Review
Insects as models to study the epigenetic basis of disease
Krishnendu Mukherjee
a, Richard M. Twyman
b, Andreas Vilcinskas
a,c,*aFraunhofer Institute for Molecular Biology and Applied Ecology, Department of Bioresources, Winchester Str. 2, 35394 Giessen, Germany bTRM Ltd, PO Box 93, York YO43 3WE, United Kingdom
cInstitute of Phytopathology and Applied Zoology, Justus-Liebig University of Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany
a r t i c l e i n f o
Article history:
Available online 14 March 2015 Keywords: Metamorphosis Insect epigenetics DNA methylation Histone acetylation miRNA Model host
a b s t r a c t
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, harmful chemicals and other stress factors. There is increasing evidence that transcriptional reprogramming caused by epigenetic modifi-cations can be passed from parents to offspring. Indeed, diseases such as cancer can occur in the offspring due to epigenetically-inherited gene 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 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 mecha-nisms (DNA methylation, histone acetylation and the expression of microRNAs) are evolutionarily conserved. Insects offer a number of advantages over mammalian models including ethical acceptability, short generation times and the potential to investigate complex interacting parameters such as fecundity, longevity, gender ratio, and resistance to pathogens, parasites and environmental stress.
© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Contents 1. Introduction . . . 69 2. Epigenetic mechanisms . . . 70 2.1. DNA methylation . . . .. . . 70 2.2. Histone acetylation . . . 70 2.3. MicroRNAs . . . 71
3. Advantages of insect models in epigenetic research . . . 72
4. Development, aging and longevity . . . 73
5. Neurodegenerative disorders . . . 74
6. Cancer . . . 74
7. Inflammation and sepsis . . . 75
8. Conclusions . . . 76
Acknowledgments . . . 76
References . . . 76
1. Introduction
Epigenetics is a rapidly expandingfield of research addressing hereditary mechanisms that do not involve changes to the DNA sequence but which instead involve the reprogramming of gene expression in response to endogenous and environmental stimuli * Corresponding author. Institute of Phytopathology and Applied Zoology,
Justus-Liebig University of Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany. Tel.:þ49 641 99 37600; fax: þ49 641 99 37609.
E-mail addresses:[email protected](K. Mukherjee),
[email protected](R.M. Twyman),[email protected]
(A. Vilcinskas).
Contents lists available atScienceDirect
Progress in Biophysics and Molecular Biology
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m/ l o c a t e / p b i o m o l b io
http://dx.doi.org/10.1016/j.pbiomolbio.2015.02.009
(Jaenisch and Bird, 2003). 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 methylation. There are also several heri-table mechanisms of post-transcriptional gene regulation, the most prevalent of which is the synthesis of non-coding microRNAs (miRNAs) that bind to corresponding messenger RNAs (mRNAs) and degrade them or inhibit translation (Bushati and Cohen, 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 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 (changes in gene expression that are heritable through mitosis or meiosis) and epigenetic mechanisms (the underlying molecular mechanisms, which are not necessarily heritable).
Insects are ideal models for the investigation of epigenetic in-heritance 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, but-terflies 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 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, 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 mediated by histone modification, which ultimately leads to heritable changes in DNA methylation (Chittka and Chittka, 2010).
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. Thefield of epigenetics aims to determine how epigenetic mechanisms trans-late environmental stimuli into transcriptional reprogramming to create different phenotypes, 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 shock and starvation.
2. 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 an-imals) 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 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 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 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 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 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 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 million CpG sites but only 70,000 (0.7%) are methylated (Lyko et al., 2010) compared to 70% occu-pancy 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 redflour beetle (Tribolium castaneum) with the greatest degree of CpG methylation found in the embryos (Drewell et al., 2014; Feliciello et al., 2013). 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 conformational changes in the proteins that make up chromatin, i.e. the complex of DNA and protein which is the 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 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, 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 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 deacety-lases (HDACs). It is unclear whether particular histone modification states are heritable per se, but there is significant crosstalk among histone modification, DNA methylation and even post-transcriptional regulation which helps to reinforce and perpet-uate the consequences of histone-based epigenetic mechanisms.
