insects as models to study the epigenetic basis of disease

Accepted Manuscript Insects as models to study the epigenetic basis of disease Krishnendu Mukherjee, Richard M Twyman, Andreas Vilcinskas PII: S0079-6107(15)00034-6 DOI: 10.1016/j.pbiomolbio.2015.02.009 Reference: JPBM 993 To appear in: Progress in Biophysics and Molecular Biology Received Date: September 2014 Revised Date: January 2015 Accepted Date: 23 February 2015 Please cite this article as: Mukherjee, K., Twyman, R.M, Vilcinskas, A., Insects as models to study the epigenetic basis of disease, Progress in Biophysics and Molecular Biology (2015), doi: 10.1016/ j.pbiomolbio.2015.02.009 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain ACCEPTED MANUSCRIPT Insects as models to study the epigenetic basis of disease Krishnendu Mukherjee1, Richard M Twyman2 and Andreas Vilcinskas1, 3* Fraunhofer Institute for Molecular Biology and Applied Ecology, Department of RI PT Bioresources, Winchester Str 2, 35394 Giessen, Germany Institute of Phytopathology and Applied Zoology, Justus-Liebig University of Giessen, SC TRM Ltd, PO Box 93, York YO43 3WE, United Kingdom M AN U Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany Tel: ++49 641 99 37600 Fax: ++49 641 99 37609 TE D Email: krishnendu.mukherjee@agrar.uni-giessen.de Email: richard@twymanrm.com EP Email: andreas.vilcinskas@agrar.uni-giessen.de AC C *Corresponding author ACCEPTED MANUSCRIPT Abstract Epigenetic inheritance refers to changes in gene expression that are heritable across generations but are not caused by changes in the DNA sequence Many environmental factors are now known to cause epigenetic changes, including the presence of pathogens, parasites, RI PT harmful chemicals and other stress factors There is increasing evidence that transcriptional reprograming caused by epigenetic modifications can be passed from parents to offspring Indeed, diseases such as cancer can occur in the offspring due to epigenetically-inherited gene SC expression profiles induced by stress experienced by the parent Empirical studies to investigate the role of epigenetics in trans-generational gene regulation and disease require M AN U appropriate model organisms In this review, we argue that selected insects can be used as models for human diseases with an epigenetic component because the underlying molecular mechanisms (DNA methylation, histone acetylation and the expression of microRNAs) are evolutionarily conserved Insects offer a number of advantages over mammalian models TE D including ethical acceptability, short generation times and the potential to investigate complex interacting parameters such as fecundity, longevity, gender ratio, and resistance to pathogens, EP parasites and environmental stress AC C Keywords: Metamorphosis; Insect epigenetics; DNA methylation; Histone acetylation; miRNA; Model host; ACCEPTED MANUSCRIPT Introduction Epigenetics is a rapidly expanding field of research addressing hereditary mechanisms that not involve changes to the DNA sequence but which instead involve the reprograming of gene expression in response to endogenous and environmental stimuli (Jaenisch and Bird, 2003) RI PT Gene expression can be regulated before the initiation of transcription by the chemical modification of DNA or the proteins (predominantly histones) that maintain DNA as chromatin The major mechanisms are DNA methylation, histone acetylation and histone SC methylation There are also several heritable mechanisms of post-transcriptional gene regulation, the most prevalent of which is the synthesis of non-coding microRNAs (miRNAs) M AN U that bind to corresponding messenger RNAs (mRNAs) and degrade them or inhibit translation (Bushati et al., 2007) The precise definition of epigenetics is contested because it is used in different contexts Waddington (1942) coined the term to describe how genotypes produce phenotypes by interacting with the environment, but in contemporary usage its meaning has TE D been refined to cover both the concept of heritable changes in the absence of mutation, and the underlying mechanisms themselves, which may not strictly be heritable (Ledford, 2008) Herein we distinguish between these concepts by using the terms epigenetic inheritance EP (changes in gene