic developmental phenotypes. Here we report the map-based cloning of mop1, an RNA-dependent RNA polymerase gene (RDRP), most similar to the RDRP in plants that is associated with the production of short interfering RNA (siRNA) targeting chromatin. Nuclear run-on assays reveal that the tandem repeats required for b1 paramutation are transcribed from both strands, but siRNAs were not detected. We propose that the mop1 RDRP is required to maintain a threshold level of repeat RNA, which functions in trans to establish and maintain the heritable chromatin states associated with paramutation.