From the cauldron of conflict: Endogenous gene regulation by piRNA and other modes of adaptation enabled by selfish transposable elements.

Transposable elements (TEs) provide a prime example of genetic conflict because they can proliferate in genomes and populations even if they harm the host. However, numerous studies have shown that TEs, though typically harmful, can also provide fuel for adaptation. This is because they code functional sequences that can be useful for the host in which they reside. In this review, I summarize the "how" and "why" of adaptation enabled by the genetic conflict between TEs and hosts. In addition, focusing on mechanisms of TE control by small piwi-interacting RNAs (piRNAs), I highlight an indirect form of adaptation enabled by conflict. In this case, mechanisms of host defense that regulate TEs have been redeployed for endogenous gene regulation. I propose that the genetic conflict released by meiosis in early eukaryotes may have been important because, among other reasons, it spurred evolutionary innovation on multiple interwoven trajectories - on the part of hosts and also embedded genetic parasites. This form of evolution may function as a complexity generating engine that was a critical player in eukaryotic evolution.

Off-target piRNA gene silencing in Drosophila melanogaster rescued by a transposable element insertion.

Transposable elements (TE) are selfish genetic elements that can cause harmful mutations. In Drosophila, it has been estimated that half of all spontaneous visible marker phenotypes are mutations caused by TE insertions. Several factors likely limit the accumulation of exponentially amplifying TEs within genomes. First, synergistic interactions between TEs that amplify their harm with increasing copy number are proposed to limit TE copy number. However, the nature of this synergy is poorly understood. Second, because of the harm posed by TEs, eukaryotes have evolved systems of small RNA-based genome defense to limit transposition. However, as in all immune systems, there is a cost of autoimmunity and small RNA-based systems that silence TEs can inadvertently silence genes flanking TE insertions. In a screen for essential meiotic genes in Drosophila melanogaster, a truncated Doc retrotransposon within a neighboring gene was found to trigger the germline silencing of ald, the Drosophila Mps1 homolog, a gene essential for proper chromosome segregation in meiosis. A subsequent screen for suppressors of this silencing identified a new insertion of a Hobo DNA transposon in the same neighboring gene. Here we describe how the original Doc insertion triggers flanking piRNA biogenesis and local gene silencing. We show that this local gene silencing occurs in cis and is dependent on deadlock, a component of the Rhino-Deadlock-Cutoff (RDC) complex, to trigger dual-strand piRNA biogenesis at TE insertions. We further show how the additional Hobo insertion leads to de-silencing by reducing flanking piRNA biogenesis triggered by the original Doc insertion. These results support a model of TE-mediated gene silencing by piRNA biogenesis in cis that depends on local determinants of transcription. This may explain complex patterns of off-target gene silencing triggered by TEs within populations and in the laboratory. It also provides a mechanism of sign epistasis among TE insertions, illuminates the complex nature of their interactions and supports a model in which off-target gene silencing shapes the evolution of the RDC complex.

Paramutation-like Epigenetic Conversion by piRNA at the Telomere of Drosophila virilis.

First discovered in maize, paramutation is a phenomenon in which one allele can trigger an epigenetic conversion of an alternate allele. This conversion causes a genetically heterozygous individual to transmit alleles that are functionally the same, in apparent violation of Mendelian segregation. Studies over the past several decades have revealed a strong connection between mechanisms of genome defense against transposable elements by small RNA and the phenomenon of paramutation. For example, a system of paramutation in Drosophila melanogaster has been shown to be mediated by piRNAs, whose primary function is to silence transposable elements in the germline. In this paper, we characterize a second system of piRNA-mediated paramutation-like behavior at the telomere of Drosophila virilis. In Drosophila, telomeres are maintained by arrays of retrotransposons that are regulated by piRNAs. As a result, the telomere and sub-telomeric regions of the chromosome have unique regulatory and chromatin properties. Previous studies have shown that maternally deposited piRNAs derived from a sub-telomeric piRNA cluster can silence the sub-telomeric center divider gene of Drosophila virilis in trans. In this paper, we show that this silencing can also be maintained in the absence of the original silencing allele in a subsequent generation. The precise mechanism of this paramutation-like behavior may be explained by either the production of retrotransposon piRNAs that differ across strains or structural differences in the telomere. Altogether, these results show that the capacity for piRNAs to mediate paramutation in trans may depend on the local chromatin environment and proximity to the uniquely structured telomere regulated by piRNAs. This system promises to provide significant insights into the mechanisms of paramutation.