expression that are heritable through mitosis or meiosis) and epigenetic AC C mechanisms (the underlying molecular mechanisms, which are not necessarily heritable) Insects are ideal models for the investigation of epigenetic inheritance because many insect species demonstrate phenotypic plasticity, i.e they occur in two or more morphologically distinct phenotypes defined by the same genotype, such as male and female adults, eusocial castes, winged and non-winged aphids, and the larvae and imagoes of holometabolous insects such as flies, butterflies and beetles (Simpson et al., 2011; Srinivasan and Brisson, 2012) In such species, the phenotype is often determined by environmental stimuli For example, female honeybee larvae (Apis mellifera) can develop into either long-lived queens responsible ACCEPTED MANUSCRIPT for reproduction or short-lived, sterile workers whose function is to feed, clean and protect the colony Workers initially feed the larvae with royal jelly produced by the queen but usually switch to pollen once the larvae have reached a certain size, which produces more workers (Chittka and Chittka, 2010) However, if they feed the larvae with royal jelly continuously, RI PT this results in the development of a new queen The royal jelly contains phenyl butyrate, which is an inhibitor of histone deacetylase The decision between worker and queen development therefore depends on diet-induced epigenetic transcriptional reprogramming methylation (Chittka and Chittka, 2010) SC mediated by histone modification, which ultimately leads to heritable changes in DNA M AN U In the above example, the epigenetic mechanism is controlled by the diet, but other stimuli that have epigenetic effects include temperature, illumination and different forms of stress The field of epigenetics aims to determine how epigenetic mechanisms translate environmental stimuli into transcriptional reprogramming to create different phenotypes, TE D which are in some cases heritable across generations This review describes the development and application of insects as models to investigate the causes and consequences of epigenetic inheritance in response to pathogens, chemicals and environmental stress factors such as heat AC C EP shock and starvation Epigenetic mechanisms 2.1 DNA methylation DNA methylation in eukaryotes typically involves the addition of methyl groups to cytidine residues in the sequence CpG (in animals) and CpNpG (in plants) to create 5-methylcytidine, which behaves as normal in terms of DNA base-pairing but changes the way DNA interacts with proteins thus providing a mechanism for gene regulation This form of methylation occurs at sites with a two-fold rotational axis of symmetry, so the methylation mark can be ACCEPTED MANUSCRIPT passed to daughter cells during DNA replication because one strand remains methylated in the daughter duplex, thus explaining how the epigenetic state can be inherited The transfer of methyl groups to DNA is mediated by several evolutionarily-conserved enzymes collectively known as DNA methyltransferases (DNMTs) These can be divided RI PT further into maintenance methyltransferases, which complete the symmetrical methylation marks on newly-replicated DNA by recognizing the hemimethylated sequences inherited from each parent, and de novo methyltransferases, which establish new methylation marks on SC unmethylated DNA (Bestor, 2000; Klose and Bird, 2006) In mammals, DNMT1 is classified as a maintenance methyltransferase, DNMT3 is a de novo methyltransferase and DNMT2 was M AN U initially misclassified and is now known to methylate transfer RNA (tRNA), which carries a number of constitutive DNA modifications (Goll et al., 2006) Whole-genome methylation analysis in insects such as the honeybee, silkworm moth (Bombyx mori) and parasitic wasp (Nasonia vitripennis) has shown that 5-methylcytosine is by far the TE D most common form of programmed DNA modification (Beeler et al., 2014; Cingolani et al., 2013; Xiang et al., 2013) However, the proportion of methylated CpG is much lower in EP insects than in humans, and the occupied sites in the insect genome are primarily restricted to exons (Glastad et al., 2011) For example, the honeybee genome contains more than 10 AC C million CpG sites