GBE Signatures of DNA Methylation across Insects Suggest Reduced DNA Methylation Levels in Holometabola Panagiotis Provataris1, Karen Meusemann1,2,3, Oliver Niehuis1,2,SonjaGrath4,*, and Bernhard Misof1,* 1Center for Molecular Biodiversity Research, Zoological Research Museum Alexander Koenig, Bonn, Germany 2Evolutionary Biology and Ecology, Institute of Biology I (Zoology), Albert Ludwig University Freiburg, Freiburg (Brsg.), Germany 3Australian National Insect Collection, CSIRO National Research Collections Australia, Acton, Australian Capital Territory, Australia Downloaded from https://academic.oup.com/gbe/article-abstract/10/4/1185/4943971 by guest on 13 December 2019 4Division of Evolutionary Biology, Faculty of Biology, Ludwig-Maximilians-Universitat€ Mu¨ nchen, Planegg, Germany *Corresponding authors: E-mails: [email protected]; [email protected]. Accepted: March 17, 2018 Abstract It has been experimentally shown that DNA methylation is involved in the regulation of gene expression and the silencing of transposable element activity in eukaryotes. The variable levels of DNA methylation among different insect species indicate an evolutionarily flexible role of DNA methylation in insects, which due to a lack of comparative data is not yet well-substantiated. Here, we use computational methods to trace signatures of DNA methylation across insects by analyzing transcriptomic and genomic sequence data from all currently recognized insect orders. We conclude that: 1) a functional methylation system relying exclusively on DNA methyltransferase 1 is widespread across insects. 2) DNA meth- ylation has potentially been lost or extremely reduced in species belonging to springtails (Collembola), flies and relatives (Diptera), and twisted-winged parasites (Strepsiptera). 3) Holometabolous insects display signs of reduced DNA methylation levels in protein-coding sequences compared with hemimetabolous insects. 4) Evolutionarily conserved insect genes asso- ciated with housekeeping functions tend to display signs of heavier DNA methylation in comparison to the genomic/tran- scriptomic background. With this comparative study, we provide the much needed basis for experimental and detailed comparative analyses required to gain a deeper understanding on the evolution and function of DNA methylation in insects. Key words: DNA methylation, DNMT1, DNMT3, Tet, Hexapoda, CpG o/e. Introduction methylation are not evolutionarily conserved. In vertebrates, Methylation of cytosine residues constitutes a common epi- and especially in mammals, CpG dinucleotides are heavily genetic modification among eukaryotes. It is functionally as- methylated genome-wide, with the exception of CpG islands. sociated with the regulation of expression of genomic CpG islands typically overlap with promoter regions and re- elements (Zemach et al. 2010). For example, promoter- main mostly unmethylated (Schu¨ beler 2015). In contrast, proximate methylation is linked to the transcriptional repres- invertebrates show intermediate or even negligible levels of sion of associated genes (Jones 2012; Schu¨ beler 2015). DNA methylation at CpG sites, which is typically targeted to a Methylation of repetitive noncoding DNA elements also has subset of gene bodies (the term gene body refers to the tran- a repressive effect, limiting the expression, and thus, the ge- scribed part of a gene, comprised of exons and introns; Suzuki nomic expansion of these elements (Schu¨ beler 2015). In con- and Bird 2008; Feng et al. 2010; Zemach et al. 2010; but see trast, intragenic methylation is associated with active Wang et al. 2014; Kao et al. 2016). transcription (Feng et al. 2010; Zemach et al. 2010; Jones In insects, the levels of gene body methylation vary consid- 2012), but a “cause and effect” relationship has not been erably (Glastad et al. 2011). On one hand, model organisms established in this context (Schu¨ beler 2015). Although DNA like the fruit fly, Drosophila melanogaster, and the red flour methylation is widely present among eukaryotes, the levels beetle, Tribolium castaneum, do not display notable DNA (i.e., the proportion of methylated cytosines or CpG sites in a methylation levels in their genomes (Zemach et al. 2010; given genome), patterns, and genomic targets of DNA Bewick et al. 2017). On the other hand, nutritionally regulated ß The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense(http://creativecommons.org/licenses/by-nc/4.0/),whichpermitsnon- commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Genome Biol. Evol. 10(4):1185–1197. doi:10.1093/gbe/evy066 Advance Access publication March 19, 2018 1185 Provataris