Evolutionary Dynamics of the Pericentromeric Heterochromatin in Drosophila virilis and Related Species.

Pericentromeric heterochromatin in Drosophila generally consists of repetitive DNA, forming the environment associated with gene silencing. Despite the expanding knowledge of the impact of transposable elements (TEs) on the host genome, little is known about the evolution of pericentromeric heterochromatin, its structural composition, and age. During the evolution of the Drosophilidae, hundreds of genes have become embedded within pericentromeric regions yet retained activity. We investigated a pericentromeric heterochromatin fragment found in D. virilis and related species, describing the evolution of genes in this region and the age of TE invasion. Regardless of the heterochromatic environment, the amino acid composition of the genes is under purifying selection. However, the selective pressure affects parts of genes in varying degrees, resulting in expansion of gene introns due to TEs invasion. According to the divergence of TEs, the pericentromeric heterochromatin of the species of virilis group began to form more than 20 million years ago by invasions of retroelements, miniature inverted repeat transposable elements (MITEs), and Helitrons. Importantly, invasions into the heterochromatin continue to occur by TEs that fall under the scope of piRNA silencing. Thus, the pericentromeric heterochromatin, in spite of its ability to induce silencing, has the means for being dynamic, incorporating the regions of active transcription.

Taming the Turmoil Within: New Insights on the Containment of Transposable Elements.

Transposable elements (TEs) are mobile genetic parasites that can exponentially increase their genomic abundance through self-propagation. Classic theoretical papers highlighted the importance of two potentially escalating forces that oppose TE spread: regulated transposition and purifying selection. Here, we review new insights into mechanisms of TE regulation and purifying selection, which reveal the remarkable foresight of these theoretical models. We further highlight emergent connections between transcriptional control enacted by small RNAs and the contribution of TE insertions to structural mutation and host-gene regulation. Finally, we call for increased comparative analysis of TE dynamics and fitness effects, as well as host control mechanisms, to reveal how interconnected forces shape the differential prevalence and distribution of TEs across the tree of life.

Hybrid dysgenesis in Drosophila virilis results in clusters of mitotic recombination and loss-of-heterozygosity but leaves meiotic recombination unaltered.

BACKGROUND: Transposable elements (TEs) are endogenous mutagens and their harmful effects are especially evident in syndromes of hybrid dysgenesis. In Drosophila virilis, hybrid dysgenesis is a syndrome of incomplete gonadal atrophy that occurs when males with multiple active TE families fertilize females that lack active copies of the same families. This has been demonstrated to cause the transposition of paternally inherited TE families, with gonadal atrophy driven by the death of germline stem cells. Because there are abundant, active TEs in the male inducer genome, that are not present in the female reactive genome, the D. virilis syndrome serves as an excellent model for understanding the effects of hybridization between individuals with asymmetric TE profiles. RESULTS: Using the D. virilis syndrome of hybrid dysgenesis as a model, we sought to determine how the landscape of germline recombination is affected by parental TE asymmetry. Using a genotyping-by-sequencing approach, we generated a high-resolution genetic map of D. virilis and show that recombination rate and TE density are negatively correlated in this species. We then contrast recombination events in the germline of dysgenic versus non-dysgenic F1 females to show that the landscape of meiotic recombination is hardly perturbed during hybrid dysgenesis. In contrast, hybrid dysgenesis in the female germline increases transmission of chromosomes with mitotic recombination. Using a de novo PacBio assembly of the D. virilis inducer genome we show that clusters of mitotic recombination events in dysgenic females are associated with genomic regions with transposons implicated in hybrid dysgenesis. CONCLUSIONS: Overall, we conclude that increased mitotic recombination is likely the result of early TE activation in dysgenic progeny, but a stable landscape of meiotic recombination indicates that either transposition is ameliorated in the adult female germline or that regulation of meiotic recombination is robust to ongoing transposition. These results indicate that the effects of parental TE asymmetry on recombination are likely sensitive to the timing of transposition.

