Exploratory analysis of transposable elements expression in the C. elegans early embryo

Background: Transposable Elements (TE) are mobile sequences that make up large portions of eukaryote genomes. The functions they play within the complex cellular architecture are still not clearly understood, but it is becoming evident that TE have a role in several physiological and pathological processes. In particular, it has been shown that TE transcription is necessary for the correct development of mice embryos and that their expression is able to finely modulate transcription of coding and non-coding genes. Moreover, their activity in the central nervous system (CNS) and other tissues has been correlated with the creation of somatic mosaicisms and with pathologies such as neurodevelopmental and neurodegenerative diseases as well as cancers. Results: We analyzed TE expression among different cell types of the Caenorhabditis elegans (C. elegans) early embryo asking if, where and when TE are expressed and whether their expression is correlated with genes playing a role in early embryo development. To answer these questions, we took advantage of a public C. elegans embryonic single-cell RNA-seq (sc-RNAseq) dataset and developed a bioinformatics pipeline able to quantify reads mapping specifically against TE, avoiding counting reads mapping on TE fragments embedded in coding/non-coding transcripts. Our results suggest that i) canonical TE expression analysis tools, which do not discard reads mapping on TE fragments embedded in annotated transcripts, may over-estimate TE expression levels, ii) Long Terminal Repeats (LTR) elements are mostly expressed in undifferentiated cells and might play a role in pluripotency maintenance and activation of the innate immune response, iii) non-LTR are expressed in differentiated cells, in particular in neurons and nervous system-Associated tissues, and iv) DNA TE are homogenously expressed throughout the C. elegans early embryo development. Conclusions: TE expression appears finely modulated in the C. elegans early embryo and different TE classes are expressed in different cell types and stages, suggesting that TE might play diverse functions during early embryo development

Comparative Study of the Distribution of Repetitive DNA in Model Organisms

Repetitive DNA elements are abundant in the genome of a wide range of organisms. In mammals, repetitive elements comprise about 40-50% of the total genomes. However, their biological functions remain largely unknown. Analysis of their abundance and distribution may shed some light on how they affect genome structure, function, and evolution. We conducted a detailed comparative analysis of repetitive DNA elements across ten different eukaryotic organisms, including chicken (G. gallus), zebrafish (D. rerio), Fugu (T. rubripes), fruit fly (D. melanogaster), and nematode worm (C. elegans), along with five mammalian organisms: human (H. sapiens), mouse (M. musculus), cow (B. taurus), rat (R. norvegicus), and rhesus (M. mulatta). Our results show that repetitive DNA content varies widely, from 7.3% in the Fugu genome to 52% in the zebrafish, based on RepeatMasker data. The most frequently observed transposable elements (TEs) in mammals are SINEs (Short Interspersed Nuclear Elements), followed by LINEs (Long Interspersed