The genome sequence of the fruitfly (Drosophila melanogaster) revealed the presence of HATs belonging to the MYST, GNAT and K. Mukherjee et al. / Progress in Biophysics and Molecular Biology 118 (2015) 69e78
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 fruitfly whereas only a class I HDAC has been found in the greater wax moth. 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 such as cancer and multiple sclerosis, which are known to have an epigenetic component (Szyf, 2009). 2.3. MicroRNAs
MicroRNAs (miRNAs) are non-coding RNAs 18e22 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 number of diseases (Ambros, 2004; Bartel, 2004; Lu and Liston, 2009). The genes encoding miRNAs are found individually and as polycistronic clusters (Lagos-Quintana et al., 2001). In the nucleus, the tran-scription of miRNA genes by RNA polymerase II/III produces double-stranded transcripts known as primary miRNAs
(pri-miRNAs). These are trimmed by the RNase III enzyme Drosha to form double-stranded stem-loop precursor miRNAs (pre-miRNAs) that are transported to the cytoplasm by Exportin-5. Pre-miRNAs are further processed into unstable, 18e22-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 con-taining 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 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, indi-vidual miRNAs can target multiple mRNAs as long as they each contain the matching complementary sequence, and 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 miRNAs are limited to particular species and sophisticated bioin-formatics algorithms are needed to identify them de novo, which can be expensive and time consuming. If a complete genome sequence is not available, the differential expression of miRNAs can Fig. 1. Chromatin modifications mediated by DNA methylation and histone acetylation. 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 cytidine residues in the dinucleotide motif CpG forms 5-methylcytidine, 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 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.
be established using microarray technology and validated by quantitative RT-PCR. This has been highly effective for the com-parison of greater wax moth miRNAs that are active at different developmental stages or induced by pathogens, but it is important to validate the sequences and to control for non-specific hybridi-zation (Mukherjee and Vilcinskas, 2014).
3. Advantages of insect models in epigenetic research
The epigenetic inheritance of transcriptional reprogramming can be investigated using model 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 sys-tem wherever possible, but cell lines do 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 regula-tions concerning the use of animals in scientific procedures are less restrictive for insects. The 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, inflam-matory/autoimmune diseases and metabolic disorders (Szyf, 2008). The 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. up to 5% of all drugs can potentially interfere with histone acetylation in humans (L€otsch et al., 2013).
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 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 hostepathogen interactions, to include epigenetic mechanisms, particularly those which translate envi-ronmental stimuli into transcriptional reprogramming across multiple generations (Srinivasan and Brisson, 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 10e12 generations to be studied in one year. Second, model insects have morphologically distinct developmental stages (eggs, larvae, pupae and imagoes) so that factors affecting developmental progress can be measured objectively (e.g. using metrics such as the percentage of a popula-tion that transforms from last-instar larvae into pupae). Insect models can therefore be used to determine the impact of envi-ronmental factors on longevity and developmental progress, which are complex parameters that cannot be measured using cell lines (Grünwald et al., 2013).
Fecundity is another complex parameter that is easy to study in insects. For example, a female greater wax moth lays up to 500 eggs and a female redflour beetle lays up to 1000 eggs, allowing the quantitative effects of environmental stimuli to be measured accurately across multiple generations. Further complex parame-ters that are easy to measure in insects include gender ratio and
body weight, because both can be averaged over hundreds of in-dividuals to avoid statistical anomalies. All three of these parame-ters are influenced by environmental triggers and regulated by epigenetic mechanisms that are conserved with mammals. Indeed, not only do insects share the same epigenetic mechanisms as mammals, but those mechanisms also regulate the same patho-physiological processes.