but only 70,000 (0.7%) are methylated (Lyko et al., 2010) compared to 70% occupancy in humans (Strichman-Almashanu et al., 2002) Interestingly, the methylated honeybee genes are predominantly those conserved among arthropods rather than restricted to this species, although 550 genes show caste-specific differential methylation between queens and workers (Lyko et al., 2010) There is also a dynamic cycle of methylation and demethylation during the life cycle of model insects such as the honeybee and the red flour beetle (Tribolium castaneum) with the greatest degree of CpG methylation found in the embryos (Drewell et al., 2014; Felliciello et al., 2013) ACCEPTED MANUSCRIPT 2.2 Histone acetylation Gene expression in eukaryotes is regulated by specific proteins known as transcription factors which influence the manner in which DNA interacts with transcriptional apparatus As well as these specific interactions, the behavior of DNA can be influenced more generally by RI PT conformational changes in the proteins that make up chromatin, i.e the complex of DNA and protein which is structural basis of chromosomes in the eukaryotic cell nucleus (Fig 1) The basic repeat element of chromatin is the nucleosome, which comprises DNA wrapped around SC an octamer of core histones (two each of H2A, H2B, H3 and H4) The nucleosomes are linked together like beads on a string by segments of linker DNA paired with linker histones (H1, M AN U H5) The structure of chromatin, and hence the accessibility of the DNA, can be controlled by modulating the positive charge density of the core histones, principally by the addition or removal of acetyl groups (Fig 1) Acetylated histones form a loose and accessible type of chromatin that promotes gene expression, whereas deacetylated histones bind DNA more TE D tightly and render it inaccessible and transcriptionally silent This property of histones is controlled by the opposing activities of two families of enzymes: histone acetyltransferases (HATs) and histone deacetylases (HDACs) It is unclear whether particular histone EP modification states are heritable per se, but there is significant crosstalk among histone AC C modification, DNA methylation and even post-transcriptional regulation which helps to reinforce and perpetuate the consequences of histone-based epigenetic mechanisms The genome sequence of the fruit fly (Drosophila melanogaster) revealed the presence of HATs belonging to the MYST, GNAT and CBP/p300 gene families, and similar enzyme repertoires have been identified by transcriptomic analysis in insects lacking a complete genome sequence, such as the greater wax moth Galleria mellonella (Mukherjee et al., 2012) Four classes of HDACs have been identified in humans, and three of these have also been found in the fruit fly whereas only a class I HDAC has been found in the greater wax moth ACCEPTED MANUSCRIPT The balance between HAT and HDAC activity has a significant impact on gene regulation during development and in disease, as established by the use of specific HAT and HDAC inhibitors in both mammalian and insect disease models (Mukherjee et al., 2012) Accordingly, several such inhibitors are undergoing clinical trials in humans for indications RI PT such as cancer and multiple sclerosis, which are known to have an epigenetic component (Szyf, 2009) SC 2.3 MicroRNAs M AN U MicroRNAs (miRNAs) are non-coding RNAs 18–22 nucleotides in length which regulate gene expression at the post-transcriptional level by binding to complementary mRNAs They are involved in the regulation of gene expression during many physiological processes (e.g development, immunity, cell cycle progression and apoptosis) and are also associated with a TE D number of diseases (Ambros, 2004; Bartel, 2004; Lu and Linston, 2009) The genes encoding miRNAs are found individually and as polycistronic clusters (Lagos-Quintana et al., 2001) In the nucleus, the transcription of miRNA genes by RNA polymerase II/III produces double- EP stranded transcripts known as primary miRNAs (pri-miRNAs) These are trimmed by the RNase III enzyme Dorsha to form double-stranded stem-loop precursor miRNAs (pre- AC C miRNAs) that are transported to the cytoplasm by Exportin-5 Pre-miRNAs are further processed into unstable, 