Tolerance thresholds underlie responses to DNA damage during germline development.

Selfish DNA modules like transposable elements (TEs) are particularly active in the germline, the lineage that passes genetic information across generations. New TE insertions can disrupt genes and impair the functionality and viability of germ cells. However, we found that in P-M hybrid dysgenesis in Drosophila, a sterility syndrome triggered by the P-element DNA transposon, germ cells harbor unexpectedly few new TE insertions despite accumulating DNA double-strand breaks (DSBs) and inducing cell cycle arrest. Using an engineered CRISPR-Cas9 system, we show that generating DSBs at silenced P-elements or other noncoding sequences is sufficient to induce germ cell loss independently of gene disruption. Indeed, we demonstrate that both developing and adult mitotic germ cells are sensitive to DSBs in a dosage-dependent manner. Following the mitotic-to-meiotic transition, however, germ cells become more tolerant to DSBs, completing oogenesis regardless of the accumulated genome damage. Our findings establish DNA damage tolerance thresholds as crucial safeguards of genome integrity during germline development.

The Transposition Rate Has Little Influence on the Plateauing Level of the P-element.

The popular trap model assumes that the invasions of transposable elements (TEs) in mammals and invertebrates are stopped by piRNAs that emerge after insertion of the TE into a piRNA cluster. It remains, however, still unclear which factors influence the dynamics of TE invasions. The activity of the TE (i.e., transposition rate) is one frequently discussed key factor. Here we take advantage of the temperature-dependent activity of the P-element, a widely studied eukaryotic TE, to test how TE activity affects the dynamics of a TE invasion. We monitored P-element invasion dynamics in experimental Drosophila simulans populations at hot and cold culture conditions. Despite marked differences in transposition rates, the P-element reached very similar copy numbers at both temperatures. The reduction of the insertion rate upon approaching the copy number plateau was accompanied by similar amounts of piRNAs against the P-element at both temperatures. Nevertheless, we also observed fewer P-element insertions in piRNA clusters than expected, which is not compatible with a simple trap model. The ping-pong cycle, which degrades TE transcripts, becomes typically active after the copy number plateaued. We generated a model, with few parameters, that largely captures the observed invasion dynamics. We conclude that the transposition rate has at the most only a minor influence on TE abundance, but other factors, such as paramutations or selection against TE insertions are shaping the TE composition.

[The P-Element Has Not Significant Effect on the Drosophila simulans Viability].

Cases of horizontal transfer of transposable elements (TEs) between species are known for the Drosophilidae family. In the middle of the last century, the case of horizontal transfer of the P-element from the Drosophila willistoni to the D. melanogaster was described. A novel P-element invasion into the D. simulans genome from D. melanogaster occurred approximately 10 years ago. Currently, the P-element has spread across all D. melanogaster population and 30% of D. simulans populations in Europe, Africa and America. In this paper, we investigated the presence of the P-element in D. simulans lines caught in different years in three Asian populations (Tashkent, Nalchik and Sakhalin Island). We also examined the physiological characteristics (cytotype, lifespan, fecundity and locomotor activity) of D. simulans lines with and without the P-element to determine the significance of this new mobile element in the genome. The P-element was found in lines isolated from nature after 2012. The number of P-element copies per genome (two-to-three dozen according to fluorescence in situ hybridization data) was greater than in the American and comparable to the African populations. There were signs of intraspecific hybrid dysgenesis for some pairs of lines. However, in general the presence of the P-element did not adversely affect the physiological characteristics. Either adaptation to the new TE occurs very quickly, or the rate of movement of the P-element is so insignificant that its appearance in the genome remains unnoticed.

Tirant Stealthily Invaded Natural Drosophila melanogaster Populations during the Last Century.

