A Quantitative, Genome-Wide Analysis in Drosophila Reveals Transposable Elements' Influence on Gene Expression Is Species-Specific.

Transposable elements (TEs) are parasite DNA sequences that are able to move and multiply along the chromosomes of all genomes. They can be controlled by the host through the targeting of silencing epigenetic marks, which may affect the chromatin structure of neighboring sequences, including genes. In this study, we used transcriptomic and epigenomic high-throughput data produced from ovarian samples of several Drosophila melanogaster and Drosophila simulans wild-type strains, in order to finely quantify the influence of TE insertions on gene RNA levels and histone marks (H3K9me3 and H3K4me3). Our results reveal a stronger epigenetic effect of TEs on ortholog genes in D. simulans compared with D. melanogaster. At the same time, we uncover a larger contribution of TEs to gene H3K9me3 variance within genomes in D. melanogaster, which is evidenced by a stronger correlation of TE numbers around genes with the levels of this chromatin mark in D. melanogaster. Overall, this work contributes to the understanding of species-specific influence of TEs within genomes. It provides a new light on the considerable natural variability provided by TEs, which may be associated with contrasted adaptive and evolutionary potentials.

DrosOmics: A Browser to Explore -omics Variation Across High-Quality Reference Genomes From Natural Populations of Drosophila melanogaster.

The advent of long-read sequencing technologies has allowed the generation of multiple high-quality de novo genome assemblies for multiple species, including well-known model species such as Drosophila melanogaster. Genome assemblies for multiple individuals of the same species are key to discover the genetic diversity present in natural populations, especially the one generated by transposable elements, the most common type of structural variant. Despite the availability of multiple genomic data sets for D. melanogaster populations, we lack an efficient visual tool to display different genome assemblies simultaneously. In this work, we present DrosOmics, a population genomic-oriented browser currently containing 52 high-quality reference genomes of D. melanogaster, including annotations from a highly reliable set of transposable elements, and functional transcriptomics and epigenomics data for 26 genomes. DrosOmics is based on JBrowse 2, a highly scalable platform, which allows the visualization of multiple assemblies at once, key to unraveling structural and functional features of D. melanogaster natural populations. DrosOmics is an open access browser and is freely available at http://gonzalezlab.eu/drosomics.

Gene expression differences consistent with water loss reduction underlie desiccation tolerance of natural Drosophila populations.

BACKGROUND: Climate change is one of the main factors shaping the distribution and biodiversity of organisms, among others by greatly altering water availability, thus exposing species and ecosystems to harsh desiccation conditions. However, most of the studies so far have focused on the effects of increased temperature. Integrating transcriptomics and physiology is key to advancing our knowledge on how species cope with desiccation stress, and these studies are still best accomplished in model organisms. RESULTS: Here, we characterized the natural variation of European D. melanogaster populations across climate zones and found that strains from arid regions were similar or more tolerant to desiccation compared with strains from temperate regions. Tolerant and sensitive strains differed not only in their transcriptomic response to stress but also in their basal expression levels. We further showed that gene expression changes in tolerant strains correlated with their physiological response to desiccation stress and with their cuticular hydrocarbon composition, and functionally validated three of the candidate genes identified. Transposable elements, which are known to influence stress response across organisms, were not found to be enriched nearby differentially expressed genes. Finally, we identified several tRNA-derived small RNA fragments that differentially targeted genes in response to desiccation stress. CONCLUSIONS: Overall, our results showed that basal gene expression differences across individuals should be analyzed if we are to understand the genetic basis of differential stress survival. Moreover, tRNA-derived small RNA fragments appear to be relevant across stress responses and allow for the identification of stress-response genes not detected at the transcriptional level.

The genomic basis of copper tolerance in Drosophila is shaped by a complex interplay of regulatory and environmental factors.

BACKGROUND: Escalation in industrialization and anthropogenic activity have resulted in an increase of pollutants released into the environment. Of these pollutants, heavy metals such as copper are particularly concerning due to their bio-accumulative nature. Due to its highly heterogeneous distribution and its dual nature as an essential micronutrient and toxic element, the genetic basis of copper tolerance is likely shaped by a complex interplay of genetic and environmental factors. RESULTS: In this study, we utilized the natural variation present in multiple populations of Drosophila melanogaster collected across Europe to screen for variation in copper tolerance. We found that latitude and the degree of urbanization at the collection sites, rather than any other combination of environmental factors, were linked to copper tolerance. While previously identified copper-related genes were not differentially expressed in tolerant vs. sensitive strains, genes involved in metabolism, reproduction, and protease induction contributed to the differential stress response. Additionally, the greatest transcriptomic and physiological responses to copper toxicity were seen in the midgut, where we found that preservation of gut acidity is strongly linked to greater tolerance. Finally, we identified transposable element insertions likely to play a role in copper stress response. CONCLUSIONS: Overall, by combining genome-wide approaches with environmental association analysis, and functional analysis of candidate genes, our study provides a unique perspective on the genetic and environmental factors that shape copper tolerance in natural D. melanogaster populations and identifies new genes, transposable elements, and physiological traits involved in this complex phenotype.