One recent example concerns the epigenetic regulation of innate immunity. In both mammals and insects, one of the hall-marks of the innate immune response is the expression of anti-microbial peptides (AMPs). Histone acetylation promotes AMP expression in the greater wax moth (Mukherjee et al., 2012) whereas histone demethylation induced by the histone demethy-lase JMJD3 promotes AMP expression in mammalian keratinocytes (Gschwandtner et al., 2014). This indicates that innate immunity in both insects and mammals is regulated by histone modification, but mammalian cell lines can provide only molecular and cellular data, whereas insect models can be investigated for complex parameters such as those listed above. Phenotypic changes in whole insects can be induced by the deliberate disruption of epigenetic mechanisms, e.g. HDAC and HAT inhibitors have been shown to affect the rate of metamorphosis in greater wax moth larvae, with the former causing the developmental clock to accelerate and the latter causing it to slow down. Similarly, as discussed in Section7, the dietary administration of pathogenic or non-pathogenic bacteria has opposing effects on fecundity in the same species, which is also mediated by changes in the balance of HDAC and HAT activity (Fig. 2).
Finally, insects provide an accessible system for the experi-mental manipulation of distinct genetic and epigenetic mecha-nisms in order to study their inheritance through meiosis, e.g. following exposure to pathogens, chemicals and other environ-mental insults. Insects generally have smaller and more euchro-matic genomes than mammals, which makes the genome-wide analysis of DNA methylation more practical. Similarly, insects such as the fruitfly, which have polytene chromosomes in their salivary glands, allow the direct visualization of modified histones (Boros, 2012). The epigenetic mechanisms investigated in key insect models are relevant to the equivalent processes in mammals and can therefore be used to investigate the epigenetic basis of human diseases (Fig. 3). We consider the use of insect models for the analysis of epigenetic processes underlying human development,
Fig. 2. 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 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 ento-mopathogenic bacterium S. entomophila (discussed in Section3). Data are means of three independent experiments± standard deviations (*p < 0.05).
K. Mukherjee et al. / Progress in Biophysics and Molecular Biology 118 (2015) 69e78 72
aging, neurodegeneration, cancer and infectious diseases as rele-vant case studies.
4. Development, aging and longevity
Development is the process by which a complex multicellular organism arises from a single cell, involving growth, an increase in cell number, progressive cellular differentiation and pattern for-mation, and morphogenesis (i.e. the creation of shapes and struc-tures). Aging is often considered a continuation of development in which cells progressively undergo senescence and the organism as a whole becomes less capable of managing stress and homeostasis and therefore more prone to disease (Dillin et al., 2014). Aging is a complex process in mammals which involves multiple genetic, molecular and environmental processes that are incompletely un-derstood. They are difficult to separate and investigate in isolation and the best current models are based on simpler organisms, pri-marily yeast, nematodes and insects (Parrella and Longo, 2010).
At the molecular level, aging is influenced by energy meta-bolism, the electron transport chain and increased signaling through the insulin/insulin-like growth factor I (IGF-I) pathway (Parrella and Longo, 2010). This affects cellular processes such as senescence (Campisi, 2013), telomere shortening (Kaszubowska, 2008), mTOR-dependent autophagy (Vellai et al., 2009), and the activity of Sirtuins, which are class III HDACs (Greiss and Gartner, 2009). Investigations in model organisms have highlighted the importance of epigenetic mechanisms including DNA methylation, histone acetylation and the expression of miRNAs in aging,
longevity and fecundity, casting doubt on earlier theories of aging based on factors such as oxidative stress (De Loof, 2011). Therefore, the identification of epigenetic markers of aging in eukaryotic cells is an important step towards the development of strategies to in-crease longevity in humans. The fruitfly has become a promising model in this regard (Brandt and Vilcinskas, 2013).