It was long thought that solely three different transposable elements (TEs)-the I-element, the P-element, and hobo-invaded natural Drosophila melanogaster populations within the last century. By sequencing the "living fossils" of Drosophila research, that is, D. melanogaster strains sampled from natural populations at different time points, we show that a fourth TE, Tirant, invaded D. melanogaster populations during the past century. Tirant likely spread in D. melanogaster populations around 1938, followed by the I-element, hobo, and, lastly, the P-element. In addition to the recent insertions of the canonical Tirant, D. melanogaster strains harbor degraded Tirant sequences in the heterochromatin which are likely due to an ancient invasion, likely predating the split of D. melanogaster and D. simulans. These degraded insertions produce distinct piRNAs that were unable to prevent the novel Tirant invasion. In contrast to the I-element, P-element, and hobo, we did not find that Tirant induces any hybrid dysgenesis symptoms. This absence of apparent phenotypic effects may explain the late discovery of the Tirant invasion. Recent Tirant insertions were found in all investigated natural populations. Populations from Tasmania carry distinct Tirant sequences, likely due to a founder effect. By investigating the TE composition of natural populations and strains sampled at different time points, insertion site polymorphisms, piRNAs, and phenotypic effects, we provide a comprehensive study of a natural TE invasion.

Optogenetics in Drosophila.

The fruit fly Drosophila melanogaster, an insect 4 mm long, has served as the experimental subject in a wide range of biological research, including neuroscience. In this chapter, we briefly introduce optogenetic applications in Drosophila neuroscience research. First, we describe the development of Drosophila from egg to adult. In fly neuroscience, temperature-controlled perturbation of neural activity, sometimes called "thermogenetics," has been an invaluable tool that predates the advent of optogenetics. After briefly introducing this perturbation technique, we describe the process of generating transgenic flies that express optogenetic probes in a specific group of cells. Transgenic techniques are crucial in the application of optogenetics in Drosophila neuroscience; here we introduce the transposon P-elements, ϕC31 integrase, and CRISPR-Cas9 methods. As for cell-specific gene expression techniques, the binary expression systems utilizing Gal4-UAS, LexA-lexAop, and Q-system are described. We also present a short and basic optogenetic experiment with Drosophila larvae as a practical example. Finally, we review a few recent studies in Drosophila neuroscience that made use of optogenetics. In this overview of fly development, transgenic methods, and applications of optogenetics, we present an introductory background to optogenetics in Drosophila.

piRNA-DQ722010 contributes to prostate hyperplasia of the male offspring mice after the maternal exposed to microcystin-leucine arginine.

BACKGROUND: Microcystin-leucine arginine (MC-LR) could disrupt prostate development and cause prostate hyperplasia. But whether and how maternal and before-weaning MC-LR exposure causes prostate hyperplasia in male offspring by changing expression profile of P-element-induced wimpy (PIWI)-interacting RNAs (piRNAs) have not yet been reported. METHODS: From the 12th day in the embryonic period to the 21st day after offspring birth, three groups of pregnant mice that were randomly assigned were exposed to 0, 10, and 50 µg/L of MC-LR through drinking water followed by the analyses of their male offspring. Abortion rate and litter size of maternal mice were recorded. The prostate histopathology was observed. Differential expressed piRNAs of prostate were screened by piRNA microarray analysis. Murine prostate cancer cell line (RM-1) was used for further mechanism study. Luciferase report assay was used to determine the relationship between piRNA-DQ722010 and polypeptide 3 (Pik3r3). RESULTS: The downregulated expression of piRNA-DQ722010 was the most significant in piRNA microarray analysis in 10 µg/L MC-LR treated group, while Pik3r3 was significantly upregulated, consistent with the results that a distinct prostatic epithelial hyperplasia was observed and phosphoinositide-3-kinase (PI3K)/protien kinase B (AKT) signaling pathway was activated. Pik3r3 was verified as the target gene of piRNA-DQ722010. In addition, we found MC-LR decreased the expression of PIWI-like RNA-mediated gene silencing 2 (Piwil2) and 4 (Piwil4) both in vivo and in vitro, and both Piwil4 and Piwil2 could regulate the expression of DQ722010. CONCLUSION: MC-LR caused downregulation of piRNA-DQ722010 and PIWI proteins, while piRNA-DQ722010 downregulation promoted activation of PI3K/AKT signaling pathway inducing prostate hyperplasia by upregulating the expression of Pik3r3. In contrast, piRNA-DQ722010 downregulation may be attributed to PIWI proteins downregulation.