A Transposon Story: From TE Content to TE Dynamic Invasion of Drosophila Genomes Using the Single-Molecule Sequencing Technology from Oxford Nanopore.

Transposable elements (TEs) are the main components of genomes. However, due to their repetitive nature, they are very difficult to study using data obtained with short-read sequencing technologies. Here, we describe an efficient pipeline to accurately recover TE insertion (TEI) sites and sequences from long reads obtained by Oxford Nanopore Technology (ONT) sequencing. With this pipeline, we could precisely describe the landscapes of the most recent TEIs in wild-type strains of Drosophila melanogaster and Drosophila simulans. Their comparison suggests that this subset of TE sequences is more similar than previously thought in these two species. The chromosome assemblies obtained using this pipeline also allowed recovering piRNA cluster sequences, which was impossible using short-read sequencing. Finally, we used our pipeline to analyze ONT sequencing data from a D. melanogaster unstable line in which LTR transposition was derepressed for 73 successive generations. We could rely on single reads to identify new insertions with intact target site duplications. Moreover, the detailed analysis of TEIs in the wild-type strains and the unstable line did not support the trap model claiming that piRNA clusters are hotspots of TE insertions.

Transposable elements contribute to the genomic response to insecticides in Drosophila melanogaster.

Most of the genotype-phenotype analyses to date have largely centred attention on single nucleotide polymorphisms. However, transposable element (TE) insertions have arisen as a plausible addition to the study of the genotypic-phenotypic link because of to their role in genome function and evolution. In this work, we investigate the contribution of TE insertions to the regulation of gene expression in response to insecticides. We exposed four Drosophila melanogaster strains to malathion, a commonly used organophosphate insecticide. By combining information from different approaches, including RNA-seq and ATAC-seq, we found that TEs can contribute to the regulation of gene expression under insecticide exposure by rewiring cis-regulatory networks. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.

Dynamic Interactions Between the Genome and an Endogenous Retrovirus: Tirant in Drosophila simulans Wild-Type Strains.

All genomes contain repeated sequences that are known as transposable elements (TEs). Among these are endogenous retroviruses (ERVs), which are sequences similar to retroviruses and are transmitted across generations from parent to progeny. These sequences are controlled in genomes through epigenetic mechanisms. At the center of the epigenetic control of TEs are small interfering RNAs of the piRNA class, which trigger heterochromatinization of TE sequences. The tirant ERV of Drosophila simulans displays intra-specific variability in copy numbers, insertion sites, and transcription levels, providing us with a well-suited model to study the dynamic relationship between a TE family and the host genome through epigenetic mechanisms. We show that tirant transcript amounts and piRNA amounts are positively correlated in ovaries in normal conditions, unlike what was previously described following divergent crosses. In addition, we describe tirant insertion polymorphism in the genomes of three D. simulans wild-type strains, which reveals a limited number of insertions that may be associated with gene transcript level changes through heterochromatin spreading and have phenotypic impacts. Taken together, our results participate in the understanding of the equilibrium between the host genome and its TEs.

An attempt to select non-genetic variation in resistance to starvation and reduced chill coma recovery time in Drosophila melanogaster.

Phenotypic variance is attributed to genetic and non-genetic factors, and only the former are presumed to be inherited and thus suitable for the action of selection. Although increasing amounts of data suggest that non-genetic variability may be inherited, we have limited empirical data in animals. Here, we performed an artificial selection experiment using Drosophila melanogaster inbred lines. We quantified the response to selection for a decrease in chill coma recovery time and an increase in starvation resistance. We observed a weak response to selection in the inbred and outbred lines, with variability across lines. At the end of the selection process, differential expression was detected for some genes associated with epigenetics, the piRNA pathway and canalization functions. As the selection process can disturb the canalization process and increase the phenotypic variance of developmental traits, we also investigated possible effects of the selection process on the number of scutellar bristles, fluctuating asymmetry levels and fitness estimates. These results suggest that, contrary to what was shown in plants, selection of non-genetic variability is not straightforward in Drosophila and appears to be strongly genotype dependent.

In silico analysis of regulatory and structural motifs of the ovine HSP90AA1 gene.