18–22-nt duplex structures by the RNase III enzyme Dicer, which also initiates the formation of an RNA-induced silencing complex (RISC) One strand of this duplex, representing a mature miRNA, is then incorporated into the RISC and guides it to the target mRNA sequence, causing the silencing of gene expression The RISC is a ribonucleoprotein complex containing an Argonaute (Ago) family protein, whose endonuclease activity is directed against mRNA strands that are complementary to the bound miRNA fragment If the sequence complementarity is perfect the mRNA target is cleaved and ACCEPTED MANUSCRIPT degraded, whereas imperfectly-matched mRNAs are deadenylated or arrested in the complex resulting in translational repression (Bartel, 2009; Macfarlane and Murphy, 2010) Because the function of miRNAs is sequence-dependent, individual miRNAs can target multiple mRNAs as long as they each contain the matching complementary sequence, and RI PT individual mRNAs can be regulated by multiple miRNAs if the mRNA contains more than one target sequence The detailed comparison of genome sequences has revealed a surprising degree of conservation in the miRNA repertoires of insects and mammals, but many further SC miRNAs are limited to particular species and sophisticated bioinformatics algorithms are needed to identify them de novo, which can be expensive and time consuming If a complete M AN U genome sequence is not available, the differential expression of miRNAs can be established using microarray technology and validated by quantitative RT-PCR This has been highly effective for the comparison of greater wax moth miRNAs that are active at different developmental stages or induced by pathogens, but it is important to validate the sequences TE D and to control for non-specific hybridization (Mukherjee and Vilcinskas, 2014) EP Advantages of insect models in epigenetic research The epigenetic inheritance of transcriptional reprogramming can be investigated using model AC C mammals (usually mice) but this requires large numbers of animals to be housed, monitored and subjected to invasive testing and therefore raises both economic and ethical concerns Mammalian cell lines are used as a surrogate system wherever possible, but cell lines not allow the investigation of trans-generational effects, i.e the epigenetic transmission of gene regulation states through meiosis Insects provide an ideal way to bridge this gap Large numbers of insects can be maintained easily and inexpensively over multiple generations, and experiments on insects are ethically acceptable National and international regulations concerning the use of animals in scientific procedures are less restrictive for insects The ACCEPTED MANUSCRIPT development of insect models for the investigation of epigenetic plasticity and inheritance in human diseases will therefore reduce the costs of basic and preclinical research Insects can be used to screen for novel drugs that reverse aberrant gene expression profiles associated with cancer, inflammatory/autoimmune diseases and metabolic disorders (Szyf, 2008) The RI PT demand for alternative and inexpensive whole-animal high-throughput testing systems has become acute given the recent recognition that many common drugs can interfere with epigenetic mechanisms and can induce negative side-effects in subsequent generations, e.g SC up to 5% of all drugs can potentially interfere with histone acetylation in humans (Lötsch et al., 2013) M AN U Insect species with completely sequenced genomes, such as the fruit fly and red flour beetle, are well established as models to explore the molecular basis of human diseases (Brandt and Vilcinskas, 2013; Prüßing et al., 2013; Tipping and Perrimon, 2014; Lee and Lee, 2014) Other insects such as the greater wax moth have emerged as useful model hosts for human TE D pathogens (Arvanitis et al., 2013; Kavanagh and Reeves, 2004) However, the use of such models is expanding beyond the genetic analysis of disease etiology and host–pathogen interactions to include epigenetic mechanisms, particularly those which translate EP environmental stimuli into transcriptional reprogramming across multiple generations AC C (Srinivasan and Brison, 2012) Insects are ideal models for the analysis of