Age‐associated de‐repression of retrotransposons in the Drosophila fat body,its potential cause and consequence

Eukaryotic genomes contain transposable elements (TE) that can move into new locations upon activation. Since uncontrolled transposition of TEs, including the retrotransposons and DNA transposons, can lead to DNA breaks and genomic instability, multiple mechanisms, including heterochromatin‐mediated repression, have evolved to repress TE activation. Studies in model organisms have shown that TEs become activated upon aging as a result of age‐associated deregulation of heterochromatin. Considering that different organisms or cell types may undergo distinct heterochromatin changes upon aging, it is important to identify pathways that lead to TE activation in specific tissues and cell types. Through deep sequencing of isolated RNAs, we report an increased expression of many retrotransposons in the old Drosophila fat body, an organ equivalent to the mammalian liver and adipose tissue. This de‐repression correlates with an increased number of DNA damage foci and decreased level of Drosophila lamin‐B in the old fat body cells. Depletion of the Drosophila lamin‐B in the young or larval fat body results in a reduction of heterochromatin and a corresponding increase in retrotransposon expression and DNA damage. Further manipulations of lamin‐B and retrotransposon expression suggest a role of the nuclear lamina in maintaining the genome integrity of the Drosophila fat body by repressing retrotransposons.     

Epigenetic telomere protection by Drosophila DNA damage response pathways

Analysis of terminal deletion chromosomes indicates that a sequence-independent mechanism regulates protection of Drosophila telomeres. Mutations in Drosophila DNA damage response genes such as atm/tefu, mre11, or rad50 disrupt telomere protection and localization of the telomere-associated proteins HP1 and HOAP, suggesting that recognition of chromosome ends contributes to telomere protection. However, the partial telomere protection phenotype of these mutations limits the ability to test if they act in the epigenetic telomere protection mechanism. We examined the roles of the Drosophila atm and atr-atrip DNA damage response pathways and the nbs homolog in DNA damage responses and telomere protection. As in other organisms, the atm and atr-atrip pathways act in parallel to promote telomere protection. Cells lacking both pathways exhibit severe defects in telomere protection and fail to localize the protection protein HOAP to telomeres. Drosophila nbs is required for both atm- and atr-dependent DNA damage responses and acts in these pathways during DNA repair. The telomere fusion phenotype of nbs is consistent with defects in each of these activities. Cells defective in both the atm and atr pathways were used to examine if DNA damage response pathways regulate telomere protection without affecting telomere specific sequences. In these cells, chromosome fusion sites retain telomere-specific sequences, demonstrating that loss of these sequences is not responsible for loss of protection. Furthermore, terminally deleted chromosomes also fuse in these cells, directly implicating DNA damage response pathways in the epigenetic protection of telomeres. We propose that recognition of chromosome ends and recruitment of HP1 and HOAP by DNA damage response proteins is essential for the epigenetic protection of Drosophila telomeres. Given the conserved roles of DNA damage response proteins in telomere function, related mechanisms may act at the telomeres of other organisms.     

Somatic recombination in adult tissues: What is there to learn?

Somatic recombination is essential to protect genomes of somatic cells from DNA damage but it also has important clinical implications, as it is a driving force of tumorigenesis leading to inactivation of tumor suppressor genes. Despite this importance, our knowledge about somatic recombination in adult tissues remains very limited. Our recent work, using the Drosophila adult midgut has demonstrated that spontaneous events of mitotic recombination accumulate in aging adult intestinal stem cells and result in frequent loss of heterozygosity (LOH). In this Extra View article, we provide further data supporting long-track chromosome LOH and discuss potential mechanisms involved in the process. In addition, we further discuss relevant questions surrounding somatic recombination and how the mechanisms and factors influencing somatic recombination in adult tissues can be explored using the Drosophila midgut model.     

