Vertebrate DNA Research Articles

N 6-Hydroxymethyladenine: a hydroxylation derivative of N6-methyladenine in genomic DNA of mammals.

In addition to DNA cytosine methylation (5-methyl-2′-deoxycytidine, m5dC), DNA adenine methylation (N6-methyl-2′-deoxyadenosine, m6dA) is another DNA modification that has been discovered in eukaryotes. Recent studies demonstrated that the content and distribution of m6dA in genomic DNA of vertebrates and mammals exhibit dynamic regulation, indicating m6dA may function as a potential epigenetic mark in DNA of eukaryotes besides m5dC. Whether m6dA undergoes the further oxidation in a similar way to m5dC remains elusive. Here, we reported the existence of a new DNA modification, N6-hydroxymethyl-2′-deoxyadenosine (hm6dA), in genomic DNA of mammalian cells and tissues. We found that hm6dA can be formed from the hydroxylation of m6dA by the Fe2+- and 2-oxoglutarate-dependent ALKBH1 protein in genomic DNA of mammals. In addition, the content of hm6dA exhibited significant increase in lung carcinoma tissues. The increased expression of ALKBH1 in lung carcinoma tissues may contribute to the increase of hm6dA in DNA. Taken together, our study reported the existence and formation of hm6dA in genomic DNA of mammals.

Environmental DNA from avian residual saliva in fruits and its potential uses in population genetics

Buccal cells are a valid source of vertebrate DNA for genetic analysis, typically collected with mouth swabs on captured individuals. Yet, advances in DNA storage and processing now permit recovering genomic material from traces of saliva in food remains. For example, the feeding habits of canopy-dwelling frugivorous birds, e.g. large macaws (Psittaciformes), could present an ideal opportunity to obtain environmental DNA (eDNA) from partially consumed fruits. We tested this by collecting tropical almond (Terminalia catappa) fruits, eaten and discarded by scarlet macaws (Ara macao), and processed them using three different collection/storage methods. We successfully isolated DNA molecules from macaw residual saliva in fruits. This genetic material allowed the amplification of 7 microsatellite markers and of the CHD region of the avian sex chromosomes using two different primer pairs. Macaw eDNA concentration (mean 12 ng/µl) was similar to the reported in other avian studies using buccal swabs but overall microsatellite (60% success) and CHD-gen (20% success) amplification was low. The best results were obtained for samples preserved in ethanol. We conclude that saliva eDNA in partially consumed food items is an underused source of non-invasive bird DNA for genetic analyses. Based on our results, we recommend that a combination of specialized collection swabs or ethanol stored swabs along with commercial DNA extraction kits be used. Protocols should be modified accordingly to reach a consistent level of individual identification and gender determination that closely matches traditional sampling.

Barcoding blood meals: New vertebrate-specific primer sets for assigning taxonomic identities to host DNA from mosquito blood meals.

The transmission dynamics of mosquito-vectored pathogens are, in part, mediated by mosquito host-feeding patterns. These patterns are elucidated using blood meal analysis, a collection of serological and molecular techniques that determine the taxonomic identities of the host animals from which blood meals are derived. Modern blood meal analyses rely on polymerase chain reaction (PCR), DNA sequencing, and bioinformatic comparisons of blood meal DNA sequences to reference databases. Ideally, primers used in blood meal analysis PCRs amplify templates from a taxonomically diverse range of vertebrates, produce a short amplicon, and avoid co-amplification of non-target templates. Few primer sets that fit these requirements are available for the cytochrome c oxidase subunit I (COI) gene, the species identification marker with the highest taxonomic coverage in reference databases. Here, we present new primer sets designed to amplify fragments of the DNA barcoding region of the vertebrate COI gene, while avoiding co-amplification of mosquito templates, without multiplexed or nested PCR. Primers were validated using host vertebrate DNA templates from mosquito blood meals of known origin, representing all terrestrial vertebrate classes, and field-collected mosquito blood meals of unknown origin. We found that the primers were generally effective in amplifying vertebrate host, but not mosquito DNA templates. Applied to the sample of unknown mosquito blood meals, > 98% (60/61) of blood meals samples were reliably identified, demonstrating the feasibility of identifying mosquito hosts with the new primers. These primers are beneficial in that they can be used to amplify COI templates from a diverse range of vertebrate hosts using standard PCR, thereby streamlining the process of identifying the hosts of mosquitoes, and could be applied to next generation DNA sequencing and metabarcoding approaches.

Structure, evolution and phylogenetic informativeness of eelpouts (Cottoidei: Zoarcales) mitochondrial control region sequences

Control region (CR) is a major non-coding domain of mitochondrial DNA in vertebrates which contains the promoters for replication and transcription of mitochondrial genome along with the binding sites for metabolic machinery and, hence, is a vital element for the integrity of mitochondrial genome as a biological replicator. The origin and diversity of structural elements within CR have been intensively studied in recent years with the involvement of new diverse taxa. In this paper, we provide new data on the nucleotide and structural patterns of CR evolution and phylogenetic suitability among eelpouts (Cottoidei: Zoarcales). To achieve this, we carried out a comparative phylogenetic and structural analysis of 29 CR sequences belonging to the long shanny Stichaeus grigorjewi together with nine sequences of other eelpouts taxa representing four families in contrast to mitochondrial protein-coding fragments. The CR organization within S. grigorjewi, as well as in all other eelpouts, is consistent with the common three-domain structure known from most vertebrates. We found a hidden CR variation constrains on the landscape level and a lack of nucleotide saturation. Finally, our results demonstrate the advantage of the length variation in CR sequences for phylogenetic reconstructions among eelpouts.

Extracts for Analysis of DNA Replication in a Nucleus-Free System.

Frog egg extracts represent a powerful approach with which to dissect molecular mechanisms of vertebrate DNA replication and repair. In the classical approach, sperm chromatin is added to a crude egg lysate to form replication-competent nuclei. We subsequently described a procedure that bypasses the requirement for nuclear assembly in DNA replication. In this method, DNA is first added to a high-speed supernatant (HSS) of egg lysate, which mimics the G1 phase of the cell cycle in that it supports replication licensing. Subsequent addition of a concentrated nucleoplasmic extract (NPE) leads to replication initiation followed by a single complete round of DNA replication. The advantage of the nucleus-free system is that it supports efficient replication of model DNA templates such as plasmids and lambda DNA that can be modified with specific features such as LacI arrays, DNA protein cross-links, or DNA interstrand cross-links. Here, we describe our current protocol for preparation of HSS and NPE. Methods for their use in DNA replication and repair are described elsewhere.

Ancient DNA from latrines in Northern Europe and the Middle East (500 BC–1700 AD) reveals past parasites and diet

High-resolution insight into parasitic infections and diet of past populations in Northern Europe and the Middle East (500 BC- 1700 AD) was obtained by pre-concentration of parasite eggs from ancient latrines and deposits followed by shotgun sequencing of DNA. Complementary profiling of parasite, vertebrate and plant DNA proved highly informative in the study of ancient health, human-animal interactions as well as animal and plant dietary components. Most prominent were finding of soil-borne parasites transmitted directly between humans, but also meat-borne parasites that require consumption of raw or undercooked fish and pork. The detection of parasites for which sheep, horse, dog, pig, and rodents serves as definitive hosts are clear markers of domestic and synanthropic animals living in closer proximity of the respective sites. Finally, the reconstruction of full mitochondrial parasite genomes from whipworm (Ascaris lumbricoides) and roundworm species (Trichuris trichiura and Trichuris muris) and estimates of haplotype frequencies elucidates the genetic diversity and provides insights into epidemiology and parasite biology.

Arabidopsis Zinc-Finger-Like Protein ASYMMETRIC LEAVES2 (AS2) and Two Nucleolar Proteins Maintain Gene Body DNA Methylation in the Leaf Polarity Gene ETTIN (ARF3).

Arabidopsis ASYMMETRIC LEAVES2 (AS2) plays a critical role in leaf adaxial-abaxial partitioning by repressing expression of the abaxial-determining gene ETTIN/AUXIN RESPONSE FACTOR3 (ETT/ARF3). We previously reported that six CpG dinucleotides in its exon 6 are thoroughly methylated by METHYLTRASFERASE1, that CpG methylation levels are inversely correlated with ETT/ARF3 transcript levels and that methylation levels at three out of the six CpG dinucleotides are decreased in as2-1. All these imply that AS2 is involved in epigenetic repression of ETT/ARF3 by gene body DNA methylation. The mechanism of the epigenetic repression by AS2, however, is unknown. Here, we tested mutations of NUCLEOLIN1 (NUC1) and RNA HELICASE10 (RH10) encoding nucleolus-localized proteins for the methylation in exon 6 as these mutations enhance the level of ETT/ARF3 transcripts in as2-1. Methylation levels at three specific CpGs were decreased in rh10-1, and two of those three overlapped with those in as2-1. Methylation levels at two specific CpGs were decreased in nuc1-1, and one of those three overlapped with that in as2-1. No site was affected by both rh10-1 and nuc1-1. One specific CpG was unaffected by these mutations. These results imply that the way in which RH10, NUC1 and AS2 are involved in maintaining methylation at five CpGs in exon 6 might be through at least several independent pathways, which might interact with each other. Furthermore, we found that AS2 binds specifically the sequence containing CpGs in exon 1 of ETT/ARF3, and that the binding requires the zinc-finger-like motif in AS2 that is structurally similar to the zinc finger-CxxC domain in vertebrate DNA methyltransferase1.

Affinity-Based Enrichment Techniques for the Genome-Wide Analysis of 5-Hydroxymethylcytosine.

Since its initial characterization in 2009 there has been a great degree of interest in comparative profiling of 5-hydroxymethylcytosine (5hmC) nucleotides in vertebrate DNA. Through a host of genome-wide studies the distribution of 5hmC has been mapped in a range of cell lines, tissue types and organisms; the majority of which have been generated through affinity-based methods for 5hmC enrichment. Although recent advances in the field have resulted in the ability to investigate the levels of both methylated and hydroxymethylated cytosines at single base resolution, such studies are still relatively cost-prohibitive as well as technically challenging. As such affinity-based methods for the enrichment of 5hmC-modified DNA fragments represent a cost-effective and highly informative method for profiling 5hmC residency in genomic DNA. Here we will outline protocols for two independent affinity based methods to generate 5hmC enriched fractions for subsequent locus specific and genome-wide analysis; immunoprecipitation using highly specific 5hmC antibodies as well as a chemical capture based method.

Host-Feeding Patterns of Culex stigmatosoma (Diptera: Culicidae) in Southern California.

Knowledge of the blood-feeding patterns exhibited by arthropod vectors is essential for understanding the complex dynamics of vector-borne disease transmission. Some species of mosquitoes belonging to the genus Culex have been implicated as having major roles in the transmission of arboviruses such as West Nile virus, Saint Louis encephalitis virus, and Western equine encephalitis virus. Although the host-feeding patterns for many of these Culex species are well studied, the host-feeding patterns of Culex stigmatosoma Dyar are relatively poorly studied, even though this species is suspected to be an important maintenance vector for West Nile virus and other arboviruses. In the current study, bloodmeals from 976 blood-engorged Cx. stigmatosoma, collected from 30 sites in southern California from 2009-2012, were processed for vertebrate host identification by nucleotide sequencing following polymerase chain reaction to amplify portions of the cytochrome oxidase I and cytochrome b genes of vertebrate animals. Vertebrate DNA was amplified, sequenced, and identified from a total of 647 Cx. stigmatosoma bloodmeals, revealing that 98.6% of bloodmeals were from birds, 1.2% from three mammal species, and a single bloodmeal was from a reptile species. In total, 40 different host species were identified. The greatest number of bloodmeals identified was from domestic chicken (Gallus gallus domesticus L.) (38% of bloodmeals), house sparrow (Passer domesticus L.) (23%), house finch (Haemorhous mexicanus Müller) (17%), American crow (Corvus brachyrhynchos L.) (4%), and mourning dove (Zenaida macroura L.) (3%). However, chicken bloodmeals were identified almost entirely from a single site where mosquito collection devices were placed in the near vicinity of confined domestic chickens. The strongly ornithophilic feeding behavior shown in this study for Cx. stigmatosoma supports the hypothesis that this mosquito species may be an important maintenance (or endemic) vector for arboviruses that circulate among susceptible birds.

Carrion fly-derived DNA metabarcoding is an effective tool for mammal surveys: Evidence from a known tropical mammal community.

Metabarcoding of vertebrate DNA derived from carrion flies has been proposed as a promising tool for biodiversity monitoring. To evaluate its efficacy, we conducted metabarcoding surveys of carrion flies on Barro Colorado Island (BCI), Panama, which has a well-known mammal community, and compared our results against diurnal transect counts and camera trapping. We collected 1,084 flies in 29 sampling days, conducted metabarcoding with mammal-specific (16S) and vertebrate-specific (12S) primers, and sequenced amplicons on Illumina MiSeq. For taxonomic assignment, we compared blast with the new program protax, and we found that protax improved species identifications. We detected 20 mammal, four bird, and one lizard species from carrion fly metabarcoding, all but one of which are known from BCI. Fly metabarcoding detected more mammal species than concurrent transect counts (29 sampling days, 13 species) and concurrent camera trapping (84 sampling days, 17 species), and detected 67% of the number of mammal species documented by 8years of transect counts and camera trapping combined, although fly metabarcoding missed several abundant species. This study demonstrates that carrion fly metabarcoding is a powerful tool for mammal biodiversity surveys and has the potential to detect a broader range of species than more commonly used methods.

Structure of a DNA glycosylase that unhooks interstrand cross-links

DNA glycosylases are important editing enzymes that protect genomic stability by excising chemically modified nucleobases that alter normal DNA metabolism. These enzymes have been known only to initiate base excision repair of small adducts by extrusion from the DNA helix. However, recent reports have described both vertebrate and microbial DNA glycosylases capable of unhooking highly toxic interstrand cross-links (ICLs) and bulky minor groove adducts normally recognized by Fanconi anemia and nucleotide excision repair machinery, although the mechanisms of these activities are unknown. Here we report the crystal structure of Streptomyces sahachiroi AlkZ (previously Orf1), a bacterial DNA glycosylase that protects its host by excising ICLs derived from azinomycin B (AZB), a potent antimicrobial and antitumor genotoxin. AlkZ adopts a unique fold in which three tandem winged helix-turn-helix motifs scaffold a positively charged concave surface perfectly shaped for duplex DNA. Through mutational analysis, we identified two glutamine residues and a β-hairpin within this putative DNA-binding cleft that are essential for catalytic activity. Additionally, we present a molecular docking model for how this active site can unhook either or both sides of an AZB ICL, providing a basis for understanding the mechanisms of base excision repair of ICLs. Given the prevalence of this protein fold in pathogenic bacteria, this work also lays the foundation for an emerging role of DNA repair in bacteria-host pathogenesis.

CRL2Lrr1 promotes unloading of the vertebrate replisome from chromatin during replication termination.

A key event during eukaryotic replication termination is the removal of the CMG helicase from chromatin. CMG unloading involves ubiquitylation of its Mcm7 subunit and the action of the p97 ATPase. Using a proteomic screen in Xenopus egg extracts, we identified factors that are enriched on chromatin when CMG unloading is blocked. This approach identified the E3 ubiquitin ligase CRL2Lrr1, a specific p97 complex, other potential regulators of termination, and many replisome components. We show that Mcm7 ubiquitylation and CRL2Lrr1 binding to chromatin are temporally linked and occur only during replication termination. In the absence of CRL2Lrr1, Mcm7 is not ubiquitylated, CMG unloading is inhibited, and a large subcomplex of the vertebrate replisome that includes DNA Pol ε is retained on DNA. Our data identify CRL2Lrr1 as a master regulator of replisome disassembly during vertebrate DNA replication termination.

Assessing the feasibility of fly based surveillance of wildlife infectious diseases.

Monitoring wildlife infectious agents requires acquiring samples suitable for analyses, which is often logistically demanding. A possible alternative to invasive or non-invasive sampling of wild-living vertebrates is the use of vertebrate material contained in invertebrates feeding on them, their feces, or their remains. Carrion flies have been shown to contain vertebrate DNA; here we investigate whether they might also be suitable for wildlife pathogen detection. We collected 498 flies in Taï National Park, Côte d’Ivoire, a tropical rainforest and examined them for adenoviruses (family Adenoviridae), whose DNA is frequently shed in feces of local mammals. Adenoviral DNA was detected in 6/142 mammal-positive flies. Phylogenetic analyses revealed that five of these sequences were closely related to sequences obtained from local non-human primates, while the sixth sequence was closely related to a murine adenovirus. Next-generation sequencing-based DNA-profiling of the meals of the respective flies identified putative hosts that were a good fit to those suggested by adenoviral sequence affinities. We conclude that, while characterizing the genetic diversity of wildlife infectious agents through fly-based monitoring may not be cost-efficient, this method could probably be used to detect the genetic material of wildlife infectious agents causing wildlife mass mortality in pristine areas.

Gene and transposable element methylation in great tit (Parus major) brain and blood.

BackgroundStudies on vertebrate DNA methylomes have revealed a regulatory role of tissue specific DNA methylation in relation to gene expression. However, it is not well known how tissue-specific methylation varies between different functional and structural components of genes and genomes. Using whole-genome bisulfite sequencing data we here describe both CpG and non-CpG methylation profiles of whole blood and brain tissue in relation to gene features, CpG-islands (CGIs), transposable elements (TE), and their functional roles in an ecological model species, the great tit (Parus major).ResultsWe show that hypomethylation at the transcription start site (TSS) is enriched in genes with functional classes that relate directly to processes specific to each tissue type. We find that 6877 (~21 %) of the CGIs are differentially methylated between blood and brain, of which 1186 and 2055 are annotated to promoter and intragenic regions, respectively. We observe that CGI methylation in promoter regions is more conserved between tissues compared to CGI methylation in intra and inter-genic regions. Differentially methylated CGIs in promoter and intragenic regions are overrepresented in genomic loci linked to development, suggesting a distinct role for CGI methylation in regulating expression during development. Additionally, we find significant non-CpG methylation in brain but not in blood with a strong preference for methylation at CpA dinucleotide sites. Finally, CpG hypermethylation of TEs is significantly stronger in brain compared to blood, but does not correlate with TE activity. Surprisingly, TEs showed significant hypomethylation in non-CpG contexts which was negatively correlated with TE expression.ConclusionThe discovery that TSS methylation levels are directly linked to functional classes related to each tissue provides new insights in the regulatory role of DNA-methylation patterns. The dominant sequence motifs for brain non-CpG methylation, similar to those found in mammals, suggests that a conserved non-CpG regulatory mechanism was already present in the amniote ancestor. The negative correlation between brain non-CpG methylation and TE activity (not found for CpG methylation) suggests that non-CpG is the dominant regulatory form of methylation in TE silencing.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2653-y) contains supplementary material, which is available to authorized users.

Column-free magnetic enrichment of pDCs yields significant contamination with DNA-containing dead cells that affect the activation status of the isolated cells

Abstract Despite their role in the induction of adaptive immune responses, plasmacytoid dendritic cells (pDC) play also a crucial role in innate immune defense by recognizing pathogen derived molecular patterns. Unmethylated CpG motifs of bacterial DNA bind specifically endosomal and lysosomal TLR9 in pDCs, thereby inducing secretion of type I IFN or proinflammatory cytokines and classical DC maturation, respectively. However, TLR9 also recognizes CpG motifs in vertebrate DNA of damaged cells as danger-associated molecular patterns (DAMPs) that activate pDCs. For in-vitro research pDCs may be isolated by indirect magnetic cell separation using either Miltenyi Biotec’s column-based or a column-free system from other suppliers. Here we describe the influence of the isolation method on the functionality of the purified pDCs. In addition to the significantly better separation performance, our column-based method enriched 10- and 30-fold less DNA-containing dead cells and debris, respectively. Although both methods yielded pDCs with a comparable phenotype instantly after isolation, after 24h of culture without stimulation pDCs isolated with the column-free system appeared pre-activated as demonstrated by elevated expression of CD80, −83, −86 and HLA-DR and by significantly higher basal IFN-α secretion. Moreover, upon stimulation with CpG-B pDCs isolated with the column-free method induced extraordinary high amounts of IFN-α, whereas pDCs isolated with our system exhibited normal responses to CpG-A and CpG-B. These results clearly demonstrate that unspecific enrichment of dead cells and debris may significantly influence the activation status of the isolated pDCs and therefore affect the results of downstream applications.

Identification of Hepatozoon erhardovae Krampitz, 1964 from bank voles (Myodes glareolus) and fleas in Southern Hungary.