trans-generational effects First, the short generation time facilitates the rapid and quantitative assessment of multi-generational responses to stress, e.g the red flour beetle completes its life cycle in ~30 days (depending on temperature) which allows 10–12 generations to be studied in one year Second, model insects have morphologically distinct developmental stages (egg, larvae, pupae and imagoes) so that factors affecting developmental progress can be measured objectively (e.g using metrics such as the percentage of a population that transforms from last-instar larvae into pupae) Insect ACCEPTED MANUSCRIPT Cingolani, P., Cao, X., Khetani, R.S., Chen, C.C., Coon, M., Sammak, A., Bollig-Fischer, A., Land, S., Huang, Y., Hudson, M.E., Garfinkel, M.D., Zhong, S., Robinson, G.E., Ruden, D.M., 2013 Intronic non-CG DNA hydroxymethylation and alternative mRNA splicing in honey bees BMC Genomics 14:666 RI PT Da Ros, V.G., Gutierrez-Perez, I., Ferres-Marco, D., Dominguez, M., 2013 Dampening the signals transduced through hedgehog via microRNA miR-7 facilitates notch-induced tumourigenesis PLoS Biol 11, e1001554 SC De Loof, A., 2011 Longevity and aging in insects: Is reproduction costly; cheap; beneficial or M AN U irrelevant? A critical evaluation of the "trade-off" concept J Insect Physiol 57, 1-11 Dillin, A., Gottschling, D.E., Nyström, T., 2014 The good and the bad of being connected: the integrons of aging Curr Opin Cell Biol 26, 107–112 Drewell, R.A., Bush, E.C., Remnant, E.J., Wong, G.T., Beeler, S.M., Stringham, J.L., Lim, J., TE D Oldroyd, B.P., 2014 The dynamic DNA methylation cycle from egg to sperm in the honey bee Apis mellifera Development 141, 2702-11 Feliciello, I., Parazajder, J., Akrap, I., Ugarković, D., 2013 First evidence of DNA EP methylation in insect Tribolium castaneum: environmental regulation of DNA methylation AC C within heterochromatin Epigenetics 8: 534-41 Ferres-Marco, D., Gutierrez-Garcia, I., Vallejo, D.M., Bolivar, J., Gutierrez-Aviño, F.J., Dominguez, M., 2006 Epigenetic silencers and Notch collaborate to promote malignant tumours by Rb silencing Nature 439, 430-6 Freitak, D., Knorr, E., Vogel, H., Vilcinskas, A., 2012 Gender- and stressor-specific microRNA expression in Tribolium castaneum Biol Lett 8, 860-3 23 ACCEPTED MANUSCRIPT Freitak, D., Schmidtberg, H., Dickel, F., Lochnit, G., Vogel, H., Vilcinskas, A., 2014 The maternal transfer of bacteria can mediate trans-generational immune priming in insects Virulence 5, 547-54 Ge, W., Chen, Y W., Weng, R., Lim, S F., Buescher, M., Zhang, R., Cohen, S M., 2012 RI PT Overlapping functions of microRNAs in control of apoptosis during Drosophila embryogenesis Cell Death Differ 19, 839-46 Ghayad, S.E., Cohen, P.A., 2010 Inhibitors of the PI3K/Akt/mTOR pathway: new hope for SC breast cancer patients Recent Pat Anticancer Drug Discov 5, 29-57 M AN U Glastad, K.M., Hunt, B G., Yi, S.V., Goodisman, M.A., 2011 DNA methylation in insects: on the brink of the epigenomic era Insect Mol Biol 20, 553-65 Glavis-Bloom, J., Muhammed, M., Mylonakis, E., 2012 Of model hosts and man: using Caenorhabditis elegans, Drosophila melanogaster and Galleria mellonella as model hosts for TE D infectious disease research Adv Exp Med Biol 710, 11-7 Goll, M.G., Kirpekar, F., Maggert, K.A., Yoder, J.A., Hsieh, C.L., Zhang, X., Golic, K.G., Jacobsen, S.E., Bestor, T.H., 2006 Methylation of tRNAAsp by the DNA methyltransferase EP homolog Dnmt2 Science 311, 395-8 AC C Gómez-Díaz, E., Jordà, M., Peinado, M.A., Rivero, A., 2012 Epigenetics of host-pathogen interactions: the road ahead and the road behind PLoS Pathog 8, e1003007 Gomez-Orte, E., Belles, X., 2009 MicroRNA-dependent metamorphosis in hemimetabolan insects Proc Natl Acad Sci U S A 106, 21678-82 Greiss, S., Gartner, A., 2009 Sirtuin/Sir2 phylogeny, evolutionary considerations and structural conservation Mol Cells 28, 407-15 Grünwald, S., Stellzig, J., Adam, I.V., Weber, K., Binger, S., Boll, M., Knorr, E., Twyman, R.M., Vilcinskas, A., Wenzel, U., 2013 Longevity in the red flour beetle Tribolium 24 ACCEPTED MANUSCRIPT castaneum is enhanced by broccoli and depends on nrf-2, jnk-1 and foxo-1 homologous genes Genes Nutr 8, 439-48 Gschwandtner, M., Zhong, S., Tschachler, A., Mlitz, V., Karner, S., Elbe-Bürger, A., Mildner, M., 