Diet has a profound impact on development, aging and longevity. As discussed earlier, the development of female honey-bees is influenced by feeding on royal jelly, which contains the HDAC inhibitor phenyl butyrate, leading to a net increase in histone acetylation (Lyko et al., 2010). The modification of histones is not thought to be heritable, but HDACs act in concert with DNMTs so that deacetylated histones tend to become associated with meth-ylated DNA, thus creating a heritable epigenetic marker. Accord-ingly, the silencing of DNMT3 in honeybee larvae mimics the effect of royal jelly and promotes the development of queens instead of workers (Kucharski et al., 2008). Similarly, the silencing of DNMT1 in the silkworm moth dramatically reduces hatchability, again showing that epigenetic mechanisms influence the fecundity of insects (Xiang et al., 2013). In the context of aging, resveratrol found in red grape skins has been shown to increase the lifespan of fruit flies and mice by promoting Sirtuin activity (Wood et al., 2004; Baur et al., 2006), and glucoraphanin as a component of broccoli increases the lifespan of the redflour beetle even under heat stress by inducing stress-tolerance pathways involving Nrf-2, Jnk-1 and Foxo-1, independent of Sirtuins (Grünwald et al., 2013).
Even complex parameters such as memory formation appear to be directly regulated by histone acetylation, e.g. the development of Fig. 3. Thefive 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 tractability, phenotypic plasticity, conservation of epigenetic, developmental and immunity-related pathways with mammals, conservation of disease phenotypes and ability to host human pathogens.
long-term memory in honeybees involves histone acetylation at specific lysine residues (Merschbaecher et al., 2012). However, these processes can also be decoupled, as shown in fruitflies where exposure to the HDAC inhibitor phenyl butyrate increased longevity and delayed aging without reducing locomotor vigor, stress resistance or fecundity (Kang et al., 2002).
Aging is also influenced by miRNAs, which regulate genes in the insulin/IGF-I and mTOR pathways as well as the targets of growth hormone signaling (Gomez-Orte and Belles, 2009). For example, miR-71 is a developmentally-regulated miRNA in the greater wax moth which targets the genes required for cell cycle progression, and miR-2002b plays an important role in larval development and adult fecundity in the cotton bollworm (Helicoverpa armigera) (Mukherjee and Vilcinskas, 2014). A synthetic miR-2002b analog fed to the larvae caused a loss of fecundity and the development of morphologically-aberrant pupae (Jayachandran et al., 2012). In the fruitfly, miR-14 and miR-9a play important roles in metamorphosis particularly by regulating the expression of the ecdysone receptor gene and the LIM-domain-only protein, dLMO, which also has a role in cancer (Varghese and Cohen, 2007; Biryukova et al., 2009). 5. Neurodegenerative disorders
Some disorders can be thought of as aberrant forms of devel-opment, e.g. neurodegeneration, which involves the progressive loss of neuronal structure and function. The epigenetic component of neurodegenerative disorders is difficult to unravel because all such diseases are characterized by complex etiology and progres-sion, and vary significantly in severity and penetrance (Jakovcevski and Akbarian, 2012). Insect models which allow the dissection of neurodegenerative disorders are therefore useful to investigate the epigenetic aspects in isolation.
As an example, the fruitfly is already recognized as a behavioral model for sleep disorders associated with Alzheimer's disease, the most common form of dementia in humans, which is characterized by a progressive decline in cognitive function, including short-term and long-term memory loss, loss of physical functions and ulti-mately death (Prüßing et al., 2013). By modeling the disease in fruit flies,Pirooznia and Elefant (2013)discovered that the HAT Tip60 interacts with the pathological form of the amyloid precursor protein (APP) to regulate the production of neuropeptide dispersing factor (PDF) by small ventrolateral neuronal pacemaker cells, and that the expression of Tip60 can be used to stabilize sleep patterns. Tip60 also regulates the development of the embryonic nervous system by maintaining synaptic plasticity and preventing the elimination of neurons by apoptosis (Pirooznia and Elefant, 2013). The fruitfly has also been used to investigate strategies to address the microtubule defects in Alzheimer's disease patients caused by phosphorylation of the neuronal microtubule-associated protein tau. The microtubule defects found in Alzheimer's disease can be induced in fruitfly neurons and muscle cells by expressing the human tau protein, but this defect can be rescued by deleting the gene encoding HDAC6 (Xiong et al., 2013). HDAC6 is therefore being investigated as a potential drug target for the treatment of Alzheimer's disease in humans.