Nuclear Elements). In contrast, LINEs, DNA transposons, simple repeats, and low complexity repeats are the most frequently observed repeat classes in the chicken, zebrafish, fruit fly, and nematode worm genomes, respectively. LTRs (Long Terminal Repeats) have significant genomic coverage and diversity, which may make them suitable for regulatory roles. With the exception of the nematode worm and fruit fly, the frequency of the repetitive elements follows a log-normal distribution, characterized by a few highly prevalent repeats in each organism. In mammals, SINEs are enriched near genic regions, and LINEs are often found away from genes. We also identified many LTRs that are specifically enriched in promoter regions, some with a strong bias towards the same strand as the nearby gene. This raises the possibility that the LTRs may play a regulatory role. Surprisingly, most intronic repeats, with the exception of DNA transposons, have a strong tendency to be on the opposite DNA strand as the host gene. One possible explanation is that intronic RNAs which result from splicing may contribute to retrotransposition to the original intronic loci. Moreover, our observations of repetitive DNA elements enrichment near genic regions and, specifically, the promoter region of genes, raise the question as to whether repetitive DNA elements have a significant impact on gene expression in both human and mouse genomes. In order to investigate the impact of these repeats on gene expression, we calculate the total number of base pairs (bp) for these repeats in two different locations upstream from the genes — namely, the 2kbp and 20kbp promoter regions. In addition to that, we quantified the gene expression levels in both human and mouse tissues using RNAseq analysis. Then, we used different statistical modeling approaches to investigate the association between repetitive DNA elements and gene expression in two different promoter regions. Although most transposable elements are primarily involved in reduced gene expression, our model\u27s results showed that Alu elements in both human and mouse are significantly associated with higher average expression in the promoter region. Furthermore, we found that the B2 in both mouse 2kbp and 20kbp and hAT.Charlie elements in the human 20kbp, are also significantly associated with up-regulated gene expression in the 2kpb promoter. In addition to Alu and B2 in 2kbp, we found that the ERV1 have a significant association with higher average expression in the 20kbp promoter in mouse tissues. We also found that L1 and Simple_repeat elements are significantly associated with lower average expression in both human and mouse tissues. Furthermore, in the human, we found that the MIR is also associated with lower average expression. The effects of Alu elements in both human and mouse are stronger at 2kbp than at 20kbp. In contrast, the L1 effect at 20kbp is stronger than at 2kbp. Our results indicate that comparative studies of repetitive DNA elements in multiple organisms can provide insights into their evolution and expansion, and lead to the elucidation of their potential functions. The non-random distribution of repeats across multiple organisms adds to the existing evidence that some repetitive DNA elements are drivers of genome evolution, rather than just “junk” DNA