Birth, School, Work, Death, and Resurrection: The Life Stages and Dynamics of Transposable Element Proliferation.

Transposable elements (TEs) can be maintained in sexually reproducing species even if they are harmful. However, the evolutionary strategies that TEs employ during proliferation can modulate their impact. In this review, I outline the different life stages of a TE lineage, from birth to proliferation to extinction. Through their interactions with the host, TEs can exploit diverse strategies that range from long-term coexistence to recurrent movement across species boundaries by horizontal transfer. TEs can also engage in a poorly understood phenomenon of TE resurrection, where TE lineages can apparently go extinct, only to proliferate again. By determining how this is possible, we may obtain new insights into the evolutionary dynamics of TEs and how they shape the genomes of their hosts.

Aging in the Drosophila ovary: contrasting changes in the expression of the piRNA machinery and mitochondria but no global release of transposable elements.

BACKGROUND: Evolutionary theory indicates that the dynamics of aging in the soma and reproductive tissues may be distinct. This difference arises from the fact that only the germline lineage establishes future generations. In the soma, changes in the landscape of heterochromatin have been proposed to have an important role in aging. This is because redistribution of heterochromatin during aging has been linked to the derepression of transposable elements and an overall loss of somatic gene regulation. A role for changes in the chromatin landscape in the aging of reproductive tissues is less well established. Whether or not epigenetic factors, such as heterochromatin marks, are perturbed in aging reproductive tissues is of interest because, in special cases, epigenetic variation may be heritable. Using mRNA sequencing data from late-stage egg chambers in Drosophila melanogaster, we characterized the landscape of altered gene and transposable element expression in aged reproductive tissues. This allowed us to test the hypothesis that reproductive tissues may differ from somatic tissues in their response to aging. RESULTS: We show that age-related expression changes in late-stage egg chambers tend to occur in genes residing in heterochromatin, particularly on the largely heterochromatic 4th chromosome. However, these expression differences are seen as both decreases and increases during aging, inconsistent with a general loss of heterochromatic silencing. We also identify an increase in expression of the piRNA machinery, suggesting an age-related increased investment in the maintenance of genome stability. We further identify a strong age-related reduction in the expression of mitochondrial transcripts. However, we find no evidence for global TE derepression in reproductive tissues. Rather, the observed effects of aging on TEs are primarily strain and family specific. CONCLUSIONS: These results identify unique responses in somatic versus reproductive tissue with regards to aging. As in somatic tissues, female reproductive tissues show reduced expression of mitochondrial genes. In contrast, the piRNA machinery shows increased expression during aging. Overall, these results also indicate that global loss of TE control observed in other studies may be unique to the soma and sensitive to genetic background and TE family.

Invasion of the P elements: Tolerance is not futile.

Organisms are locked in an eternal struggle with parasitic DNA sequences that live inside their genomes and wreak havoc on their host's chromosomes as they spread through populations. To combat these parasites, host species have evolved elaborate mechanisms of resistance that suppress their activity. A new study in Drosophila indicates that, prior to the acquisition of resistance, individuals can vary in their ability to tolerate the activity of these genomic parasites, ignoring or repairing the damage they induce. This tolerance results from variation at genes involved in germline development and DNA damage checkpoints and suggests that these highly conserved cellular processes may be influenced by current and historical intragenomic parasite loads.

RNAi Doxxes Segregation Distorters on the X.

Species with chromosomal sex determination are susceptible to an evolutionary tug-of-war over sex chromosome segregation. RNA silencing has been proposed to play a role in this intragenomic conflict. Reporting in Developmental Cell, Lin et al. (2018) demonstrate that RNA interference is key to this conflict as a genome defender.

Spontaneous gain of susceptibility suggests a novel mechanism of resistance to hybrid dysgenesis in Drosophila virilis.