The disruption of HDAC activity is associated with other neurodegenerative disorders including Parkinson's disease, so HDAC inhibitors are being investigated as a new therapeutic approach in this context too. Parkinson's disease in humans in-volves the progressive loss of dopaminergic neurons concomitant with the expression of the small neuronal protein,
a
-synuclein. The fruitfly and silkworm have been explored as potential models of epigenetic plasticity associated with Parkinson's disease (Tabunoki et al., 2013; Bayersdorfer et al, 2010). For example, inhibitors of HDAC6 and Sirtuin-2 suppress Parkinson-like phenotypes inducedby
a
-synuclein in insects by forming inclusion bodies that sequester the protein and limit its toxicity. Furthermore, sodium butyrate inhibits class I and II HDACs except HDAC6, and this re-duces the degeneration of dopaminergic neurons in transgenic fruit flies expressing a pathological mutant version ofa
-synuclein (Chuang et al., 2009). The study used pesticides to chemically induce Parkinson's disease in the insects, and confirmed that HDAC inhibitors had the therapeutic potential to rectify the associated locomotor impairment.Class I and II HDAC inhibitors such as suberoylanilide hydroxa-mic acid (SAHA), phenyl butyrate and sodium butyrate can also be used to treat models of Huntington's disease established in fruit flies and mice (Pe~na-Altamira et al., 2013; Lee et al., 2013; Steinert et al., 2012). Huntington's disease is caused by a CAG trinucleotide repeat expansion within the coding region of the HTT gene, but pathogenesis is mediated by a disturbance in the normal balance of HDAC and HAT activities in the cell, reducing the acetylation of histone H3 and H4 and disrupting gene regulation (Sadri-Vakili et al., 2007). The investigation of this mechanism in insect models has suggested a novel therapeutic approach for the treat-ment of Huntington's disease based on the inhibition of NADþ -dependent class III HDACs.
6. Cancer
Cancer is another group of diseases which can be thought of as ‘development-gone-wrong’ because it involves deregulated and uncontrolled cell proliferation, unprogrammed differentiation and even the formation of aberrant tissues, e.g. during neo-vascularization. Many genes that are active in development are also ectopically activated in tumors. The fruitfly, which was one of the pioneering models of developmental biology, is therefore the most widely used insect model for the investigation of epigenetic mechanisms controlling tumor formation and metastasis (Tipping and Perrimon, 2014).
In one example, the deacetylation of lysine 16 on histone H4 by HDAC3 has been shown to antagonize tumor growth in the fruitfly by inducing the expression of the insulin receptor, phosphoinosi-tide 3-kinase (PI3K) or S6 kinase (Lv et al., 2012). This is relevant because hyperactivation of the PI3K pathway and the loss of H4K16 acetylation have been reported in human cancers (Ghayad and Cohen, 2010; Martelli et al., 2009). Similarly, the analysis of DNA methylation using methylation-sensitive restriction enzymes and bisulfite sequencing showed that the Retinoblastoma (Rb) tumor-suppressor gene is hypermethylated in human tumors, leading to the activation of oncogenes and the loss of cell cycle control, thus promoting tumor growth and metastasis (Ferres-Marco et al., 2006). Hypermethylation within the promoter region of the gene encoding cytoplasmic polyadenylation element binding protein 1 (CPEBP 1) caused it to be downregulated in fruitfly gastric cancer cell lines, but this could be reversed by applying pharmacological inhibitors of DNA methylation (Caldeira et al., 2012).