A rapid change in P-element-induced hybrid dysgenesis status in Ukrainian populations of Drosophila melanogaster.

The Drosophila melanogaster P-transposable element is an example of mobile DNA transferred horizontally and known to have spread globally over the last 50-60 years. In Drosophila, the P-element causes a syndrome known as 'P-M hybrid dysgenesis' that obstructs normal ovary development in the female progeny of susceptible populations. Despite extensive research, the stability and global population dynamics of P-M dysgenic phenotypes remain poorly understood. Here, we report a recent and rapid transition in the P-M status of D. melanogaster populations from Ukraine. We demonstrate that these populations are currently dominated by the P'-cytotype characterized by active genomic P-elements and unknown from Ukraine just two decades ago. Our results suggest a recent invasion of the P-element in Ukraine, a pattern that matches recent discoveries from Turkey.

The recent invasion of natural Drosophila simulans populations by the P-element.

The P-element is one of the best understood eukaryotic transposable elements. It invaded Drosophila melanogaster populations within a few decades but was thought to be absent from close relatives, including Drosophila simulans. Five decades after the spread in D. melanogaster, we provide evidence that the P-element has also invaded D. simulans. P-elements in D. simulans appear to have been acquired recently from D. melanogaster probably via a single horizontal transfer event. Expression data indicate that the P-element is processed in the germ line of D. simulans, and genomic data show an enrichment of P-element insertions in putative origins of replication, similar to that seen in D. melanogaster. This ongoing spread of the P-element in natural populations provides a unique opportunity to understand the dynamics of transposable element spread and the associated piwi-interacting RNAs defense mechanisms.

Rationally subdividing the fly nervous system with versatile expression reagents.

The ability to image and manipulate specific cell populations in Drosophila enables the investigation of how neural circuits develop and coordinate appropriate motor behaviors. Gal4 lines give genetic access to many types of neurons, but the expression patterns of these reagents are often complex. Here, we present the generation and expression patterns of LexA lines based on the vesicular neurotransmitter transporters and Hox transcription factors. Intersections between these LexA lines and existing Gal4 collections provide a strategy for rationally subdividing complex expression patterns based on neurotransmitter or segmental identity.

P elements and the determinants of hybrid dysgenesis have different dynamics of propagation in Drosophila melanogaster populations.

Intraspecific hybrid dysgenesis (HD) appears after some strains of D. melanogaster are crossed. The predominant idea is that the movement of transposable P elements causes HD. It is believed that P elements appeared in the D. melanogaster genome in the middle of the last century by horizontal transfer, simultaneously with the appearance of HD determinants. A subsequent simultaneous expansion of HD determinants and P elements occurred. We analyzed the current distribution of HD determinants in natural populations of D. melanogaster and found no evidence of their further spread. However, full-sized P elements were identified in the genomes of all analyzed natural D. melanogaster strains independent of their cytotypes. Thus, the expansion of P elements does not correlate with the expansion of HD determinants. We found that the ovaries of dysgenic females did not contain germ cells despite the equal number of primordial germ cells in early stages in dysgenic and non-dysgenic embryos. We propose that HD does not result from DNA damage caused by P element transposition, but it would be the disruption in the regulation of dysgenic ovarian formation that causes the dysgenic phenotypes.

piRNA-Guided Transposon Silencing and Response to Stress in Drosophila Germline.

Transposons are integral genome constituents that can be domesticated for host functions, but they also represent a significant threat to genome stability. Transposon silencing is especially critical in the germline, which is dedicated to transmitting inherited genetic material. The small Piwi-interacting RNAs (piRNAs) have a deeply conserved function in transposon silencing in the germline. piRNA biogenesis and function are particularly well understood in Drosophila melanogaster, but some fundamental mechanisms remain elusive and there is growing evidence that the pathway is regulated in response to genotoxic and environmental stress. Here, we review transposon regulation by piRNAs and the piRNA pathway regulation in response to stress, focusing on the Drosophila female germline.