Gene promoters are essential regions of DNA where the transcriptional molecular machinery to produce RNA molecules is recruited. In this process, DNA epigenetic modifications can acquire a fundamental role in the regulation of gene expression. Recently, in a previous work of our group, functional features and DNA methylation involved in the ovine HSP90AA1 gene expression regulation have been observed. In this work, we report a combination of methylation analysis by bisulfite sequencing in several tissues and at different developmental stages together with in silico bioinformatic analysis of putative regulating factors in order to identify regulative mechanisms both at the promoter and gene body. Our results show a "hybrid structure" (TATA box + CpG island) of the ovine HSP90AA1 gene promoter both in somatic and non-differentiated germ tissues, revealing the ability of the HSP90AA1 gene to be regulated both in an inducible and constitutive fashion. In addition, in silico analysis showed that several putative alternative spliced regulatory motifs, exonic splicing enhancers (ESEs), and G-quadruplex secondary structures were somehow related to the DNA methylation pattern found. The results obtained here could help explain the differences in cell-type transcripts, tissue expression rate, and transcription silencing mechanisms found in this gene.

Ovine HSP90AA1 gene promoter: functional study and epigenetic modifications.

When environmental temperatures exceed a certain threshold, the upregulation of the ovine HSP90AA1 gene is produced to cope with cellular injuries caused by heat stress. It has been previously pointed out that several polymorphisms located at the promoter region of this gene seem to be the main responsible for the differences in the heat stress response observed among alternative genotypes in terms of gene expression rate. The present study, focused on the functional study of those candidate polymorphisms by electrophoretic mobility shift assay (EMSA) and in vitro luciferase expression assays, has revealed that the observed differences in the transcriptional activity of the HSP90AA1 gene as response to heat stress are caused by the presence of a cytosine insertion (rs397514115) and a C to G transversion (rs397514116) at the promoter region. Next, we discovered the presence of epigenetic marks at the promoter and along the gene body founding an allele-specific methylation of the rs397514116 mutation in DNA extracted from blood samples. This regulatory mechanism interacts synergistically to modulate gene expression depending on environmental circumstances. Taking into account the results obtained, it is suggested that the transcription of the HSP90AA1 ovine gene is regulated by a cooperative action of transcription factors (TFs) whose binding sites are polymorphic and where the influence of epigenetic events should be also taken into account.

Looking for adaptive footprints in the HSP90AA1 ovine gene.

BACKGROUND: Climatic factors play an important role in determining species distributions and phenotypic variation of populations over geographic space. Since domestic sheep is managed under low intensive systems animals could have retained some genome adaptive footprints. The gene encoding the Hsp90α has been extensively studied in sheep and some polymorphisms located at its promoter have been associates with differences in the transcription rate of the gene depending on climatic conditions. In this work the relationships among the distribution and frequencies of 11 polymorphisms of the ovine HSP90AA1 gene promoter in 31 sheep breeds and the climatic and geographic variables prevailing in their regions of origin have been studied. Also the promoter sequence has been characterized in 9 species of the Caprinae subfamily. RESULTS: Correlations among several climatic variables and allele frequencies of the polymorphisms of the HSP90AA1 gene promoter linked with differences in the transcription activity of the gene under heat stress conditions have been assessed. A group of breeds reared in semi dry climates have high frequencies of the insertion allele of the g.667-668insC associated with the heat stress response. Other group of breeds native to semi arid conditions showed very low frequencies of this same allele. However, in some cases, this previous correlation has not been achieved, revealing the high levels of gene flow among populations occurred following domestication. The Bayesian Test of Beaumont and Balding identified two outlier loci, the g.522A > G and g.703_704del(2)A candidates to balancing and directional selection, respectively. Polymorphisms detected in O. aries are also present in several species of the Caprinae subfamily being C. hircus, O. musimon and O. moschatus those sharing the highest number of them with O. aries. CONCLUSIONS: Despite domestication, sheep breeds showed some genetic footprints related to climatic variables. Adaptation of breeds to heat climates can suppose a selective advantage to cope with global warming caused by climatic change. Polymorphisms of the HSP90AA1 gene detected in the Ovis aries species are also present in wild species from the Caprinae subfamily, indicating a great antiquity of these mutations and its importance in the adaptation of species to past climatic conditions existing in its native environments.

Differences in the ovine HSP90AA1 gene expression rates caused by two linked polymorphisms at its promoter affect rams sperm DNA fragmentation under environmental heat stress conditions.