Gene expression during tumorigenesis in the fruitfly is also regulated by miRNAs. For example, the overexpression of miR-8, which is homologous to the miR-200 family of miRNAs in humans, suppresses tumor formation and metastasis by regulating the Notch signaling pathway (Vallejo et al., 2011). In contrast, the Notch pathway can be activated to promote tumorigenesis by the overexpression of miR-7 (Da Ros et al., 2013). Another develop-mentally regulated miRNA in the fruitfly is miR-34, which is ho-mologous to human miRNAs associated with several different cancers, including miR-310/13 which regulates the cancer-associated Wnt pathway (represented by the Wingless pathway in the fruit fly) and interferes with the function of the major pathway effector
b
-catenin, which plays a significant role in the K. Mukherjee et al. / Progress in Biophysics and Molecular Biology 118 (2015) 69e78progression of cancers in humans (Pancratov et al., 2013). The fruit fly has also been used for the functional characterization of miRNAs such as miR-14, bantam, miR-2, miR-13, miR-278 and miR-11, which act as inhibitors of cell death in cancer models (Nelson et al., 2014; Herranz et al., 2012; Ge et al., 2012; Boutla et al., 2003; Nairz et al., 2006; Truscott et al., 2011).
7. Inflammation and sepsis
Epigenetic mechanisms are known to mediate the transcrip-tional reprogramming of development and immunity-related genes which are necessary to maintain beneficial microorganisms while keeping the harmful ones at bay. However, pathogens have co-evolved to hijack these defense mechanisms for their own benefit by epigenetically reprogramming not only their own transcriptome but also gene expression profiles in the host. Histone and other chromatin modifications as well as small RNAs are known to control the switch between the replicative and non-replicative stages of pathogens such as Toxoplasma gondii (Bougdour et al., 2009). Recent evidence indicates that DNA methylation causes Candida albicans to switch from the yeast to the hyphal form (Mishra et al., 2011). Bac-terial pathogens lack chromatin but they have evolved a range of effector molecules and virulence factors that allow them to over-come host defenses, e.g. by reprogramming the host epigenome to suppress anti-inflammatory responses (Mujtaba et al., 2013). Such effectors represent a novel spectrum of drug targets, aiming to prevent disease by blocking the ability of human pathogens to manipulate host epigenetic mechanisms for their own advantage. As an example, the human pathogen Yersinia enterocolitica expresses a cysteine protease (YopJ) that inhibits the ubiquitinylation of I
k
B (an inhibitor of the NF-k
B pathway) and TRAF6, and acetylates IKKa
, thereby preventing the nuclear translocation of NF-k
B and effec-tively blocking NF-k
B signaling. The identification of epigenetic markers that regulate hostepathogen interactions therefore pro-vides a novel approach in the development of alternative anti-infective strategies (Gomez-Díaz et al., 2012).The extent to which pathogens epigenetically manipulate host immunity to influence survival and development is unclear. The larval stage of the greater wax moth has been widely used to identify novel virulence factors, infection mechanisms and anti-infectives for human pathogens such as Staphylococcus aureus, Ba-cillus cereus, C. albicans, Cryptococcus neoformans and Listeria monocytogenes (Wang et al., 2013; Glavis-Bloom et al., 2012; Browne and Kavanagh, 2013) because it can be reared at 37C and
thus supports the analysis of pathogens in a near native state. Therefore, this model can be used to investigate single or multiple virulence genes to determine their relevance in human infections, and to highlight conserved infection strategies such as brain infection by L. monocytogenes (Mukherjee et al., 2010, 2011, 2013). Recently, we investigated the role of histone acetylation and miRNA expression in the greater wax moth when it was infected with the human pathogen L. monocytogenes, the entomopathogenic fungus Metarhizium anisopliae or a non-pathogenic strain of Escherichia coli (Mukherjee et al., 2012). We found a profound difference in epigenetic behavior when comparing the host interaction with pathogenic and non-pathogenic species. The interaction with E. coli was characterized by a rapid restoration of the pre-infection HDACeHAT balance and accelerated metamorphosis, whereas in-fections with L. monocytogenes or M. anisopliae caused the disrup-tion of relative HDAC and HAT activities, delayed metamorphosis and was ultimately lethal. Septic shock induces hyperactivity of the immune system in both mammals and insects, but the adminis-tration of HDAC inhibitors such as SAHA in a greater wax moth injury model improved survival as was previously established in mice (Mukherjee et al., 2012).