CAF-1 is required for efficient replication of euchromatic DNA in <Emphasis Type="Italic">Drosophila</Emphasis> larval endocycling cells

The endocycle constitutes an effective strategy for cell growth during development. In contrast to the mitotic cycle, it consists of multiple S-phases with no intervening mitosis and lacks a checkpoint ensuring the replication of the entire genome. Here, we report an essential requirement of chromatin assembly factor-1 (CAF-1) for Drosophila larval endocycles. This complex promotes histone H3–H4 deposition onto newly synthesised DNA in vitro. In metazoans, the depletion of its large subunit leads to the rapid accumulation of cells in S-phase. However, whether this slower S-phase progression results from the activation of cell cycle checkpoints or whether it reflects a more direct requirement of CAF-1 for efficient replication in vivo is still debated. Here, we show that, strikingly, Drosophila larval endocycling cells depleted for the CAF-1 large subunit exhibit normal dynamics of progression through endocycles, although accumulating defects, such as perturbation of nucleosomal organisation, reduction of the replication efficiency of euchromatic DNA and accumulation of DNA damage. Given that the endocycle lacks a checkpoint ensuring the replication of the entire genome, the biological context of Drosophila larval development offered a unique opportunity to highlight the requirement of CAF-1 for chromatin organisation and efficient replication processes in vivo, independently of checkpoint activation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

1.688 g/cm3 satellite‐related repeats: a missing link to dosage compensation and speciation

Despite the important progress that has been made on dosage compensation (DC), a critical link in our understanding of the X chromosome recognition mechanisms is still missing. Recent studies in Drosophila indicate that the missing link could be a family of DNA repeats populating the euchromatin of the X chromosome. In this opinion article, I discuss how these findings add a new fresh twist on the DC problem. In the following sections, I first summarize our understanding of DC in Drosophila and integrate these recent discoveries into our knowledge of the X chromosome recognition problem. Next, I introduce a model according to which, 1.688 g/cm³ satellite‐related (SR) repeats would be the primary recognition elements for the dosage compensation complex. Contrary to the current belief, I suggest that the DC system in Drosophila is not conserved and static, but it is continuously co‐evolving with the target SR repeats. The potential role of the SR repeats in hybrid incompatibilities and speciation is also discussed.     

Efficient RNA virus control in Drosophila requires the RNA methyltransferase Dnmt2

Drosophila use small‐interfering RNA mechanisms to limit the amplification of viral genomes. However, it is unclear how small RNA interference components recognize and separate viral from cellular RNA. Dnmt2 enzymes are highly conserved RNA methyltransferases with substrate specificity towards cellular tRNAs. We report here that Dnmt2 is required for efficient innate immune responses in Drosophila. Dnmt2 mutant flies accumulate increasing levels of Drosophila C virus and show activated innate immune responses. Binding of Dnmt2 to DCV RNA suggests that Dnmt2 contributes to virus control directly, possibly by RNA methylation. These observations demonstrate a role for Dnmt2 in antiviral defence.     

Listeria monocytogenes induces host DNA damage and delays the host cell cycle to promote infection

Listeria monocytogenes (Lm) is a human intracellular pathogen widely used to uncover the mechanisms evolved by pathogens to establish infection. However, its capacity to perturb the host cell cycle was never reported. We show that Lm infection affects the host cell cycle progression, increasing its overall duration but allowing consecutive rounds of division. A complete Lm infectious cycle induces a S-phase delay accompanied by a slower rate of DNA synthesis and increased levels of host DNA strand breaks. Additionally, DNA damage/replication checkpoint responses are triggered in an Lm dose-dependent manner through the phosphorylation of DNA-PK, H2A.X, and CDC25A and independently from ATM/ATR. While host DNA damage induced exogenously favors Lm dissemination, the override of checkpoint pathways limits infection. We propose that host DNA replication disturbed by Lm infection culminates in DNA strand breaks, triggering DNA damage/replication responses, and ensuring a cell cycle delay that favors Lm propagation.     

Cellular and humoral immune interactions between Drosophila and its parasitoids

The immune interactions occurring between parasitoids and their host insects, especially in Drosophila–wasp models, have long been the research focus of insect immunology and parasitology. Parasitoid infestation in Drosophila is counteracted by its multiple natural immune defense systems, which include cellular and humoral immunity. Occurring in the hemocoel, cellular immune responses involve the proliferation, differentiation, migration and spreading of host hemocytes and parasitoid encapsulation by them. Contrastingly, humoral immune responses rely more heavily on melanization and on the Toll, Imd and Jak/Stat immune pathways associated with antimicrobial peptides along with stress factors. On the wasps’ side, successful development is achieved by introducing various virulence factors to counteract immune responses of Drosophila. Some or all of these factors manipulate the host's immunity for successful parasitism. Here we review current knowledge of the cellular and humoral immune interactions between Drosophila and its parasitoids, focusing on the defense mechanisms used by Drosophila and the strategies evolved by parasitic wasps to outwit it.     