Syndromes of hybrid dysgenesis (HD) have been critical for our understanding of the transgenerational maintenance of genome stability by piRNA. HD in D. virilis represents a special case of HD since it includes simultaneous mobilization of a set of TEs that belong to different classes. The standard explanation for HD is that eggs of the responder strains lack an abundant pool of piRNAs corresponding to the asymmetric TE families transmitted solely by sperm. However, there are several strains of D. virilis that lack asymmetric TEs, but exhibit a "neutral" cytotype that confers resistance to HD. To characterize the mechanism of resistance to HD, we performed a comparative analysis of the landscape of ovarian small RNAs in strains that vary in their resistance to HD mediated sterility. We demonstrate that resistance to HD cannot be solely explained by a maternal piRNA pool that matches the assemblage of TEs that likely cause HD. In support of this, we have witnessed a cytotype shift from neutral (N) to susceptible (M) in a strain devoid of all major TEs implicated in HD. This shift occurred in the absence of significant change in TE copy number and expression of piRNAs homologous to asymmetric TEs. Instead, this shift is associated with a change in the chromatin profile of repeat sequences unlikely to be causative of paternal induction. Overall, our data suggest that resistance to TE-mediated sterility during HD may be achieved by mechanisms that are distinct from the canonical syndromes of HD.

Horizontal Transfer Can Drive a Greater Transposable Element Load in Large Populations.

Genomes are comprised of contrasting domains of euchromatin and heterochromatin, and transposable elements (TEs) play an important role in defining these genomic regions. Therefore, understanding the forces that control TE abundance can help us understand the chromatin landscape of the genome. What determines the burden of TEs in populations? Some have proposed that drift plays a determining role. In small populations, mildly deleterious TE insertion alleles are allowed to fix, leading to increased copy number. However, it is not clear how the rate of exposure to new TE families, via horizontal transfer (HT), can contribute to broader patterns of genomic TE abundance. Here, using simulation and analytical approaches, we show that when the effects of drift are weak, exposure rate to new TE families via HT can be an important determinant of genomic copy number. If population exposure rate is proportional to population size, larger populations are expected to have a higher rate of exposure to rare HT events. This leads to the counterintuitive prediction that larger populations may carry a higher TE load. We also find that increased rates of recombination can lead to greater probabilities of TE establishment. This work has implications for our understanding of the evolution of chromatin landscapes, genome defense by RNA silencing, and recombination rates.

What Drives Positive Selection in the Drosophila piRNA Machinery? The Genomic Autoimmunity Hypothesis.

In animals, PIWI-interacting RNAs (piRNAs) play a crucial role in genome defense. Moreover, because piRNAs can be maternally transmitted, they contribute to the epigenetic profile of inheritance. Multiple studies, especially in Drosophila, have demonstrated that the machinery of piRNA biogenesis is often the target of positive selection. Because transposable elements (TEs) are a form of genetic parasite, positive selection in the piRNA machinery is often explained by analogy to the signatures of positive selection commonly observed in genes that play a role in host-parasite dynamics. However, the precise mechanisms that drive positive selection in the piRNA machinery are not known. In this review, we outline several mechanistic models that might explain pervasive positive selection in the piRNA machinery of Drosophila species. We propose that recurrent positive selection in the piRNA machinery can be partly explained by an ongoing tension between selection for sensitivity required by genome defense and selection for specificity to avoid the off-target effects of maladaptive genic silencing by piRNA.

Holding it together: rapid evolution and positive selection in the synaptonemal complex of Drosophila.

BACKGROUND: The synaptonemal complex (SC) is a highly conserved meiotic structure that functions to pair homologs and facilitate meiotic recombination in most eukaryotes. Five Drosophila SC proteins have been identified and localized within the complex: C(3)G, C(2)M, CONA, ORD, and the newly identified Corolla. The SC is required for meiotic recombination in Drosophila and absence of these proteins leads to reduced crossing over and chromosomal nondisjunction. Despite the conserved nature of the SC and the key role that these five proteins have in meiosis in D. melanogaster, they display little apparent sequence conservation outside the genus. To identify factors that explain this lack of apparent conservation, we performed a molecular evolutionary analysis of these genes across the Drosophila genus. RESULTS: For the five SC components, gene sequence similarity declines rapidly with increasing phylogenetic distance and only ORD and C(2)M are identifiable outside of the Drosophila genus. SC gene sequences have a higher dN/dS (ω) rate ratio than the genome wide average and this can in part be explained by the action of positive selection in almost every SC component. Across the genus, there is significant variation in ω for each protein. It further appears that ω estimates for the five SC components are in accordance with their physical position within the SC. Components interacting with chromatin evolve slowest and components comprising the central elements evolve the most rapidly. Finally, using population genetic approaches, we demonstrate that positive selection on SC components is ongoing. CONCLUSIONS: SC components within Drosophila show little apparent sequence homology to those identified in other model organisms due to their rapid evolution. We propose that the Drosophila SC is evolving rapidly due to two combined effects. First, we propose that a high rate of evolution can be partly explained by low purifying selection on protein components whose function is to simply hold chromosomes together. We also propose that positive selection in the SC is driven by its sex-specificity combined with its role in facilitating both recombination and centromere clustering in the face of recurrent bouts of drive in female meiosis.