2014 Fetal human keratinocytes produce large amounts of antimicrobial peptides: RI PT involvement of histone-methylation processes J Invest Dermatol 134, 2192-201 Herranz, H., Hong, X., Hung, N T., Voorhoeve, P M., Cohen, S M., 2012 Oncogenic cooperation between SOCS family proteins and EGFR identified using a Drosophila SC epithelial transformation model Genes Dev 26, 1602-11 M AN U Jaenisch, R., Bird, A., 2003 Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals Nat Genet 33, 245–254 Jakovcevski, M., Akbarian, S., 2012 Epigenetic mechanisms in neurological disease Nat Med 18, 1194-204 TE D Jayachandran, B., Hussain, M., Asgari, S., 2012 An insect trypsin-like serine protease as a target of microRNA: utilization of microRNA mimics and inhibitors by oral feeding Insect Biochem Mol Biol 43, 398-406 EP Kaszubowska, L., 2008 Telomere shortening and ageing of the immune system J Physiol AC C Pharmacol 9, 169-86 Kang, H.L., Benzer, S., Min, K.T., 2002 Life extension in Drosophila by feeding a drug Proc Natl Acad Sci U S A 99, 838-43 Kavanagh, K., Reeves, E.P., 2004 Exploiting the potential of insects for in vivo pathogenicity testing of microbial pathogens FEMS Microbiol Rev 28, 101-12 Klose, R.J., Bird, A.P., 2006 Genomic DNA methylation: the mark and its mediators Trends Biochem Sci 31, 89-97 25 ACCEPTED MANUSCRIPT Kucharski, R., Maleszka, J., Foret, S., Maleszka, R., 2008 Nutritional control of reproductive status in honeybees via DNA methylation Science 319: 1827–1830 Lagos-Quintana, M., Rauhut, R., Lendeckel, W., Tuschl, T., 2001 Identification of novel Ledford, H., 2008 Disputed definitions Nature 455, 1023–1028 RI PT genes coding for small expressed RNAs Science 294, 853-8 Lee, J., Hwang, Y.J., Kim, K.Y., Kowall, N.W., Ryu, H., 2013 Epigenetic mechanisms of SC neurodegeneration in Huntington's disease Neurotherapeutics 10, 664-76 Lee, K.A., Lee, W.J., 2014 Drosophila as a model for intestinal dysbiosis and chronic M AN U inflammatory diseases Dev Comp Immunol 42, 102-10 Lötsch, J., Schneider, G., Reker, D., Parnham, M., Schneider, P., Geisslinger, G., Doehring, A., 2013 Common non-epigenetic drugs as epigenetic modulators Trends Mol Med 19, 742-753 TE D Lu, L.F., Liston, A., 2009 MicroRNA in the immune system, microRNA as an immune system Immunology 127, 291–298 EP Lv, W.W., Wei, H.M., Wang, D.L., Ni, J.Q., Sun, F.L., 2012 Depletion of histone deacetylase antagonizes PI3K-mediated overgrowth of Drosophila organs through the AC C acetylation of histone H4 at lysine 16 J Cell Sci 125, 5369-78 Lyko, F., Foret, S., Kucharski, R., Wolf, S., Falckenhayn, C., Maleszka, R., 2010 The honey bee epigenomes: differential methylation of brain DNA in queens and workers PLoS Biol 8, e1000506 Macfarlane, L.A., Murphy, P.R., 2010 MicroRNA: Biogenesis, Function and Role in Cancer Curr Genomics 11, 537-61 26 ACCEPTED MANUSCRIPT Martelli, A.M., Evangelisti, C., Chiarini, F., Grimaldi, C., Manzoli, L., McCubrey, J.A., 2009 Targeting the PI3K/AKT/mTOR signaling network in acute myelogenous leukemia Expert Opin Investig Drugs 18, 1333-49 RI PT Merschbaecher, K., Haettig, J., Mueller, U., 2012 Acetylation-mediated suppression of transcription-independent memory: bidirectional modulation of memory by acetylation PLoS One 7, e45131 SC Mishra, P.K., Baum, M., Carbon, J., 2011 DNA methylation regulates phenotype-dependent transcriptional activity in Candida albicans Proc Natl Acad Sci U S A 108, 11965-70 M AN U Mujtaba, S., Winer, B.Y., Jaganathan, A., Patel, J., Sgobba, M., Schuch, R., Gupta, Y.K , Haider, S., Wang, R., Fischetti, V.A., 2013 Anthrax SET protein: a potential virulence determinant that epigenetically represses NF-κB activation in infected macrophages J Biol Chem 288, 23458-72 TE D Mukherjee, K., Altincicek, B., Hain, T., Domann, E., Vilcinskas, A., Chakraborty, T., 2010 Galleria mellonella as a model system for studying Listeria pathogenesis Appl Environ EP Microbiol 76, 310-7 Mukherjee, K., Abu Mraheil, M., Silva, S., Müller, D., Cemic, F., Hemberger, J., Hain, T., AC C Vilcinskas, A., Chakraborty, T., 2011 Anti-Listeria activities of Galleria mellonella hemolymph proteins Appl Environ Microbiol 77, 4237-40 Mukherjee, K., Hain, T., Fischer, R., Chakraborty, T., Vilcinskas, A., 