Nutritional supplement chromium picolinate generates chromosomal aberrations and impedes progeny development in Drosophila melanogaster

Chromium picolinate, [Cr(pic)₃], is a popular nutritional supplement found in a variety of consumer products. Despite its popularity, safety concerns over its use have arisen. The supplement has been shown to generate clastogenic damage, mitochondrial damage, oxidative damage, and mutagenic effects in cultured cells and oxidative DNA damage and lipid peroxidation in rats. Recently [Cr(pic)₃] has been demonstrated to generate heritable genetic change and delays in progeny development in Drosophila melanogaster. Based on the damage to chromosomes of cultured cells and of animal models, similar chromosome damage appeared to be a likely source of the mutagenic effects of the supplement in Drosophila. The current three-part study examines the effects of several chromium-containing supplements and their components on hatching and eclosion rates and success of development of first generation progeny of adult Drosophila fed food containing these compounds. It further examines the effects of the compounds on longevity of virgin male and female adults. Finally, the chromosomes in the salivary glands of Drosophila late in the third instar larval stage, which were the progeny of Drosophila whose diets were supplemented with nutritional levels of [Cr(pic)₃], are shown to contain on average over one chromosomal aberration per two identifiable chromosomal arms. No aberrations were observed in chromosomes of progeny of untreated flies. The results suggest that human consumption of the supplement should be a matter of concern and continued investigation to provide insight into the requirements of chromium-containing supplements to give rise to genotoxic effects.     

The mechanism of telomere protection: a comparison between Drosophila and humans

Drosophila telomeres are maintained by transposition of specialized retrotransposons rather than by telomerase activity, and their stability is independent of the sequence of DNA termini. Recent studies have identified several proteins that protect Drosophila telomeres from fusion events. These proteins include the telomere capping factors HP1/ORC-associated protein (HOAP) and heterochromatin protein 1 (HP1), the Rad50 and Mre11 DNA repair proteins that are required for HOAP and HP1 localization at telomeres, and the ATM kinase. Another telomere-protecting factor identified in Drosophila is UbcD1, a polypeptide highly homologous to class I ubiquitin-conjugating E2 enzymes. In addition, it has been shown that HP1 and both components of the Drosophila Ku70/80 heterodimer act as negative regulators of telomere length. Except for HOAP, all these proteins are conserved in humans and are associated with human telomeres. Collectively, these results indicate that Drosophila is an excellent model system for the analysis of the mechanisms of telomere maintenance. In past and current studies, 15 Drosophila genes have been identified that prevent telomeric fusion, and it has been estimated that the Drosophila genome contains at least 40 genes required for telomere protection. We believe that the molecular characterization of these genes will lead to identification of many novel human genes with roles in telomere maintenance.     

A lucid review of Helicobacter pylori‐induced DNA damage in gastric cancer

Helicobacter pylori (H pylori) is the main risk factor for gastric cancer (GC). In recent years, many studies have addressed the effects of H pylori itself and of H pylori‐induced chronic inflammation on DNA damage. Unrepaired or inappropriately repaired DNA damage is one possible carcinogenic mechanism. We may conclude that H pylori‐induced DNA damage is one of the carcinogenic mechanisms of GC. In this review, we summarize the interactions between H pylori and DNA damage and the effects of H pylori‐induced DNA damage on GC. Then, focusing on oxidative stress, we introduce the application of antioxidants in GC. At the end of this review, we discuss the outlook for further research on H pylori‐induced DNA damage.     

Autonomous replication in Drosophila melanogaster tissue culture cells

This study addresses the ability of DNA fragments from various sources to mediate autonomous DNA replication in cultured Drosophila melanogaster cells. We created a series of plasmids containing genomic DNA fragments from the Ultrabithorax gene of Drosophila and tested them for autonomous replication after transfection into Schneider line 2 cells. We found that all plasmids containing Drosophila DNA fragments were able to replicate autonomously, as were plasmids containing random human and Escherichia coli genomic DNA fragments. Most of the plasmids were detectable 18 days after transfection in the absence of selection, suggesting that transfected DNA is maintained in Drosophila cells without rapid loss or degradation. The finding that all plasmids containing Drosophila, human, or bacterial DNA replicate autonomously in Drosophila cells suggests that the signals that direct autonomous replication in Drosophila contain a low degree of sequence specificity. A two-dimensional gel analysis of initiation on one of the plasmids was consistent with many dispersed initiation sites. Low sequence specificity and dispersed initiation sites also characterize autonomous replication in human cells and Senopus eggs and may be general properties of autonomous replication in animal cells.     

