Characterizing the transcriptional expression and in situ localization of the Dnmt2 gene in Drosophila willistoni

Background: Organisms that have only the DNA methyltransferase 2 (Dnmt2) to mediate the DNA methylation are called "Dnmt2-only" and they have been investigated in recent surveys. Drosophila is one of the “Dnmt2-only” organisms and is also an ideal model for Dnmt2 research. However, the biological function of the Dnmt2 protein is still uncertain. Some studies have pointed to a putative role during the early stages of invertebrate development. In this work, we present our findings on the Dnmt2 expression in D. willistoni, a neotropical species of large ecological versatility and peculiar molecular features. Results: By RT-PCR and in situ hybridization we demonstrate here the presence of transcripts not only in the early stages of development, but also during the oogenesis. Using qPCR analysis, we verify that Dnmt2 transcription level is higher during early stages of development, though transcription levels are subtly higher in D. willistoni adults than in D. melanogaster levels found in previous studies. We also mapped the Dnmt2 on the IIL chromosome arm (Muller’s B element) of D. willistoni, near at the end of the singular telomeric region. Conclusions: Our findings give insights on the possible biological function of Dnmt2-related processes associated with the development and differentiation of oocytes since germinative tissue formation seems to require a higher expression of Dnmt2. The Dnmt2 localization in the subtelomeric region brings up a series of issues that involve the peculiar characteristics of D. willistoni Dnmt2 enzyme, like evolutionary patterns and the epigenetic phenomena of sex-specific methylation.


Background
Cytosine-5 RNA methylation is one among more than 100 distinct RNA modifications that has been already detected in tRNA, rRNA and mRNA [1], representing an important epigenetic modification that regulates gene expression in eukaryotes, with a standing role 3 on their development and for the etiology of human diseases and mediating chromatin organization [2][3][4]. Elucidation of the molecular mechanisms mediating RNA and DNA methylation is crucial to understanding the roles that diverse nucleic acids play in the regulation of genetic information.
Although DNA methylation appears to be a widespread epigenetic regulatory mechanism, genomes are methylated in different ways in diverse organisms. DNA methylation in plant genomes, for instance, can occur symmetrically at cytosine nucleotides in both CG and CHG (H= A, T or C) contexts, whereas DNA methylation systems are well characterized in vertebrates [5] and occur mostly symmetrically at the cytosine of a CG dinucleotide [6,7].
The cytosine methylation is established and maintained by a family of conserved methyltransferases. In eukaryotes, there are three distinct families of DNA methyltransferases: Dnmt1, Dnmt2, and Dnmt3. Whereas Dnmt3 enzymes seem to be responsible for establishing DNA methylation patterns ( de novo methyltransferases), Dnmt1 enzymes are involved in the maintenance of the methylation patterns [8]. On the other hand, the biochemical activity and the biological function of Dnmt2 enzymes are still an open case.
Evidence for DNA methylation has been described and its importance demonstrated in different insect orders. The honeybee genome contains genes that encode orthologues of all vertebrate proteins required for DNA methylation [9,10]. In addition to Dnmt2 (also found in Diptera), two CpG-specific Dnmt family genes were identified: Dnmt1 and Dnmt3a/b genes [11]. The specificity for CpT and CpA nucleotide residues distinguishes Dnmt2 from all other known animal DNA methyltransferases and confirms our previous suggestion of predominant non-CpG methylation in Drosophila [12,13]. The Apis mellifera genome shows greater similarities to vertebrate genomes than Drosophila and Anopheles genomes for the 4 genes involved in DNA methylation, among others (The Honeybee Genome Sequencing Consortium, 2006). For these reasons, methylation in D. melanogaster and other invertebrates remains a controversial research area.
Regarding the genus Drosophila, the species of this genus are so-called " Dnmt2-only" organisms, not containing any of canonical vertebrates DNA methyltransferases homologs (Dnmt1 and Dnmt3). For the species of this genus studied so far, methylation was demonstrated at non-CpG nucleotides [13][14][15], in contrast to most organisms already analyzed. Furthermore, the specificity for CpT, CpA, and CpC nucleotide residues distinguishes Dnmt2 from all other known animal DNA methyltransferases [13,16], and the functional role of DNA methylation in Drosophila remains unclear. However, adding to that discussion, a recent study describes the importance of the methyl-CpG binding domain proteins to inhibit interspecies courtship and promoting aggression in Drosophila [17].
Another important difference between DNA methylation in mammals and Drosophila is the level of the cytosine nucleotides methylated. Whereas mammals have 2 to 10% of all cytosine residues modified to 5-methylcytosine [18,19], Drosophila melanogaster has only about 0.1 -0.6% [14,20], making it experimentally difficult to demonstrate unambiguously DNA methylation in this organism. Moreover, in contrast to the pattern of genome-wide DNA methylation in vertebrates, DNA methylation is relatively scattered in invertebrates [21].
Several attempts were made to demonstrate the DNA methylation activity of Dnmt2 in D.
melanogaster [13,22] revealing a low, but significant activity by distinct experimental methods. Overexpression of Dnmt2 in Drosophila species appears to enhance genome-wide DNA methylation from 0.2% to 0.4 -0.7%, as determined by capillary electrophoresis [13,22]. Nevertheless, in a recent study reported that Dnmt2 controls DNA methylation in early D. melanogaster embryos and provide insight into its function in control of 5 retrotransposon silencing and telomere integrity in somatic cells [23]. This report showed a clear-cut difference of methylation within Invader4 elements LTRs between wild type and Dnmt2-/-flies [23]. Dnmt2 appears to mediate methylation on D. melanogaster embryos genome, even though both this activity and its functional consequences remaining poorly understood. However, a recent study showed that cytosine methylation in the genome of Drosophila melanogaster probably is independent of Dnmt2 activity [24]. These findings show how elusive is the genomic methylation context in drosophilids.
Previous studies reported by our research group in the neotropical D. willistoni and its related species (willistoni subgroup) shows a distinct scenario. Adult flies show sex-specific patterns of rDNA genes investigated by Methylation Restriction Sensitive Endonucleases [15,25]. Comparisons of D. willistoni and D. melanogaster Dnmt2 protein sequences indicated higher primary structure conservation on motifs responsible for the catalysis of methyl transfer and great variability in the region related to specific recognition of target DNA sequences. These outcomes from the willistoni subgroup species are encouraging due to the previously reported peculiarities of this species when compared to other species of the Drosophila genus that have their genomes sequenced [26]. D. willistoni singularities correspond to overall genome size, distribution of transposable element classes, patterns of codon usage, dot chromosome lacking and unclear phylogenetic clustering placement, among others [26][27][28].
In addition to the peculiarity of having sex-specific methylation [15,25] [33]. The ambiguity arises as a consequence of the elevated level of sequence and geneorder evolution that leads to a significantly long lineage leading to D. willistoni. Most phylogenetic reconstruction software tends to force D. willistoni as an outgroup, based on the high evolutionary rates. In light of these reasons, studies are needed to map more genes in the genome of D. willistoni in order to elucidate the discrepancies found in the previous reports. New approaches, using an additional marker will not only identify more genes to confirm the ordering of scaffolds assembled so far but will also facilitate further comparative studies by increasing the database from this species.
To improve the Dnmt2 knowledge about its biological function and importance, more assays for characterization and detection of Dnmt2-dependent DNA/RNA methylation will have to be established. Genomic DNA methylation patterns need to be characterized mostly in performed the detection and revelation of hybridization signals, respectively. Slides were stained with Giemsa solution (5%) and coverslips were assembled with Entellan (Merck).
Polytene chromosomes images were acquired in phase contrast photomicroscope and analyzed according to the photomap of D. willistoni [33].