TEspeX: consensus-specific quantification of transposable element expression preventing biases from exonized fragments

Summary: Transposable elements (TEs) play key roles in crucial biological pathways. Therefore, several tools enabling the quantification of their expression were recently developed. However, many of the existing tools lack the capability to distinguish between the transcription of autonomously expressed TEs and TE fragments embedded in canonical coding/non-coding non-TE transcripts. Consequently, an apparent change in the expression of a given TE may simply reflect the variation in the expression of the transcripts containing TE-derived sequences. To overcome this issue, we have developed TEspeX, a pipeline for the quantification of TE expression at the consensus level. TEspeX uses Illumina RNA-seq short reads to quantify TE expression avoiding counting reads deriving from inactive TE fragments embedded in canonical transcripts. Availability and implementation: The tool is implemented in python3, distributed under the GNU General Public License (GPL) and available on Github at https://github.com/fansalon/TEspeX (Zenodo URL: https://doi.org/10.5281/zenodo.6800331). Supplementary information: Supplementary data are available at Bioinformatics online

Microhabitat, microbiota, mitochondria and the epigenome shape the biparental legacy of heat exposure in a tropical arthropod

As technology and policy advance towards sustainable energy sources, humanity is reaching a critical climax regarding climate change. Average global surface temperatures have risen by 1oC since pre-industrial levels, and it is estimated that temperatures will continue to rise by at least 1.5oC until at least 2100, despite mitigation efforts. The effect that such warming will have on biodiversity remains undetermined. Warming will likely have a substantial impact on tropical regions, which are a hotbed for biodiversity, and warming in tropical regions has already been associated with massive declines in species population numbers. Whether biodiversity will continue to decline, culminating in mass extinction, due to climate change depends on the ability of tropical species to adapt to new thermal limits. Tropical regions exhibit low seasonal variability, thus tropical species are adapted to little variation in environmental conditions and rapid increases in temperature may prove unsustainable for life. Tropical arthropods make up the majority of species in the tropics, with 1.3 million species described, and the true estimate at several million species. However, they are at especially high risk, since as ectotherms, arthropod metabolism increases exponentially with increases in ambient temperature. Understanding the effect of temperature on tropical arthropods is crucial to understanding biome dynamics to target conservation efforts as the planet continues to warm. Epigenetic mechanisms, or changes in gene expression without changes in the genetic sequence, provide a short-term plastic way for tropical arthropods to respond to climate change. Here,i I utilize a model ectothermic arthropod, Cordylochernes scorpioides, to investigate: (1) behavioral mechanisms for moderation of the effects of high temperature; (2) phenotypic, genetic and epigenetic effects of high temperature exposure on intergenerational inheritance, and (3) the microbial composition of individuals exposed to high temperature. This tropical arthropod has a unique reproductive biology, enabling non-invasive investigation into fitness effects and reproductive capacity, and it also exhibits high variation in the mitochondrial genome, which was exploited to determine how mitochondrial function may influence biological responses to climate change. In Chapter 1, I present the results of a study designed to determine how microclimate variation in lowland tropical rainforests impacts the abundance in C. scorpioides populations. Tropical rainforests exhibit high levels of fine-scale climatic variation, yet climate studies in these regions typically retrieve temperature data from large-scale weather stations. This provides an incomplete view of how tropical arthropods may experience temperature and change their behavior to compensate for warming. This species of arthropod inhabits decaying Ficus trees in their native Panamanian rainforests. Several Ficus trees occupied by C. scorpioides were found and microclimate variation was assessed by placing iButton temperature loggers in three types of microhabitat, frass, side/south, and top/north over two types of trees, fallen and standing, in open canopy or closed canopy habitats. C. scorpioides were then collected from the trees to estimate abundance. Reduced abundance was associated with the hottest microhabitats, top/north in open canopy trees, and high abundance was iiassociated with the coolest microhabitats, frass in closed canopy trees. Thus C. scorpioides actively manage exposure to high temperatures through behavioral mechanisms.In Chapter 2, I investigate whether phenotypes from simulated climate change can be passed to offspring intergenerationally. During nymphal development, C. scorpioides were placed in incubators programmed to diurnally fluctuate in correspondence with ambient temperature measurements from rainforest habitats found in Chapter 1. A split-brood design was employed where, upon birth, 40 pseudoscorpions were collected from each family and half were directly exposed to a control temperature regime and the other half to a high (+2.5o) temperature regime. Developmental, survival, and male and female reproductive traits were assayed. An intergenerational effects study was then carried to determine whether direct effects of high temperature