Whole-Genome Analysis of Individual Meiotic Events in Drosophila melanogaster Reveals That Noncrossover Gene Conversions Are Insensitive to Interference and the Centromere Effect.

A century of genetic analysis has revealed that multiple mechanisms control the distribution of meiotic crossover events. In Drosophila melanogaster, two significant positional controls are interference and the strongly polar centromere effect. Here, we assess the factors controlling the distribution of crossovers (COs) and noncrossover gene conversions (NCOs) along all five major chromosome arms in 196 single meiotic divisions to generate a more detailed understanding of these controls on a genome-wide scale. Analyzing the outcomes of single meiotic events allows us to distinguish among different classes of meiotic recombination. In so doing, we identified 291 NCOs spread uniformly among the five major chromosome arms and 541 COs (including 52 double crossovers and one triple crossover). We find that unlike COs, NCOs are insensitive to the centromere effect and do not demonstrate interference. Although the positions of COs appear to be determined predominately by the long-range influences of interference and the centromere effect, each chromosome may display a different pattern of sensitivity to interference, suggesting that interference may not be a uniform global property. In addition, unbiased sequencing of a large number of individuals allows us to describe the formation of de novo copy number variants, the majority of which appear to be mediated by unequal crossing over between transposable elements. This work has multiple implications for our understanding of how meiotic recombination is regulated to ensure proper chromosome segregation and maintain genome stability.

piRNAs Are Associated with Diverse Transgenerational Effects on Gene and Transposon Expression in a Hybrid Dysgenic Syndrome of D. virilis.

Sexual reproduction allows transposable elements (TEs) to proliferate, leading to rapid divergence between populations and species. A significant outcome of divergence in the TE landscape is evident in hybrid dysgenic syndromes, a strong form of genomic incompatibility that can arise when (TE) family abundance differs between two parents. When TEs inherited from the father are absent in the mother's genome, TEs can become activated in the progeny, causing germline damage and sterility. Studies in Drosophila indicate that dysgenesis can occur when TEs inherited paternally are not matched with a pool of corresponding TE silencing PIWI-interacting RNAs (piRNAs) provisioned by the female germline. Using the D. virilis syndrome of hybrid dysgenesis as a model, we characterize the effects that divergence in TE profile between parents has on offspring. Overall, we show that divergence in the TE landscape is associated with persisting differences in germline TE expression when comparing genetically identical females of reciprocal crosses and these differences are transmitted to the next generation. Moreover, chronic and persisting TE expression coincides with increased levels of genic piRNAs associated with reduced gene expression. Combined with these effects, we further demonstrate that gene expression is idiosyncratically influenced by differences in the genic piRNA profile of the parents that arise though polymorphic TE insertions. Overall, these results support a model in which early germline events in dysgenesis establish a chronic, stable state of both TE and gene expression in the germline that is maintained through adulthood and transmitted to the next generation. This work demonstrates that divergence in the TE profile is associated with diverse piRNA-mediated transgenerational effects on gene expression within populations.

Corolla is a novel protein that contributes to the architecture of the synaptonemal complex of Drosophila.

In most organisms the synaptonemal complex (SC) connects paired homologs along their entire length during much of meiotic prophase. To better understand the structure of the SC, we aim to identify its components and to determine how each of these components contributes to SC function. Here, we report the identification of a novel SC component in Drosophila melanogaster female oocytes, which we have named Corolla. Using structured illumination microscopy, we demonstrate that Corolla is a component of the central region of the SC. Consistent with its localization, we show by yeast two-hybrid analysis that Corolla strongly interacts with Cona, a central element protein, demonstrating the first direct interaction between two inner-synaptonemal complex proteins in Drosophila. These observations help provide a more complete model of SC structure and function in Drosophila females.