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The Dnmt2 gene resides in the arm IIL of the D. willistoni chromosome The location of the probe hybridization signal was determined to be at the distal region of the chromosome II left arm (IIL), specifically in section 55C ( Figure 5A and 5B biological function has been fervently discussed [40,41]. There are accumulating findings that instigate investigations in " Dnmt2-only" organisms, such as female-specific DNA methylation that was reported for D. willistoni and related species [15,25]. Recently, males that have reduced expression of dMBD-R2 (a methylated 12 cytosine binding protein), specifically octopamine (equivalent to norepinephrine) neurons, have exhibited mating behavior toward divergent interspecies such as D. virilis and D.
yakuba and a decrease in the success of co-specific mating [17]. These data strongly suggest the existence of methylation-dependent chromatin structures in Drosophila, that is, the role of methylation of genomic cytosines appears to be fundamental at various levels in these organisms. Thus, Dnmt2 studies is of great importance towards a better understanding of DNA and RNA methylation in '' Dnmt2-only'' systems, whose data are still vastly controversial and uncertain. The importance of DNA methylation in Drosophila still is an elusive case, and there are some attempts to understand the biological function in Drosophila genomes. However, when we are talking about a genus with more than 4.000 species, peculiarities may arise, like the sex-specific methylation described in D. willistoni [15,25], so generalization must be avoided. It was observed that overexpression of D. melanogaster Dnmt2 results in an extended fly life span and in overexpression of several genes [42]. Also, it is discussed whether Dnmt2 acts as DNA or tRNA methyltransferase. If Dnmt2 was described associated to a nuclear matrix, and if Drosophila is a "Dnmt2-only" organism, and if D. willistoni has sex-specific methylation in its genome, we cannot deny that the evidence suggests a duality in the activity of that enzyme in these organisms. The peculiarities of Dnmt2 enzyme rise in different organisms; in humans, for example, it is located in cytoplasmic regions [43], whereas in Dyctiostelium discoideum and Entamoeba histolytica, Dnmt2 is located only in the nuclear matrix [27,44], and guaranteeing the structural integrity of chromatin and silencing retrotransposons in Drosophila [23]. Generating Dnmt2 null mutations allowed us to correlate loss of DNA methylation, identified in early D. melanogaster embryos by bisulfite sequencing and restriction enzyme analysis. Besides that, these results suggest the Dnmt2-dependent DNA methylation during early embryonic development of D.

Our quantitative analyses on different development stages showed differences in
melanogaster has a key function in control of retrotransposon silencing in somatic cells, specifically as an initial step of the process [23]. This feature contrasts with the high local specificity of the Dnmt1 and Dnmt3 to the cell nucleus region.
On the other hand, the HIV virus is able to increase its survival in the host cell using the RNA methylation activity of Dnmt2, increasing the stability of its own genome. Through infection, Dnmt2 relocates from the nucleus to the cytoplasmic stress granules and methylates the RNA virus, promoting the post-transcriptional stability of viral RNA [45].
RNAs interference (RNAi) have a strong association with different epigenetic mechanisms, such as histone methylation/acetylation and genomic DNA methylation [46]. Doublestranded RNAs (dsRNA) trigger the process of post-transcriptional homology-dependent gene interference (RNAi) closely related to the co-suppression of viral exogenous transcripts. The dsRNAs are processed by a Dicer family of enzymes into small dsRNA sequences having 21-25 nucleotides, termed small interfering RNAs (or siRNAs).
Interestingly, it is known that Dnmt2 also interacts with Dicer-2 in response to heat shock treatments [47,48], controlling the expression and regulation of various Heat Shock proteins [49][50][51]. Finally, the activity of Dnmt2 in conjunction with NSun2 is important to promote the stability of tRNAs and protein synthesis [52]. That is, we can assume that the Dnmt2 has a multilevel biological role, inserted in an extensive network of interactions in the genetic machinery of the gene expression control.
In the present study, we performed the in situ localization of the D. willistoni Dnmt2 ( Figure   5) It is known that heterochromatin regions (centromeric and telomeric) have different patterns of evolution than those found in the euchromatin regions (gene-rich). Comparative analyses between telomeric regions of humans and chimpanzees have shown that both are very distinct, in that the human chromosomes present a large scale of rearrangements and differences in the repetitive elements present [53]. Interestingly, the chromosome ends in Drosophila melanogaster have the peculiarity of presenting transposable elements (

HeT-A
and TART) [54][55][56][57], contrasting with the human chromosome ends that are structured from the activity of the enzyme telomerase. Another interesting aspect is that most-distal regions of Drosophila subtelomeres seem to evolve rapidly between Drosophila melanogaster and its close relatives, D. simulans and D. yakuba [58]. In this region, the mutation rate of the most-distal portion is so high that the structure is likely to be different among cells of the same individual [59]. However, what is observed in terms of recombination in the telomeric regions of Drosophila melanogaster is that there is a great suppression of crossing over events, within and in regions proximal to the telomeres [60].
These aspects are in agreement with previous findings of our research group, in which D.
willistoni Dnmt2 shows the highest rate of nucleotide substitution per codon in relation to the other drosophilids and different selective pressures at specific sites involved in an interaction-driven co-evolution with other genes [61].

CONCLUSION
Epigenetic mechanisms are fundamental in the coordination of the ontogeny of organisms and in the intermediation of information from the environment to the nucleus and from the nucleus to the environment. The understanding of such mechanisms is of paramount importance in order to understand the ecological, physiological and evolutionary aspects of the different species. Among the epigenetic phenomena, cytosine methylation is the most widely studied and known. However, its role is still elusive in organisms known as " Dnmt2only", among them Drosophila.
In the present work, we verified that the expression of Dnmt2 in D. willistoni is closely related to its ontogenetic stages. In addition, it has been found that expression levels in adults of D. willistoni Dnmt2 are apparently slightly higher than in D. melanogaster. It is suggested, therefore, that this difference may be closely related to the phenomenon of sex-specific methylation, a peculiar and restricted phenomenon to the species of the subgroup willistoni. We also conduct the in situ localization of the Dnmt2 gene, revealing its presence in the arm IIL (Muller B element). The Dnmt2 is located in the subtelomeric region, and this brings up a series of issues that involve the peculiar characteristics of D. willistoni Dnmt2 enzyme, like evolutionary pattern in nucleotide substitutions rates [61], the high prevalence of basic amino acids residues in its target recognition domain surface [62] and the epigenetic phenomena of sex-specific methylation [15,25].

Availability of data and materials
All the data supporting the results of this article are included within the article. All data can be provided by request. VLSV provided expertise and feedback. GCV, CFG and RZ wrote the manuscript with input from all authors. All authors read and approved the final manuscript.
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Consent for publication
Not applicable.  Estimate of Dnmt2 transcriptional expression levels in the D. willistoni samples.
The results were normalized with β-actin expression. All data were obtained from triplicates.