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Cell Cycle 12:21, 33573365; November 1, 2013; 2013 Landes Bioscience

The mysterious presence of a 5-methylcytosine oxidase in the

Drosophila genome
Possible explanations
Thomas L Dunwell1, Liam J McGuffin2, Jim M Dunwell3, and Gerd P Pfeifer1,*
Beckman Research Institute of the City of Hope; Duarte, CA USA; 2School of Biological Sciences; University of Reading; Reading, Berkshire, UK;
School of Agriculture, Policy and Development; University of Reading; Reading, Berkshire, UK


-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

Keywords: Drosophila,
epigenetics, DNA methylation,
5-methylcytosine, dioxygenase


Submitted: 09/05/2013
Accepted: 09/18/2013
*Correspondence to: Gerd P Pfeifer;

Modified DNA bases exist in the

genomes of a wide array of organisms,
including bacteria, fungi, plants, and
animals. These modified bases usually escape detection by standard DNA
sequencing approaches, and therefore

their nature, prevalence, tissue specificity,

and DNA sequence-dependent distribution has remained largely unexplored.
All studied vertebrate genomes contain
the base 5-methylcytosine (5mC) at levels equivalent to a few % of all cytosines
present in that organism.1 However, 5mC
is present at variable and often very low
to non-detectable levels in invertebrates.
This modification arises in a post-replicative enzymatic reaction, when DNA
methyltransferase (Dnmt) proteins transfer a methyl group from the cofactor
S-adenosyl-L-methionine onto carbon 5
of cytosine in DNA. The primary target
sequence in mammalian cells is the CpG
dinucleotide. Paradoxically, even though
methylation of cytosine is highly conserved in vertebrates, its presence at CpG
dinucleotides has a substantial mutagenic
effect, which has led to a strong depletion of CpG sequences during evolution.2
When CpG sequences are unmethylated,
however, they are not mutated, are maintained in the germ line, and have accumulated in particular compartments of the
genome termed CpG islands. The evolutionary conservation of DNA methylation
despite its inherent mutagenicity attests
to its likely biological importance, which
after decades of research, is still not understood in complete detail.
DNA Methylation in Mammals
In mammalian cells, methylation of
cytosines is chiefly performed by 3 catalytically active DNA methyltransferases, the

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maintenance methyltransferase DNMT1,

which acts predominantly on hemimethylated CpG sites arising during DNA
replication, and the DNA methyltransferases DNMT3A and DNMT3B, which
operate in de novo methylation of previously unmethylated CpG sequences but
also in the maintenance of DNA methylation patterns.3,4 A catalytically inactive
DNMT3-like protein, DNMT3L, forms
heterodimers with and stimulates the de
novo methylation activity of DNMT3A
and DNMT3B.5-7 There is a fourth
DNA methyltransferase gene in mammalian genomes, termed DNMT2, which
was initially identified using homology
searches with known DNA methyltransferase sequences.8,9 It is well understood
that deletion of the Dnmt genes Dnmt1,
Dnmt3a, or Dnmt3b in the mouse results
in lethality and severe developmental
defects,10,11 whereas deletion of Dnmt2 in
mice has no recognizable phenotype.12 The
major functional role of DNA methylation in vertebrate development is thought
to be the suppression of inappropriate
transcription, which is accomplished by
different mechanisms, such as interference with transcription factor binding or
modulation of chromatin structure mediated by proteins that interact with CpGmethylated DNA.13
DNA Methylation in Insects
For well over 2 decades, there has been
much debate as to whether there is cytosine methylation present in the Drosophila melanogaster genome. Initial studies
performed in the 1980s produced conflicting results.14,15 This organism lacks
homologs of the mammalian DNA methyltransferase genes Dnmt1, Dnmt3a, or
Dnmt3b but contains a gene encoding a
homolog of Dnmt2 (also known as Mt2;
NM_058127). The Drosophila genome
also harbors a homolog of the mammalian methylated-CpG binding protein
MBD2 (dMBD2/3). The dMBD2/3
protein was shown to have the capacity to bind to CpG-methylated DNA, at
least in vitro.16,17 In the year 2000, Lyko
et al. reported that Drosophila DNA is
methylated, but that Drosophila genome
methylation is restricted to early stages of
embryo development.18 Confocal analysis