2013 Brain infection and activation of neuronal repair mechanisms by the human pathogen Listeria monocytogenes in the lepidopteran model host Galleria mellonella Virulence 4, 324-32 Mukherjee, K., Fischer, R., Vilcinskas, A., 2012 Histone acetylation mediates epigenetic regulation of transcriptional reprogramming in insects during metamorphosis, wounding and infection Front Zool 9, 25 27 ACCEPTED MANUSCRIPT Mukherjee, K., Vilcinskas, A., 2014 Development and immunity-related microRNAs of the lepidopteran model host Galleria mellonella BMC Genomics 15, 705 Nairz, K., Rottig, C., Rintelen, F., Zdobnov, E., Moser, M., Hafen, E., 2006 Overgrowth caused by misexpression of a microRNA with dispensable wild-type function Dev Biol 291, RI PT 314-24 Nelson, C., Ambros, V., Baehrecke, E H., 2014 miR-14 regulates autophagy during SC developmental cell death by targeting ip3-kinase Mol Cell 56, 376-88 Pancratov, R., Peng, F., Smibert, P., Yang, S.Jr., Olson, E.R., Guha-Gilford, C., Kapoor, A.J., M AN U Liang, F.X., Lai, E.C., Flaherty, M.S., DasgGupta, R., 2013 The miR-310/13 cluster antagonizes β-catenin function in the regulation of germ and somatic cell differentiation in the Drosophila testis Development 140, 2904-16 Parrella, E., Longo, V.D., 2010 Insulin/IGF-I and related signaling pathways regulate aging TE D in nondividing cells: from yeast to the mammalian brain ScientificWorld Journal 10, 161-77 Peña-Altamira, L.E., Polazzi, E., Monti, B., 2013 Histone post-translational modifications in Huntington's and Parkinson's diseases Curr Pharm Des.19, 5085-92 EP Pirooznia, S K., Elefant, F., 2013 A HAT for sleep?: epigenetic regulation of sleep by Tip60 AC C in Drosophila Fly (Austin) 7, 99-104 Prüßing, K., Voigt, A., Schulz, J.B., 2013 Drosophila melanogaster as a model organism for Alzheimers`s disease Mol Neurodegener 8, 35 Sadri-Vakili, G., Bouzou, B., Benn, C.L., Kim, M.O., Chawla, P., Overland, R.P., Glajch, K.E., Xia, E., Qiu, Z., Hersch, S.M., Clark, T.W., Yohrling, G.J., Cha, J.H., 2007 Histones associated with downregulated genes are hypo-acetylated in Huntington's disease models Hum Mol Genet 16, 1293-306 28 ACCEPTED MANUSCRIPT Skalsky, R.L., Cullen, B.R., 2010 Viruses, microRNAs, and host interactions Annu Rev Microbiol 64, 123–141 Skalsky, R L., Vanlandingham, D L., Scholle, F., Higgs, S., Cullen, B R., 2010 Identification of microRNAs expressed in two mosquito vectors, Aedes albopictus and Culex RI PT quinquefasciatus BMC Genomics 11:119 Simpson, S.J., Sword, G.A., Lo, N., 2011 Polyphenism in insects Curr Biol 21, R738-49 SC Srinivasan, D.G., Brisson, J.A., 2012 Aphids: a model for polyphenism and epigenetics Genet Res Int 2012:431531 M AN U Steinert, J.R., Campesan, S., Richards, P., Kyriacou, C.P., Forsythe, I.D., Giorgini, F., 2012 Rab11 rescues synaptic dysfunction and behavioural deficits in a Drosophila model of Huntington's disease Hum Mol Genet 21, 2912-22 Strichman-Almashanu, L.Z., Lee, R.S., Onyango, P.O., Perlman, E., Flam, F., Frieman, M.B., TE D Feinberg, A.P., 2002 A genome-wide screen for normally methylated human CpG islands that can identify novel imprinted genes Genome Res 12, 543-5454 Szyf, M., 2008 DNA demethylation and cancer metastasis: therapeutic implications Expert EP Opin Drug Discov 3, 519-31 AC C Szyf, M., 2009 Epigenetics, DNA methylation and chromatin modifying drugs Annu Rev Pharmacol Toxicol 49, 243-263 Tabunoki, H., Ono, H., Ode, H., Ishikawa, K., Kawana, N., Banno, Y., Shimada, T., Nakamura, Y., Yamamoto, K., Satoh, J., Bono, H., 2013 Identification of key uric acid synthesis pathway in a unique mutant silkworm Bombyx mori model of Parkinson's disease PLoS One 8, e69130 Tipping, M., Perrimon, N., 2014 Drosophila as a model for context-dependent tumorigenesis J Cell Physiol 229, 27-33 29 ACCEPTED MANUSCRIPT Truscott, M., Islam, A B., López-Bigas, N., Frolov, M V., 2011 mir-11 limits the proapoptotic function of its host gene, dE2f1 Genes Dev 25, 1820-34 Vallejo, D.M., Caparros, E., Dominguez, M., 2011 Targeting Notch signalling by the 756-69 RI PT conserved miR-8/200 microRNA family in development and cancer cells EMBO J 230, Vellai, T., Takács-Vellai, K., Sass, M., Klionsky, D.J., 2009 The regulation of aging: does SC autophagy underlie longevity? Trends Cell