exposure are transmitted to offspring maternally, paternally or through both sexes. Females and males directly exposed to the high temperature were mated to control males and females, respectively, to establish female outcross (FOUT) and male outcross (MOUT) families. Developmental, survival, and reproductive traits were then assayed on offspring. As in males directly exposed to high temperature, males with both high-temperature treated mothers and fathers had significantly reduced sperm counts, though this effect was stronger in males with high temperature mothers. Sperm counts were also significantly affected by haplotype, with A2 haplotype males producing the most sperm and B2 haplotype males the most negatively affected by high-temperature treatment of the parent. iiiFemale reproductive traits did not exhibit significant intergenerational effects, suggesting they are more robust to the effects of high temperature. However, high temperature effects on some non-reproductive traits, including reduced survivorship and reduced male offspring size, were transmitted maternally. Interestingly, although males born to high-temperature mothers exhibited reduced body size, their development time was not significantly reduced, as in the directly treated generation. By contrast, female offspring with high temperature fathers had reduced developmental time, but not significantly reduced body size, indicating an uncoupling of size-temperature dependence and developmental rate often associated with arthropod species. This is hypothesized to be due to C. scorpioides XX/XO system of sex determination, where epigenetic or genetic mutations acting on the males’ lone X chromosome make them more vulnerable to sex-linked effects of high temperature. However, males with high temperature mothers experienced some positive effects of reproduction, producing 22% more offspring than did males with control temperature mothers. The best explanation for this may be selection, where only the most genetically or epigenetically fit males were capable of siring offspring.In Chapter 3, I determined the potential for intergenerational epigenetic effects in males exposed to high temperature by assaying gene expression of protein-coding genes, transposable elements, and noncoding RNA of testicular and spermatic tissue. Transposable elements are mobile genetic elements able to excise themselves and move throughout the genome, capable of inducing genomic instability and disrupted gene expression, but they are typically ivcontrolled by epigenetic modifications to silence their expression. They are highly expressed in the germline, where suppression is achieved through targeted destruction by non-coding RNAs known as PIWI-interacting RNAs. Approximately 70% of the C. scorpioides genome consists of transposable elements/highly repetitive regions, an unusually high level for an arthropod species, making transposable element expression a likely factor in intergenerational inheritance of alternative phenotypes in males exposed to high temperature. Males exposed to high temperature were either dissected for removal of the testes or had their mating sequence interrupted to collect sperm, and RNA sequences annotated to determine levels of differential expression. Testicular protein-coding genes and transposable elements were significantly up- regulated, and testicular and spermatic piRNA expression were significantly down-regulated in males exposed to high temperature. Hence, high temperature exposure likely induces transposable element activity in the germline, and causes dysregulation of the epigenetic control mechanisms designed to silence them.Finally, in Chapter 4, I conducted microbial diversity analysis to determine whether other mechanisms may be responsible for alternative phenotypes intergenerationally inherited from exposure to high temperature. Diverse and healthy gut microbiomes play a key role in host metabolism, physiology, nutrition, immune function, and pathogen defense for many species, with differing microbial profiles associated with environmental factors, such as temperature and nutrition, and host factors, such as genotype or mitochondrial haplotype. On vthe other hand, dysbiosis, or imbalances in the gut microbiome, have been associated with a variety of diseases, including metabolic disorders, immune disorders, neurodegenerative disease, and psychological disorders. Growing evidence also suggests that crosstalk between the gut microbiome and the host is mediated by microbial-derived molecules that induce changes in the epigenetic mechanisms that regulate host gene expression. Microbial diversity of control temperature and high temperature C. scorpioides were analyzed using a factorial design of two temperature treatments and three haplotypes to investigate temperature, haplogroup, and interaction effects on the diversity and community composition of the C. scorpioides microbiome. Elevated temperature was associated with increased microbial diversity, which was a counterintuitive result as increased microbial diversity is often correlated with a healthy microbiome, yet elevated temperature induced maladaptive states in C. scorpioides. The most variation between microbial profiles was due to haplotype, with A1/A2 haplotypes having higher diversity than B2 haplotypes. Lack of appreciable temperature effects on the C. scorpioides microbiome coupled with significant haplogroup effects suggest that microbial composition changes are not responsible for detrimental phenotypic effects of a 2.5oC temperature increase found in Chapter 2, and that epigenetic mechanisms and loss of epigenetic control over transposable elements, as discovered in Chapter 3, are implicated instead

Meta-Analysis Suggests That Intron Retention Can Affect Quantification of Transposable Elements from RNA-Seq Data

Simple Summary Transposable elements (TEs) are repetitive sequences comprising more than one third of the human genome with the original ability to change their location within the genome. Owing to their repetitive nature, the quantification of TEs results often challenging. RNA-seq is a useful tool for genome-wide TEs quantification, nevertheless it also presents technical issues, including low reads mappability and erroneous quantification derived from the transcription of TEs fragments embedded in canonical transcripts. Fragments derived from TEs are found within the introns of most genes, which led to the hypothesis that intron retention (IR) can affect the unbiased quantification of TEs expression. Performing meta-analysis of public RNA-seq datasets, here we observe that IR can indeed impact the quantification of TEs by increasing the number of reads mapped on intronic TE copies. Our work highlights a correlation between IR and TEs expression measurement by RNA-seq that should be taken into account to achieve reliable TEs quantification, especially in samples characterized by extensive IR, because differential IR might be confused with differential TEs expression. Transposable elements (TEs), also known as "jumping genes", are repetitive sequences with the capability of changing their location within the genome. They are key players in many different biological processes in health and disease. Therefore, a reliable quantification of their expression as transcriptional units is crucial to distinguish between their independent expression and the transcription of their sequences as part of canonical transcripts. TEs quantification faces difficulties of different types, the most important one being low reads mappability due to their repetitive nature preventing an unambiguous mapping of reads originating from their sequences. A large fraction of TEs fragments localizes within introns, which led to the hypothesis that intron retention (IR) can be an additional source of bias, potentially affecting accurate TEs quantification. IR occurs when introns, normally removed from the mature transcript by the splicing machinery, are maintained in mature transcripts. IR is a widespread mechanism affecting many different genes with cell type-specific patterns. We hypothesized that, in an RNA-seq experiment, reads derived from retained introns can introduce a bias in the detection of overlapping, independent TEs RNA expression. In this study we performed meta-analysis using public RNA-seq data from lymphoblastoid cell lines and show that IR can impact TEs quantification using established tools with default parameters. Reads mapped on intronic TEs were indeed associated to the expression of TEs and influence their correct quantification as independent transcriptional units. We confirmed these results using additional independent datasets, demonstrating that this bias does not appear in samples where IR is not present and that differential TEs expression does not impact on IR quantification. We concluded that IR causes the over-quantification of intronic TEs and differential IR might be confused with differential TEs expression. Our results should be taken into account for a correct quantification of TEs expression from RNA-seq data, especially in samples in which IR is abundant

Ac/Ds transposition for CRISPR/dCas9-SID4x epigenome modulation in zebrafish

Due to its genetic amenability coupled with advances in genome editing, zebrafish is an excellent model to examine the function of (epi)genomic elements. Here, we repurposed the Ac/Ds maize transposition system to efficiently characterise zebrafish cis-regulated elements, also known as enhancers, in F0-microinjected embryos. We further used the system to stably express guide RNAs enabling CRISPR/dCas9-interference (CRISPRi) perturbation of enhancers without disrupting the underlying genetic sequence. In addition, we probed the phenomenon of antisense transcription at two neural crest gene loci. Our study highlights the utility of Ac/Ds transposition as a new tool for transient epigenome modulation in zebrafish

Relationships between chromatin features and genome regulation

Regulation of gene expression is an essential process for all living organisms. Transcriptional regulation, associated with chromatin, is governed by: (1) DNA sequence, which creates regulatory sites (promoters, enhancers and silencers), where sequence motifs and features (e. g. CpG) can attract transcription factors (TFs) and influence chromatin structure or RNA polymerase II (Pol II) binding, initiation and elongation; (2) non-sequence, epigenetic factors - histone modifications, TF binding, chromatin remodelling (histone placement, eviction and reconstitution), and non-coding RNA regulation. These factors interact with each other, creating a complex network of interactions. In this thesis I describe computational studies of heterochromatin factors in regulation of gene and repeat expression, an analysis of active regulatory elements, and global analyses of big datasets in C. elegans. I first show that a team of heterochromatin factors - HPL-2/HP1, LIN-13, LIN-61, LET-418/Mi-2, and H3K9me2 histone methyltransferase MET-2/SETDB1 - collaborates with piRNA and nuclear RNAi pathways to silence repetitive elements and protect the germline. I also found that the TACBGTA motif is particularly enriched on repeats and heterochromatin factors binding sites, and that repeat elements are derepressed in the soma during normal C. elegans ageing. I then describe the work on active regulatory regions. I show that CFP-1/CXXC1 binds CpG dense, nucleosome depleted promoters and, along SET-2, is required for H3K4me3 deposition at these loci. Using expression profiling I determined that the majority of CFP-1 binding targets are not significantly mis-regulated in cfp-1 mutants, but are weakly upregulated in bulk analyses. I also show that CFP-1 functionally interacts with the Sin3S/HDAC complex. In cfp-1 mutant I observed both loss and gain of SIN-3 binding, depending on chromatin context. Finally, I performed a data driven study on a large collection of ChIP-seq profiles using non-parametric sparse factor analyses (NSFA) and compared it to other, unsupervised machine learning algorithms. This study uncovered interactions and structure in genomic datasets. In addition, I present a collection of computational tools and methods I developed to facilitate processing, storage, retrieval, annotation, and analyses of large datasets in genomics

Abundant small RNAs in the reproductive tissues and eggs of the honey bee, Apis mellifera

Background: Polyandrous social insects such as the honey bee are prime candidates for parental manipulation of gene expression in offspring. Although there is good evidence for parent-of-origin effects in honey bees the epigenetic mechanisms that underlie these effects remain a mystery. Small RNA molecules such as miRNAs, piRNAs and siRNAs play important roles in transgenerational epigenetic inheritance and in the regulation of gene expression during development. Results: Here we present the first characterisation of small RNAs present in honey bee reproductive tissues: ovaries, spermatheca, semen, fertilised and unfertilised eggs, and testes. We show that semen contains fewer piRNAs relative to eggs and ovaries, and that piRNAs and miRNAs which map antisense to genes involved in DNA regulation and developmental processes are differentially expressed between tissues. tRNA fragments are highly abundant in semen and have a similar profile to those seen in the semen of other animals. Intriguingly we also find abundant piRNAs that target the sex determination locus, suggesting that piRNAs may play a role in honey bee sex determination. Conclusions: We conclude that small RNAs may play a fundamental role in honey bee gametogenesis and reproduction and provide a plausible mechanism for parent-of-origin effects on gene expression and reproductive physiology

Comparative Analysis of Small Non-Coding RNA and Messenger RNA Expression in Somatic Cell Nuclear Transfer and In Vitro-Fertilized Bovine Embryos During Early Development Through the Maternal-to-Embryonic Transition

Cloning animals using somatic cell nuclear transfer (scNT) was first successfully demonstrated with the birth of Dolly the sheep, but the process of cloning remains highly inefficient. By improving our understanding of the errors that may occur during cloned cattle embryo development, we could obtain a greater understanding of how specific molecular events contribute to successful development. The central dogma of biology refers to the process of DNA being transcribed into messenger RNA (mRNA) and the translation of mRNA into proteins, which ultimately carry out the functions encoded by genes. The epigenetic code is defined as the array of chemical modifications, or “marks”, to DNA molecules that do not change the genome sequence but do allow for control of gene expression. During early development, genome reprogramming involves the removal of epigenetic marks from the sperm and egg and re-establishment of marks for the embryonic genome that code for proper gene expression to support embryo development. The point during this process at which the embryo’s genes are turned on is known as embryonic genome activation (EGA). Small non-coding RNAs (sncRNAs), including microRNAs (miRNAs), may also contribute to the this process. For example, miRNA molecules do not code for proteins themselves, but rather bind to mRNAs and effectively block their translation into protein. We hypothesized that aberrant expression of sncRNAs in cloned embryos may lead to anomalous abundance of mRNA molecules, thus explaining poor development of cloned embryos. First, we used RNA sequencing to examine the total population of sncRNAs in cattle embryos produced by in vitro fertilization (IVF) and found a dramatic shift in populations at the EGA. Next, we collected both sncRNA and mRNA from scNT cattle embryos, and again performed sequencing of both RNA fractions. We found that few sncRNAs were abnormally expressed in scNT embryos, with all differences appearing after EGA at the morula developmental stage. However, notable differences in the populations of sncRNAs were evident when comparing embryos by developmental stage. For populations of mRNA, we observed dramatic differences when comparing scNT and IVF cattle embryos, with the highest number of changes occurring at the EGA (8-cell stage) and after (morula stage). While changes in specific miRNA molecules (miR-34a and miR-345) were negatively correlated with some of their predicted target mRNAs, this pattern was not widespread as would be expected if these sncRNAs are functionally binding to all of the predicted mRNA targets. Collectively, our observations suggest that other mechanisms leading to altered expression of mRNA in cloned embryos may be responsible for their relatively poor development