Whole genome sequencing in Drosophila virilis identifies Polyphemus, a recently activated Tc1-like transposon with a possible role in hybrid dysgenesis.

BACKGROUND: Hybrid dysgenic syndromes in Drosophila have been critical for characterizing host mechanisms of transposable element (TE) regulation. This is because a common feature of hybrid dysgenesis is germline TE mobilization that occurs when paternally inherited TEs are not matched with a maternal pool of silencing RNAs that maintain transgenerational TE control. In the face of this imbalance TEs become activated in the germline and can cause F1 sterility. The syndrome of hybrid dysgenesis in Drosophila virilis was the first to show that the mobilization of one dominant TE, the Penelope retrotransposon, may lead to the mobilization of other unrelated elements. However, it is not known how many different elements contribute and no exhaustive search has been performed to identify additional ones. To identify additional TEs that may contribute to hybrid dysgenesis in Drosophila virilis, I analyzed repeat content in genome sequences of inducer and non-inducer lines. RESULTS: Here I describe Polyphemus, a novel Tc1-like DNA transposon, which is abundant in the inducer strain of D. virilis but highly degraded in the non-inducer strain. Polyphemus expression is also increased in the germline of progeny of the dysgenic cross relative to reciprocal progeny. Interestingly, like the Penelope element, it has experienced recent re-activation within the D. virilis lineage. CONCLUSIONS: Here I present the results of a comprehensive search to identify additional factors that may cause hybrid dysgenesis in D. virilis. Polyphemus, a novel Tc1-like DNA transposon, has recently become re-activated in Drosophila virilis and likely contributes to the hybrid dysgenesis syndrome. It has been previously shown that the Penelope element has also been re-activated in the inducer strain. This suggests that TE co-reactivation within species may synergistically contribute to syndromes of hybrid dysgenesis.

An age-of-allele test of neutrality for transposable element insertions.

How natural selection acts to limit the proliferation of transposable elements (TEs) in genomes has been of interest to evolutionary biologists for many years. To describe TE dynamics in populations, previous studies have used models of transposition-selection equilibrium that assume a constant rate of transposition. However, since TE invasions are known to happen in bursts through time, this assumption may not be reasonable. Here we propose a test of neutrality for TE insertions that does not rely on the assumption of a constant transposition rate. We consider the case of TE insertions that have been ascertained from a single haploid reference genome sequence. By conditioning on the age of an individual TE insertion allele (inferred by the number of unique substitutions that have occurred within the particular TE sequence since insertion), we determine the probability distribution of the insertion allele frequency in a population sample under neutrality. Taking models of varying population size into account, we then evaluate predictions of our model against allele frequency data from 190 retrotransposon insertions sampled from North American and African populations of Drosophila melanogaster. Using this nonequilibrium neutral model, we are able to explain ∼ 80% of the variance in TE insertion allele frequencies based on age alone. Controlling for both nonequilibrium dynamics of transposition and host demography, we provide evidence for negative selection acting against most TEs as well as for positive selection acting on a small subset of TEs. Our work establishes a new framework for the analysis of the evolutionary forces governing large insertion mutations like TEs, gene duplications, or other copy number variants.

Terminator oligo blocking efficiently eliminates rRNA from Drosophila small RNA sequencing libraries.

A large number of methods are available to deplete ribosomal RNA reads from high-throughput RNA sequencing experiments. Such methods are critical for sequencing Drosophila small RNAs between 20 and 30 nucleotides because size selection is not typically sufficient to exclude the highly abundant class of 30 nucleotide 2S rRNA. Here we demonstrate that pre-annealing terminator oligos complimentary to Drosophila 2S rRNA prior to 5' adapter ligation and reverse transcription efficiently depletes 2S rRNA sequences from the sequencing reaction in a simple and inexpensive way. This depletion is highly specific and is achieved with minimal perturbation of miRNA and piRNA profiles.