Heat shock (HS) is one of the best-studied exogenous cellular stresses. Almost all tissues, cell types, metabolic pathways and biochemical reactions are affected in greater or lesser extent by HS. However, there are some especially thermo sensible cellular types such as the mammalian male germ cells. The present study examined the role of three INDELs in conjunction with the -660G/C polymorphism located at the HSP90AA1 promoter region over the gene expression rate under HS. Specially, the -668insC INDEL, which is very close to the -660G/C transversion, is a good candidate to be implied in the transcriptional regulation of the gene by itself or in a cooperative way with this SNP. Animals carrying the genotype II-668 showed higher transcription rates than those with ID-668 (FC = 3.07) and DD-668 (FC = 3.40) genotypes for samples collected under HS. A linkage between gene expression and sperm DNA fragmentation was also found. When HS conditions were present along or in some stages of the spermatogenesis, alternative genotypes of the -668insC and -660G/C mutations are involved in the effect of HS over sperm DNA fragmentation. Thus, unfavorable genotypes in terms of gene expression induction (ID-668GC-660 and DD-668GG-660) do not produce enough mRNA (stored as messenger ribonucleoprotein particles) and Hsp90α protein to cope with future thermal stress which might occur in posterior stages when transcriptional activity is reduced and cell types and molecular processes are more sensible to heat (spermatocytes in pachytene and spermatids protamination). This would result in the impairment of DNA packaging and the consequent commitment of the events occurring shortly after fertilization and during embryonic development. In the short-term, the assessment of the relationship between sperm DNA fragmentation sensitivity and ram's fertility will be of interest to a better understanding of the mechanisms of response to HS and its consequences on animal production and reproduction performance.

Influence of the temperature and the genotype of the HSP90AA1 gene over sperm chromatin stability in Manchega Rams.

The present study addresses the effect of heat stress on males' reproduction ability. For that, we have evaluated the sperm DNA fragmentation (DFI) by SCSA of ejaculates incubated at 37 °C during 0, 24 and 48 hours after its collection, as a way to mimic the temperature circumstances to which spermatozoa will be subject to in the ewe uterus. The effects of temperature and temperature-humidity index (THI) from day 60 prior collection to the date of semen collection on DFI were examined. To better understand the causes determining the sensitivity of spermatozoa to heat, this study was conducted in 60 males with alternative genotypes for the SNP G/C-660 of the HSP90AA1 promoter, which encode for the Hsp90α protein. The Hsp90α protein predominates in the brain and testis, and its role in spermatogenesis has been described in several species. Ridge regression analyses showed that days 29 to 35 and 7 to 14 before sperm collection (bsc) were the most critical regarding the effect of heat stress over DFI values. Mixed model analyses revealed that DFI increases over a threshold of 30 °C for maximum temperature and 22 for THI at days 29 to 35 and 7 to 14 bsc only in animals carrying the GG-660 genotype. The period 29-35 bsc coincide with the meiosis I process for which the effect of the Hsp90α has been described in mice. The period 7-14 bsc may correspond with later stages of the meiosis II and early stages of epididymal maturation in which the replacement of histones by protamines occurs. Because of GG-660 genotype has been associated to lower levels of HSP90AA1 expression, suboptimal amounts of HSP90AA1 mRNA in GG-660 animals under heat stress conditions make spermatozoa DNA more susceptible to be fragmented. Thus, selecting against the GG-660 genotype could decrease the DNA fragmentation and spermatozoa thermal susceptibility in the heat season, and its putative subsequent fertility gains.

Ovine HSP90AA1 expression rate is affected by several SNPs at the promoter under both basal and heat stress conditions.

The aim of this work was to investigate the association between polymorphisms located at the HSP90AA1 ovine gene promoter and gene expression rate under different environmental conditions, using a mixed model approach. Blood samples from 120 unrelated rams of the Manchega sheep breed were collected at three time points differing in environmental conditions. Rams were selected on the basis of their genotype for the transversion G/C located 660 base pairs upstream the gene transcription initiation site. Animals were also genotyped for another set of 6 SNPs located at the gene promoter. Two SNPs, G/C-660 and A/G-444, were associated with gene overexpression resulting from heat stress. The composed genotype CC-660-AG-444 was the genotype having the highest expression rates with fold changes ranging from 2.2 to 3.0. The genotype AG-522 showed the highest expression levels under control conditions with a fold change of 1.4. Under these conditions, the composed genotype CC-601-TT-524-AG-522-TT-468 is expected to be correlated with higher basal expression of the gene according to genotype frequencies and linkage disequilibrium values. Some putative transcription factors were predicted for binding sites where the SNPs considered are located. Since the expression rate of the gene under alternative environmental conditions seems to depend on the composed genotype of several SNPs located at its promoter, a cooperative regulation of the transcription of the HSP90AA1 gene could be hypothesized. Nevertheless epigenetic regulation mechanisms cannot be discarded.