Biol 19, 487-94 Varghese J, Cohen S.M., 2007 MicroRNA miR-14 acts to modulate a positive autoregulatory M AN U loop controlling steroid hormone signaling in Drosophila Genes Dev 21, 2277–2282 Wang, Y., Li, D.D., Jiang, Y.Y., Mylonakis, E., 2013 Utility of insects for studying human pathogens and evaluating new antimicrobial agents Adv Biochem Eng Biotechnol 35, 125 TE D Winter, F., Edaye, S., Hüttenhofer, A., Brunel, C., 2007 Anopheles gambiae miRNAs as actors of defence reaction against Plasmodium invasion Nucleic Acids Res 35, 6953-62 Wood, J.G., Rogina, B., Lavu, S., Howitz, K., Helfand, S.L., Tatar, M., Sinclair, D., 2004 EP Sirtuin activators mimic caloric restriction and delay ageing in metazoans Nature 430, 686-9 AC C Xiang, H., Li, X., Dai, F., Xu, X., Tan, A., Chen, L., Zhang, G., Ding, Y., Li, Q., Lian, J., Willden, A., Guo, Q., Xia, Q., Wang, J., Wang, W., 2013 Comparative methylomics between domesticated and wild silkworms implies possible epigenetic influences on silkworm domestication BMC Genomics 14, 646 Xiao, C., Rajewsky, K., 2009 MicroRNA control in the immune system: basic principles Cell 136, 26-36 30 ACCEPTED MANUSCRIPT Xiong, Y., Zhao, K., Wu, J., Xu, Z., Jin, S., Zhang, Y.Q., 2013 HDAC6 mutations rescue human tau-induced microtubule defects in Drosophila Proc Natl Acad Sci U S A 110, RI PT 4604-9 Figure legends: Fig Chromatin modifications mediated by DNA methylation and histone acetylation SC Gene expression can be regulated before the initiation of transcription by the chemical modification of DNA or associated histone proteins The addition of a methyl group (CH3) to M AN U cytidine residues in the dinucleotide motif CpG forms 5-methylcytosine, which base pairs with guanidine as normal but modifies the way in which DNA interacts with the proteins that control gene expression The chromatin structure can be also controlled by modulating the positive charge density of the core histones The removal of acetyl groups produces a compact TE D form of chromatin that is inaccessible to RNA polymerase, whereas the addition of acetyl groups produces a loose form of chromatin that favors transcriptional activity EP Fig The impact of contaminated diets on the fecundity of the greater wax moth The average number of eggs laid by adult female wax moths increased significantly if the larvae AC C were fed on a diet which was contaminated with non-pathogenic E coli but declined significantly if the larvae were fed on a diet contaminated with the entomopathogenic bacterium S entomophila (discussed in section 3) Data are means of three independent experiments ± standard deviations (* p < 0.05) 31 ACCEPTED MANUSCRIPT Fig The five most relevant insect models for epigenetic research The fruit fly (Drosophila melanogaster), the silkworm (Bombyx mori), the honeybee (Apis mellifera), the red flour beetle (Tribolium castaneum) and the greater wax moth (Galleria mellonella) have each been selected as models for epigenetic research based on features such as genetic RI PT tractability, phenotypic plasticity, conservation of epigenetic, developmental and immunityrelated pathways with mammals, conservation of disease phenotypes and ability to host human pathogens SC Fig Transcriptional analysis of genes encoding HDACs and HATs in greater wax moth larval midgut and eggs Real-time quantitative RT-PCR analysis of transcripts M AN U encoding histone deacetylase 8, histone deacetylase isoform 2, histone deacetylase complex subunit sap18, histone acetyltransferase Tip60 and histone acetyltransferase type B catalytic subunit in (A) larval midgut and (B) eggs after feeding the larvae on diets contaminated with S entomophila or E coli (discussed in section 7) The expression levels are presented as fold- TE D change values compared to controls fed on uncontaminated diets and the data are normalized against the 18S rRNA housekeeping gene methylation, EP Fig Overview of insect epigenetics: Conserved epigenetic mechanisms including DNA histone acetylation and miRNA expression regulate transcriptional AC C reprogramming in model insects affecting similar pathophysiological processes as seen in humans, aging, neurodegeneration, cancer, inflammation and sepsis 32 AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT