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-methylcytosine is an important
epigenetic modification involved in
gene control in vertebrates and many
other complex living organisms. Its presence in Drosophila has been a matter of
debate and recent bisulfite sequencing
studies of early-stage fly embryos have
concluded that the genome of Drosophila is essentially unmethylated. However, as we outline here, the Drosophila
genome harbors a well-conserved homolog of the TET protein family. The
mammalian orthologs TET1/2/3 are
known to convert 5-methylcytosine into
5-hydroxymethylcytosine. We discuss
several possible explanations for these
seemingly contradictory findings. One
possibility is that the 2 modified cytosine bases are generated in Drosophila
only at certain developmental stages and
in a cell type-specific manner during
neurogenesis. Alternatively, Drosophila
Tet and its mammalian homologs may
carry out catalytic activity-independent
functions, and the possibility that these
proteins may oxidize 5-methylcytosine
in RNA created by the methyltransferase Dnmt2 should also be strongly
considered.
Keywords: Drosophila,
epigenetics, DNA methylation,
5-methylcytosine, dioxygenase
Introduction
Submitted: 09/05/2013
Accepted: 09/18/2013
http://dx.doi.org/10.4161/cc.26540
*Correspondence to: Gerd P Pfeifer;
Email: gpfeifer@coh.org
www.landesbioscience.com
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CpG depletion is not observed in Drosophila genes, in which the observed-toexpected CpG frequency is very close to
1. Assuming that CpG depletion is caused
by mutational mechanisms, including
deamination of 5-methylcytosine,2 one
would therefore predict that the genome
of Drosophila contains little to no 5mC.
However, this prediction would only apply
to the germ line and not to somatic cells or
tissues, which might still harbor methylated sequences. CpG islands, the typical
GC-rich and not CpG-depleted areas
of vertebrate genomes, which are often
important for gene control, are not present
in the Drosophila genome, perhaps owing
to its already higher general CpG frequency.33 In contrast to flies, the observed
to expected ratio of CpGs in the honey bee
(Apis mellifera) shows a striking bimodal
distribution clearly suggesting that the
genome of bees contains methylated and
unmethylated genes.25 In mammals, different promoter classes have been identified. Among these, the so called broad
promoters have multiple transcription
start sites, are rich in CpG sequences, are
generally embedded within large CpG
islands, and are controlled often by epigenetic modifications, including DNA
methylation and histone modifications.
Other types of promoters including those
having sharp transcription start sites usually are not within CpG islands and are
less sensitive to such epigenome modifications. Surprisingly, Drosophila has both
the broad and sharp promoters, suggesting that this bimodal architecture of
promoters is conserved.33,34 This would
suggest that either (1) there is such epigenomic modulation also in Drosophila,
but we have not detected it, or (2)there
are other mechanisms that replace CpG
methylation in the regulation of those
broad promoters.
The Enigmatic Dnmt2
There are some well-studied organisms, including Saccharomyces cerevisiae
and Caenorhabditis elegans, which do
not contain any DNA methyltransferase
gene, and there is agreement that these
2 organisms also do not contain DNA
cytosine methylation. Several organisms, including Drosophila melanogaster
Volume 12 Issue 21
Figure1. Domain architecture of human TET1, TET2, and TET3 and Drosophila melanogaster dTet.
The conserved CXXC, cysteine-rich region and catalytic domain regions are indicated in blue,
orange, and green, respectively.
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Figure2. Alignment of the conserved residues from the functional domains of TET proteins from diverse multicellular organisms. (A) Alignment of the
CXXC DNA binding domains from human, Xenopus tropicalis, Mus musculus, Drosophila melanogaster, and Apis mellifera TET genes. Conserved domain
residues are highlighted in blue. The residues predicted to play a role in DNA binding from previous crystal structure data of MLL1 and Cfp1 are indicated
in brown. (B and C) Alignment of the conserved HxD and H residues of the Fe2+ and 2-oxoglutarate binding sites comprising the active site from human,
Xenopus, mouse, Drosophila, and Apis TET genes. The conserved catalytic residues are indicated in green and orange.
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Volume 12 Issue 21
unlikely that Drosophila would have maintained a protein the size of dTet if, as is
becoming more apparent in mammalian
studies, it was not playing a significant
role at one or more points in development as an epigenetic modifier. From the
alignments (Fig.2) the residues for DNA
binding and catalytic activity are all very
highly conserved, indicating that it is also
less likely that dTet would have evolved
a different function than that which has
been previously reported for mammalian
TETs. Given the obvious conservation of
important residues, domains, and protein
Figure3. 3D modeling of the metal binding domain of human TET proteins and Drosophila Tet. The 3D models of human TET1 (A), human TET2 (B),
human TET3 (C), and dTet (D) conserved sub-sequences including the metal binding residues of the catalytic domain are shown. Models were based
on the template 3s57 chain A of the human ABH2 protein and were built and evaluated using the IntFOLD, I-TASSER and ModFOLD4 servers and the
SPARKS-X and Modeler standalone programs.73-76 Details of the modeling approach are provided in the Supplementary Material. The images of models were rendered using PyMOL. The conserved metal binding residues are indicated as blue sticks; -sheets are in yellow; helices in red; and loop
regions are shown in green. (E) Root-mean-square deviation (RMSD) scores (lower is closer) and template modeling TM scores (higher is closer) indicating the structural relationship between human TET 3D models and the Drosophila Tet 3D model. The TM-align method77 was used to score the structural
relationships between models. The TM scores in Figure2E indicate that all of the human Tet models are likely to share the same fold as the Drosophila
Tet model, as they are above the 0.5 threshold therefore indicating a significant match.78 Both the RMSD and the TM scores indicate that the Human Tet3
model is perhaps the closest to the Drosophila Tet model in terms of global structural similarity.
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Figure4. Expression of Dnmt2 and dTet during embryonic stages of Drosophila development. The expression patterns of dTet and Dnmt2/Mt2 show a
significant increase of dTet expression from the fourth hour of embryo development onwards; this pattern is mirrored by the expression of Dnmt2/Mt2.
RNA-seq data were derived from Graveley et al. 59 Expression levels are given as average number of fragments (RNA-seq reads) per kilobase of transcript
per million fragments mapped.
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Figure5. Expression of Dnmt2 and dTet from various Drosophila tissues. These data show that the expression of dTet is highest in the central nervous
system. dTet can also be detected in other tissues but at a much lower level. The expression levels of Dnmt2/Mt2 are also highest in the central nervous
system though the difference relative to other tissues is smaller.79 Expression levels are given as average number of RNA-seq reads per kilobase of transcript per million fragments mapped.
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transferase (OGT).66-70 Although the precise meaning of these interactions are currently not well understood, it is possible
that analogous or even novel functions
have evolved in dTet to maintain an alternative epigenetic role of the protein in the
absence of 5mC.
Conclusion
In conclusion, we clearly show that a
Tet gene is expressed along with Dnmt2 in
the Drosophila genome in specific cells and
development states, and by inference, the
possibility for the presence of 5mC and
5hmC should be considered in those same
cells and tissues. We also suggest that any
examination of DNA methylation in Drosophila or other organisms where a TET
gene is present needs to take into account
the likely presence of 5hmC generated by
the conserved TET protein(s) and ensure
that appropriate methods for the differential examination of both 5mC and 5hmC
be used.71,72 The tissues and stages at
which Drosophila Tet is expressed would
be ideal targets for use to develop a better
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