A recent investigation revealed that parental exposure to pathogens leads to trans-generational immune priming in the greater wax moth by depositing bacterial particles in the eggs (Freitak et al., 2014). This provided thefirst experimental confir-mation that trans-generational immune priming is controlled by an epigenetic process. We found that the HDACeHAT balance was tipped in favor of HDAC activity in the larval midgut and eggs following the consumption of diet contaminated with the patho-genic bacterium Serratia entomophila whereas the HDACeHAT balance was not affected in uninfected larvae or their eggs (Fig. 4). It is now well established that miRNAs are also involved in hostepathogen interactions (Asgari, 2011; Skalsky and Cullen, 2010; Xiao and Rajewsky, 2009). Viral infections can inhibit the growth and development of insects, which provides an important link between the roles of miRNAs in immunity and metamorphosis. The expression of miRNAs in the midgut of the malaria vector mosquito (Anopheles gambiae) is modulated by the presence of the parasite Plasmodium berghei, but the functions of the down-regulated miRNAs (aga-miR-34, aga-miR-1174 and aga-miR-1175) and the upregulated miRNA aga-miR-989 remain largely un-known (Winter et al., 2007). Interestingly, the silencing of Dicer-1, which is necessary for miRNA biogenesis, can increase Plasmo-dium spp. oocyte numbers in the mosquito midgut.
Fig. 4. Transcriptional analysis of genes encoding HDACs and HATs in greater wax moth larval midgut and eggs. Real-time quantitative RT-PCR analysis of transcripts encoding histone deacetylase 8, histone deacetylase 8 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 Section7). The expression levels are presented as fold-change values compared to controls fed on uncontaminated diets and the data are normalized against the 18S rRNA housekeeping gene.
The number of miRNAs specifically induced by pathogens is likely to be small and potentially dependent on the route of infec-tion. For example, most miRNAs in the southern house mosquito (Culex quinquefasciatus) are unaffected by infection with West Nile virus and the only miRNA genes specifically induced by the virus are miR-989 and miR-92 (Skalsky et al., 2010). Microarray analysis of greater wax moth miRNAs during a natural infection with M. anisopliae revealed that only miR-210b was specifically induced (Mukherjee and Vilcinskas, 2014). However, injecting microbial elicitors into the host or experimentally inducing immune re-sponses caused the differential expression of numerous miRNAs in the redflour beetle. Interestingly, gender-specific and stress-specific miRNAs were also identified in this species (Freitak et al., 2012). 8. Conclusions
Epigenetic mechanisms such as DNA methylation, histone modification and the expression of regulatory miRNAs convert environmental stimuli into patterns of gene expression that may be heritable if an epigenetic marker can be transmitted through meiosis. In this manner, the impact of factors such as pathogens, parasites, chemicals and environmental insults can affect complex parameters such as fecundity, longevity and susceptibility to dis-ease across several generations. Insects provide many excellent examples of phenotypic plasticity controlled by epigenetic mech-anisms, producing distinct phenotypic variants from the same ge-notype by transcriptional reprogramming. They are also inexpensive to breed, have short generation times and no associ-ated ethical issues. The strong conservation between mammals and insects in terms of both epigenetic mechanisms and signaling pathways related to development, immunity and disease, means that insects are now regarded as ideal models in which to investi-gate the epigenetic trans-generational effects of environmental factors related to development, aging, neurodegeneration, cancer and infectious diseases (Fig. 5).
Acknowledgments
The authors acknowledge financial support from the Hessen State Ministry of Higher Education, Research and the Arts (HMWK) via the collaborative research projects granted under the LOEWE
programs “Insect Biotechnology” (Insektenbiotechnologie) and “Translational Pharmaceutical Research” (Angewandte Arznei-mittelforschung). AV thanks the German Research Foundation for funding of the project“the role of epigenetics in hosteparasite-coevolution” (VI 219/3-2) which is embedded within the DFG Pri-ority Programme 1399“HosteParasite Coevolution e rapid recip-rocal adaptations and its genetic basis”.
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