The Landscape of the DNA Transposons in the Genome of the Horezu_LaPeri Strain of Drosophila melanogaster.

Natural transposons (NTs) represent mobile DNA sequences found in both prokaryotic and eukaryotic genomes. Drosophila melanogaster (the fruit fly) is a eukaryotic model organism with NTs standing for about 20% of its genome and has contributed significantly to the understanding of various aspects of transposon biology. Our study describes an accurate approach designed to map class II transposons (DNA transposons) in the genome of the Horezu_LaPeri fruit fly strain, consecutive to Oxford Nanopore Technology sequencing. A whole genome bioinformatics analysis was conducted using Genome ARTIST_v2, LoRTE and RepeatMasker tools to identify DNA transposons insertions. Then, a gene ontology enrichment analysis was performed in order to evaluate the potential adaptive role of some DNA transposons insertions. Herein, we describe DNA transposon insertions specific for the Horezu_LaPeri genome and a predictive functional analysis of some insertional alleles. The PCR validation of P-element insertions specific for this fruit fly strain, along with a putative consensus sequence for the KP element, is also reported. Overall, the genome of the Horezu_LaPeri strain contains several insertions of DNA transposons associated with genes known to be involved in adaptive processes. For some of these genes, insertional alleles obtained via mobilization of the artificial transposons were previously reported. This is a very alluring aspect, as it suggests that insertional mutagenesis experiments conducting adaptive predictions for laboratory strains may be confirmed by mirroring insertions which are expected to be found at least in some natural fruit fly strains.

P-Element-Induced Wimpy Testis Proteins and P-Element-Induced Wimpy Testis-Interacting RNAs Expression in Ovarian Cancer Stem Cells.

Background: P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are a type of noncoding RNA and are predominantly expressed in germline cells. piRNAs function as gene regulators and potential biomarkers for the development of a number of malignancies. The biological importance of piRNAs in ovarian cancer is still unknown. In this study, we investigated the expression of piRNAs in ovarian cancer stem cells and compared it with that in adherent cells. Methods: To assess changes in the expression levels of PIWIL1/HIWI, PIWIL2/HILI, PIWIL3, and PIWIL4/HIWI2, we used quantitative reverse-transcription polymerase chain reaction (RT-qPCR) analysis. Changes in piRNA expression levels in ovarian cancer stem cells were analyzed using Arraystar piRNA microarray screening. Gene Ontology (GO) enrichment analysis was conducted to determine the potential functions of piRNAs. Results: Using microarray analysis, we identified a cohort of differentially expressed piRNAs. Fifteen piRNAs, including DQ570763 and DQ597396, were downregulated, and 58 piRNAs were upregulated when compared with those in adherent A2780 and SKOV3 cells (p > 0.05, >2.0, respectively). GO functions of the downregulated piRNAs (DQ570763 and DQ570797) suggest that their roles are commonly associated with the Golgi apparatus. In addition, A2780-SP and SKOV3-SP cells had higher PIWIL3 and PIWIL4 mRNA levels than adherent cells (A2780 and SKOV3). Moreover, we determined, using receiver operating characteristic plot, that the expression level of PIWIL4 was lower in responders than in nonresponders after treatment with platins in patients with ovarian cancer. Finally, in ovarian cancer, PIWIL4 expression was associated with somatic mutations of dynein axonemal heavy chain 2, signal induced proliferation associated 1 like 2, YTH N6-methyladenosine RNA-binding protein 1, TBC1 domain family member 8, and LPS responsive Beige-like anchor protein. Conclusion: Our study showed that PIWI proteins and piRNAs are potential diagnostic and prognostic biomarkers for ovarian cancer.

Regulatory architecture of housekeeping genes is driven by promoter assemblies.