et al. GBE levels of DNA methylation contribute to the ontogenetic es- demethylation and 5mC demethylation in the D. mela- tablishment of alternative castes in the honey bee, Apis melli- nogaster DNA and mRNA, respectively (Zhang et al. 2015; fera (Kucharski et al. 2008; Foret et al. 2012). This observation Delatte et al. 2016). The distribution of Tet enzymes in insects has supported the hypothesis that DNA methylation in insects and its relationship to the presence of 5mC is currently not is associated with caste development and the evolution of known. (eu)sociality, but recent empirical evidence from research on Comparative analyses using experimental data have eusocial Hymenoptera (wasps, ants, and bees) suggests that shown that the levels of gene body methylation of the silk this association is not universal (Bonasio et al. 2012; Patalano moth and the honey bee are substantially lower compared et al. 2015; Kapheim et al. 2015; Libbrecht et al. 2016; with other invertebrates (sea squirt, Ciona intestinallis,and Downloaded from https://academic.oup.com/gbe/article-abstract/10/4/1185/4943971 by guest on 13 December 2019 Standage et al. 2016). Obviously, a taxonomically representa- sea anemone, Nematostella vectensis; Sarda et al. 2012). tive description of the levels and patterns of DNA methylation These results have fueled the hypothesis that DNA methyl- is one major prerequisite to improve our understanding of the ation was reduced in the ancestors of insects (Glastad et al. evolution and, eventually, the function of DNA methylation in 2014). However, experimental and computational evidence insects. Therefore, we conducted a comparative analysis of from research on hemimetabolous lineages point to signif- DNA methylation patterns in insects by making use of recently icantly elevated DNA methylation levels in species belonging published, extensive transcriptomic (Misofetal.2014)and to Orthoptera, Phasmatodea, and Isoptera compared with publicly available genomic sequence data covering all extant certain Hymenoptera and B. mori (Krauss et al. 2009; insect orders. Falckenhayn et al. 2013; Glastad et al. 2013, 2016; Two types of DNA methyltransferases (DNMTs), DNMT1 Terrapon et al. 2014). Thus, the validity of the proposed and DNMT3, are responsible for DNA methylation in animals hypothesis on the ancestral state of DNA methylation in (Goll and Bestor 2005). In mammals, DNMTs carry out de insects is questionable. novo (DNMT3) and maintenance (DNMT1) methylation, The methylomic profiling of insects, mostly representing with functional overlap (Jeltsch and Jurkowska 2014). Hymenoptera, and to a lesser extent Lepidoptera and Another, noncanonical member of the DNMT family, Coleoptera, revealed largely similar patterns of DNA methyl- TRDMT1 (tRNA aspartic acid methyltransferase 1, most com- ation, primarily targeted to exons of protein-coding genes monly known as DNMT2), long considered a DNMT, has (Lyko et al. 2010; Xiang et al. 2010; Bonasio et al. 2012; seemingly shifted substrate and is now known to methylate Wang et al. 2013; Cunningham et al. 2015; Patalano et al. tRNA, not DNA (Goll et al.2006; Lyko 2017). It is generally 2015; Libbrecht et al. 2016; Rehan et al. 2016; Standage et al. assumed that these functions are conserved in insects (Wang 2016). Additionally, genes targeted by DNA methylation were et al. 2006). This assumption is supported by the observation ubiquitously expressed among various tissue types (Foret et al. that the absence of DNMT1 and DNMT3 is associated with 2012; Xiang et al. 2010), among different morphs in ants the loss or extreme reduction of DNA methylation in D. mel- (Bonasio et al. 2012; Libbrecht et al. 2016), and among de- anogaster (Raddatz et al. 2013). However, in contrast to velopmental stages in the parasitoid wasp, Nasonia vitripennis mammals, the DNMT toolkit of insects that show substantial (Wang et al. 2013). Gene ontology annotations showed that levels of DNA methylation in their genomes is not conserved. the majority of these genes mostly serves basic cellular func- For example, the silk moth, Bombyx mori, has empirically de- tions, exhibit a highly methylated state among species, and termined DNA methylation, but lacks copies of DNMT3 are highly conserved at sequence level (Elango et al. 2009; homologs from its genome (Xiang et al. 2010; Bewick et al. Lyko et al. 2010; Hunt et al. 2013; Wang et al. 2013; 2017). Thus, functional methylation systems in insects can be Cunningham et al. 2015; Rehan et al. 2016). These patterns realized in the absence of DNMT3. The frequency of DNMT3 are even found when comparing orthologous genes among