In order to investigate the prevalence and life cycle of apicomplexan parasites, small mammals were live-trapped with modified Sherman traps in Southern Hungary between 2010 and 2012. Altogether, 528 rodents (Apodemus flavicollis Melchior, 1834, Apodemus agrarius Pallas, 1771, Myodes glareolus Schreber, 1780, Microtus agrestis Linnaeus, 1761, Mus musculus Linnaeus, 1758 and Micromys minutus Pallas, 1771) were collected and four shrews (Sorex spp.) were by-catched. Captured animals belonging to non-protected species were euthanized, and spleen samples were preserved for histological and molecular analyses. During the examination of spleen smears, Hepatozoon parasites were observed in eight out of 48 bank voles (M. glareolus). DNA was isolated from altogether 221 spleen samples, and 18S rDNA was amplified using two different PCR protocols. The eight bank vole samples were positive with PCR, but none of the other M. glareolus spleen samples or any of the tissue samples from other species were found to be infected. Sequenced amplicons were very similar to Hepatozoon spp. detected in M. glareolus in Spain and Poland. Ectoparasites were collected from the small mammal carcasses and from the vegetation. Hepatozoon DNA was not found in the 181 ticks removed from the small mammals or in the 162 ticks collected with flagging, but was detected in all three flea species (4/43 Megabothris turbidus Rothschild, 1909, 3/10 Ctenophthalmus assimilis Taschenberg, 1880 and 7/78 Ctenophthalmus agyrtes Heller, 1896). Based on gamont morphology, vertebrate and arthropod host species and DNA sequences, the parasites in our study can be identified as Hepatozoon erhardovae.

Expanding the diversity of DNA base modifications with N⁶-methyldeoxyadenosine.

Vertebrate DNA is subjected to epigenetic base modifications that have been thought to be limited to methylated and other modified forms of cytidine. A recent study shows that methylation of adenine to form N6-methyladenine is a rare but readily detectable modification that can be mapped to distinct genomic sites in vertebrates.

Cytokine Diedel and a viral homologue suppress the IMD pathway in Drosophila

Viruses are obligatory intracellular parasites that suffer strong evolutionary pressure from the host immune system. Rapidly evolving viral genomes can adapt to this pressure by acquiring genes that counteract host defense mechanisms. For example, many vertebrate DNA viruses have hijacked cellular genes encoding cytokines or cytokine receptors to disrupt host cell communication. Insect viruses express suppressors of RNA interference or apoptosis, highlighting the importance of these cell intrinsic antiviral mechanisms in invertebrates. Here, we report the identification and characterization of a family of proteins encoded by insect DNA viruses that are homologous to a 12-kDa circulating protein encoded by the virus-induced Drosophila gene diedel (die). We show that die mutant flies have shortened lifespan and succumb more rapidly than controls when infected with Sindbis virus. This reduced viability is associated with deregulated activation of the immune deficiency (IMD) pathway of host defense and can be rescued by mutations in the genes encoding the homolog of IKKγ or IMD itself. Our results reveal an endogenous pathway that is exploited by insect viruses to modulate NF-κB signaling and promote fly survival during the antiviral response.

Characterization of How DNA Modifications Affect DNA Binding by C2H2 Zinc Finger Proteins.

Much is known about vertebrate DNA methylation and oxidation; however, much less is known about how modified cytosine residues within particular sequences are recognized. Among the known methylated DNA-binding domains, the Cys2-His2 zinc finger (ZnF) protein superfamily is the largest with hundreds of members, each containing tandem ZnFs ranging from 3 to >30 fingers. We have begun to biochemically and structurally characterize these ZnFs not only on their sequence specificity but also on their sensitivity to various DNA modifications. Rather than following published methods of refolding insoluble ZnF arrays, we have expressed and purified soluble forms of ZnFs, ranging in size from a tandem array of two to six ZnFs, from seven different proteins. We also describe a fluorescence polarization assay to measure ZnFs affinity with oligonucleotides containing various modifications and our approaches for cocrystallization of ZnFs with oligonucleotides.

Identification of methylated deoxyadenosines in vertebrates reveals diversity in DNA modifications.

Methylation of cytosine deoxynucleotides (dC5m) is a well-established epigenetic mark, but in higher eukaryotes much less is known about modifications affecting other deoxynucleotides. Here, we report the detection of N-6-methyl-deoxyadenosine (dA6m) in vertebrate DNA, specifically in Xenopus laevis, but also in other species including mouse and human. Our methylome analysis reveals that dA6m is widely distributed across the eukaryotic genome, is present in different cell types, but commonly depleted from gene exons. Thus, direct DNA modifications might be more widespread than previously thought.