Genes that are key to cell identity are generally regulated by cell-type-specific enhancer elements bound by transcription factors, some of which facilitate looping to distant gene promoters. In contrast, genes that encode housekeeping functions, whose regulation is essential for normal cell metabolism and growth, generally lack interactions with distal enhancers. We find that Ronin (Thap11) assembles multiple promoters of housekeeping and metabolic genes to regulate gene expression. This behavior is analogous to how enhancers are brought together with promoters to regulate cell identity genes. Thus, Ronin-dependent promoter assemblies provide a mechanism to explain why housekeeping genes can forgo distal enhancer elements and why Ronin is important for cellular metabolism and growth control. We propose that clustering of regulatory elements is a mechanism common to cell identity and housekeeping genes but is accomplished by different factors binding distinct control elements to establish enhancer-promoter or promoter-promoter interactions, respectively.

The need for unbiased genetic screens to dissect aggression in Drosophila melanogaster.

Aggression is an evolutionarily conserved behavior present in most animals and is necessary for survival when competing for limited resources and mating partners. Studies have shown that aggression is modulated both genetically and epigenetically, but details of how the molecular and cellular mechanisms interact to determine aggressive behavior remain to be elucidated. In recent decades, Drosophila melanogaster has emerged as a powerful model system to understand the mechanisms that regulate aggression. Surprisingly most of the findings discovered to date have not come from genetic screens despite the fly's long and successful history of using screens to unravel its biology. Here, we highlight the tools and techniques used to successfully screen for aggression-linked behavioral elements in Drosophila and discuss the potential impact future screens have in advancing our knowledge of the underlying genetic and neural circuits governing aggression.

Spatiotemporal transcription of the P element and the 412 retrotransposon during embryogenesis of Drosophila melanogaster and D. willistoni.

Transposable elements (TEs) are mobile nucleotide sequences which, through changing position in host genomes, partake in important evolutionary processes. The expression patterns of two TEs, P element transposon and 412 retrotransposon, were investigated during Drosophila melanogaster and D. willistoni embryogenesis, by means of embryo hybridization using riboprobes. Spatiotemporal transcription patterns for both TEs were similar to those of developmental genes. Although the two species shared the same P element transcription pattern, this was not so with 412 retrotransposon. These findings suggest that the regulatory sequences involved in the initial development of Drosophila spp are located in the transposable element sequences, and differences, such as in this case of the 412 retrotransposon, lead to losses or changes in their transcription patterns.

P-element Induced WImpy protein-like RNA-mediated gene silencing 2 regulates tumor cell progression, apoptosis, and metastasis in oral squamous cell carcinoma.

OBJECTIVE: P-element Induced WImpy protein-like RNA-mediated gene silencing 2 (PIWIL2) is a reported oncogene strongly associated with tumorigenesis and cancer progression. However, the potential function of PIWIL2 in oral cancer is still largely unclear. METHODS: In this study, we investigated the clinical significance of PIWIL2 expression in human oral squamous cell carcinoma (OSCC) cell lines and tissues. We also examined its function in OSCC pathogenesis by knocking down PIWIL2 expression with short hairpin RNAs, followed by phenotypic experiments focused on cell migration, invasion, proliferation, and apoptosis rates. RESULTS: We found that PIWIL2 was overexpressed in OSCC cell lines and tissues and significantly correlated with the malignancy stage. Furthermore, knockdown of PIWIL2 in a human OSCC cell line Tca8113 induced cell cycle arrest and apoptosis. Silencing PIWIL2 expression also significantly suppressed the migration and invasion abilities of Tca8113 cells. CONCLUSIONS: Collectively, our results suggest a functional role of PIWIL2 in regulating OSCC pathogenesis. Our data imply that PIWIL2 could serve as a potential therapeutic target for OSCC treatment.

Expression and role of P-element-induced wimpy testis-interacting RNA in diabetic-retinopathy in mice.