Induction of programmed lysis in <Emphasis Type="Italic">Streptomyces lividans</Emphasis> culture by the inhibitors of eukaryotic type serine/threonine protein kinases

Programmed death (PD) of the mycelium of Streptomyces lividans, namely, its delayed lysis in response to treatment with indolylmaleimide derivatives, which inhibit actinobacterial serine/threonine protein kinases (STPK), is described. Delayed lysis of mycelial cell was accompanied by DNA damage similar to PD in differentiating S. lividans mycelium. Two-dimensional electrophoresis and mass spectrometry were used to identify proteins up-regulated by a PD-inducing STPK inhibitor. Most of these proteins are known to be implicated in responses to various stress stimuli. Thus, our model of delayed cell lysis of actinobacteria upon STPK inhibition may serve for unveiling the molecular mechanisms of bacterial PD and for antimicrobial drug design.     

Stable transformation and cloning mediated by <Emphasis Type="Italic">piggyBac</Emphasis> vector and RNA interference knockdown of <Emphasis Type="Italic">Drosophila</Emphasis> ovarian cell line

An in vitro study is a powerful method for elucidating gene functions in cellular and developmental events. However, until date, no reliable in vitro transformation, cloning, or knockdown system has been reported for Drosophila cells, with the exception of S2 and Kc cells. In this study, we demonstrated that the piggyBac vector stably integrates donor DNA into ovarian somatic sheets derived from follicle stem cells. The transformed ovarian somatic sheet cells were easily cloned with a new piggyBac selection vector carrying enhanced green fluorescent protein and dihydrofolate reductase genes, egfp, and dhfr, respectively, in culture media containing methotrexate, an inhibitor of DNA synthesis. Donor egfp continued to be expressed at a high level in long-term culture. Furthermore, the translation of donor egfp was inhibited by treatment with double-stranded RNA derived from the target gene. The transfection and cloning methods mediated by the piggyBac vector would thus be useful for future analyses of gene functions in OSS cells and possibly be applicable to other Drosophila cell lines.     

The benefits of oxidative stress for tissue repair and regeneration

Recent work has strengthened Drosophila imaginal discs as a model system for regeneration studies. Evidence is accumulating that oxidative stress drives the cellular responses for repair and regeneration. Drosophila imaginal discs generate a burst of reactive oxygen species (ROS) upon damage that is necessary for the activation of the Jun N-terminal kinase (JNK) and p38 MAP kinase signaling pathways. Moreover, these pathways are pivotal in the activation of regenerative growth. A hypothetical mechanism of how the ROS are initiated, and how repair and regeneration is activated is discussed here.     

Studying the DNA damage response using in vitro model systems

Exogenous and endogenous insults continuously damage DNA. DNA damage must be detected in order to prevent loss of vital genetic information. Cells respond to DNA damage by activating checkpoint pathways that delay the progression through the cell cycle, promote DNA repair or induce cell death. A regulatory network of proteins has been identified that participate in DNA damage checkpoint pathways. Central to this network are ATM, ATR and the Mre11/Rad50/Nbs1 (MRN) complex. Detailed biochemical analysis of ATM, ATR and the MRN dependent DNA damage responses has taken advantage of several in vitro model systems to understand the detailed mechanisms underlying their function. Here we describe some recent findings obtained analysing these pathways using in vitro model systems. In particular we focus on the studies performed in the Xenopus laevis egg cell free extract, which recapitulates the DNA damage response in the context of the cell cycle.     

Teaching report: the use of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> larval thermosensitive escape behaviour as a model system to demonstrate sensory function

We describe how a novel thermosensitive escape behaviour in Drosophila larvae can be used to teach neurobiology principles to both undergraduates and school children. The assays are inexpensive, robust and reliably accurate employing apparatus readily available in most science classrooms. The use of Drosophila avoids the employment of vertebrate models and the ethical and expense issues that this entails. We use this practical to effectively teach the principals of calibration and neural sensory responses to post-16-year-old students and undergraduate students.