BACKGROUND: As one of the major microvascular complications of diabetes, diabetic retinopathy (DR) is the leading cause of blindness in the working age population. Because the extremely complex pathogenesis of DR has not been fully clarified, the occurrence and development of DR is closely related to tissue ischemia and hypoxia and neovascularization The formation of retinal neovascularization (RNV) has great harm to the visual acuity of patients. AIM: To investigate the expression of P-element-induced wimpy testis-interacting RNA (piRNA) in proliferative DR mice and select piRNA related to RNV. METHODS: One hundred healthy C57BL/6J mice were randomly divided into a normal group as control group (CG) and proliferative DR (PDR) group as experimental group (EG), with 50 mice in each group. Samples were collected from both groups at the same time, and the lesions of mice were evaluated by hematoxylin and eosin staining and retinal blood vessel staining. The retinal tissues were collected for second-generation high-throughput sequencing, and the differentially expressed piRNA between the CG and EG was detected, and polymerase chain reaction (PCR) was conducted for verification. The differentially obtained piRNA target genes and expression profiles were enrichment analysis based on gene annotation (Gene Ontology) and Kyoto Encyclopedia of Genes and Genomes. RESULTS: In the CG there was no perfusion area, neovascularization and endothelial nucleus broke through the inner boundary membrane of retinap. In the EG, there were a lot of nonperfused areas, new blood vessels and endothelial nuclei breaking through the inner boundary membrane of the retina. There was a statistically significant difference in the number of vascular endothelial nuclei breaking through the inner retinal membrane between the two groups. High-throughput sequencing analysis showed that compared with the CG, a total of 79 piRNAs were differentially expressed in EG, among which 43 piRNAs were up-regulated and 36 piRNAs were down-regulated. Bioinformatics analysis showed that the differentially expressed piRNAs were mainly concentrated in the signaling pathways of angiogenesis and cell proliferation. Ten piRNAs were selected for PCR, and the results showed that the expression of piR-MMU-40373735, piR-MMU-61121420, piR-MMU-55687822, piR-MMU-1373887 were high, and the expression of piR-MMU-7401535, piR-MMU-4773779, piR-MMU-1304999, and piR-MMU-5160126 were low, which were consistent with the sequencing results. CONCLUSION: In the EG, the abnormal expression of piRNA is involved in the pathway of angiogenesis and cell proliferation, suggesting that piRNAs have some regulatory function in proliferative diabetic-retinopathy.

Small Non-Coding-RNA in Gynecological Malignancies.

Gynecologic malignancies, which include cancers of the cervix, ovary, uterus, vulva, vagina, and fallopian tube, are among the leading causes of female mortality worldwide, with the most prevalent being endometrial, ovarian, and cervical cancer. Gynecologic malignancies are complex, heterogeneous diseases, and despite extensive research efforts, the molecular mechanisms underlying their development and pathology remain largely unclear. Currently, mechanistic and therapeutic research in cancer is largely focused on protein targets that are encoded by about 1% of the human genome. Our current understanding of 99% of the genome, which includes noncoding RNA, is limited. The discovery of tens of thousands of noncoding RNAs (ncRNAs), possessing either structural or regulatory functions, has fundamentally altered our understanding of genetics, physiology, pathophysiology, and disease treatment as they relate to gynecologic malignancies. In recent years, it has become clear that ncRNAs are relatively stable, and can serve as biomarkers for cancer diagnosis and prognosis, as well as guide therapy choices. Here we discuss the role of small non-coding RNAs, i.e., microRNAs (miRs), P-Element induced wimpy testis interacting (PIWI) RNAs (piRNAs), and tRNA-derived small RNAs in gynecological malignancies, specifically focusing on ovarian, endometrial, and cervical cancer.

Transposable elements and Piwi­interacting RNAs in hemato­oncology with a focus on myelodysplastic syndrome (Review).

Our current understanding of hematopoietic stem cell differentiation and the abnormalities that lead to leukemogenesis originates from the accumulation of knowledge regarding protein­coding genes. However, the possible impact of transposable element (TE) mobilization and the expression of P­element­induced WImpy testis­interacting RNAs (piRNAs) on leukemogenesis has been beyond the scope of scientific interest to date. The expression profiles of these molecules and their importance for human health have only been characterized recently due to the rapid progress of high­throughput sequencing technology development. In the present review, current knowledge on the expression profile and function of TEs and piRNAs was summarized, with specific focus on their reported involvement in leukemogenesis and pathogenesis of myelodysplastic syndrome.