of immunostained Drosophila embryos

provided further evidence for the methylation of embryonic DNA.19 Following this
initial work, a more region-specific distribution of 5mC in the Drosophila genome
has been suggested by studies using singlenucleotide resolution bisulfite sequencing
for DNA methylation analysis.20 However,
these results have been controversially
debated in the literature.21,22
In a very recent publication, the conclusion was reached that the genomes of
organisms that contain a Dnmt2 gene
but do not harbor Dnmt1 or Dnmt3a/b,
such as Drosophila, lack DNA methylation altogether.23 This difference in results
was attributed to a lack of specificity of
the earlier applied methods in comparison
to the highly selective and comprehensive
whole genome bisulfite sequencing used in
the latest publication. However there are
numerous reported cases of insects, including ants, aphids, bees, flies, and beetles
(Acyrthosiphon pisum, Aedes aegypti, Apis
mellifera, Camponotus floridanus, Harpegnathos saltator, Nasonia vitripennis, Tribolium castaneum) having clearly detectable
levels and specific patterns of 5mC,24-31
which would mean that the absence of
DNA methylation in Drosophila melanogaster would seemingly be unique within
this class of animals. Particularly wellstudied cases can be found with honeybees
and ants25,32 whose epigenetic machinery
has much more in common with mammals than that found in Drosophila. These
insects contain Dnmt1- and Dnmt3-like
enzymes. The Apis mellifera DNA methylation has been shown be to responsible
for the generation of the different colony
phenotypes in bees.28
CpG Dinucleotide Frequencies
within Drosophila Genes
We first asked if it is possible to obtain
clues about the presence or absence of
DNA methylation in Drosophila by considering sequence features of the Drosophila
genome. As mentioned earlier, CpG dinucleotides have eroded over an evolutionary time scale, leading to a current level
of observed vs. expected CpG frequencies
of only 0.2 to 0.25 in mammals, in other
words, around 80% of these sites have
been lost during mammalian evolution.

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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

and Schistosoma mansoni, contain only

a Dnmt2 homolog. It is now recognized
that Dnmt2 is primarily an RNA methyltransferase since it has been convincingly
shown that Dnmt2 methylates the cytosine at position 38 in tRNA Asp.12 However, earlier studies with DNA substrates
in vitro35 have revealed some limited
activity of Dnmt2 on DNA. According
to biochemical fractionation studies, the
Dnmt2 protein is present in both cytoplasm and nucleus, where it resides in
the nuclear matrix, and therefore it could
theoretically act on RNA and/or DNA.36
Dnmt2 shows little sequence homology
with known RNA methyltransferases37,38
and uses a DNA methyltransferase-type
mechanism to methylate tRNA.39 Furthermore, the catalytic active site amino
acids typical of DNA methyltransferases, including the critical adjacent prolinecysteine residues, are all present in
Dnmt2.3 Collectively, the currently available experimental evidence favors a role of
Dnmt2 in RNA methylation, although
activity on DNA cannot categorically be
TET Proteins and
The ten-11 translocation (TET ) family of genes belong to a larger family of
Fe2+ - and 2-oxoglutarate-dependent dioxygenases, which were found to be capable

of oxidizing 5-methylcytosine (5mC)

to 5-hydroxymethylcytosine (5hmC)
and subsequently of further oxidation of
5hmC to 5-formylcytosine (5foC) and terminally to 5-carboxlycytosine (5caC).40-43
Since the identification of these additional
layers of complexity upon the single original DNA modification (5mC), there has
been much interest in identifying possible roles that these new modifications
might play during cellular development
and differentiation.44-46 Much of the currently available evidence suggests that
5hmC is important in the regulation
of transcription and in developmental
remodeling of DNA methylation patterns when 5mC oxidation appears to be
an initial step toward genome-wide DNA
TET Family Proteins and Orthology
in Drosophila and Other Metazoans
It has been previously reported that
at least one member of the TET family is present in metazoans.50 Mammalian genomes encode 3 different 5mC
oxidases, TET1, TET2, and TET3.
Unexpectedly, the genome of Drosophila
melanogaster also contains a TET homolog. The TET proteins can be characterized by the presence of 2 conserved core
domains, an N-terminal CXXC zinc-finger DNA binding domain and a C-terminal Fe2+ - and 2-oxoglutarate-dependent

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.

dioxygenase (Tet_JBP) catalytic domain

(Fig.1). This excludes TET2 orthologs,
which are believed to have lost the CXXC
domain via a chromosomal inversion.51
TET3 encodes several catalytically active
isoforms that differ in the presence or
absence of the N-terminal CXXC domain
(S-G Jin and GP Pfeifer, unpublished
results). Interestingly, a TET protein
homolog is present in the basal placozoan
Tricoplax adhaerens (XM_002108929)
and the even more basal sponge Amphimedon queenslandica (XP_003384410).
Indeed, surprisingly, the sponge protein
is more similar to the human, than the
Trichoplax is to the human TET proteins. The CXXC DNA binding domain
has long been known to be responsible
for CXXC domain containing proteins
ability to bind to unmethylated cytosines
in the context of CpG dinucleotides.52
These domains are characterized by 8
conserved cysteine residues. Crystallography studies have revealed a pair of
histidine and glutamine residues, which
are responsible for the majority of interactions with CpG dinucleotides during
DNA binding, and which are also evolutionally conserved.53-55 The catalytic
domain of TET proteins can itself be
divided into an N-terminal and C-terminal portion, which contain two halves of
the conserved catalytic (HxD/H) triad
separated by a lengthy, low-complexity,
non-conserved linker region.50 The conserved core domains distinguish the TET
family of dioxygenases from other members of the dioxygenase superfamily by
the presence of the CXXC DNA binding domain, and it is the combination
of all of the domains that characterizes a
TET protein. Organisms can generally
be split into evolutionary groups based on
the number of TET genes identified in
their genomes. The first group, mammals
and vertebrates, have 3 orthologs of TET
genes, except X. tropicalis (a model organism), which only has 2 identifiable members orthologous to TET2 and TET3. The
second group is all other metazoans from
Trichoplax onwards, such as arthropods,
including the aforementioned honeybees
and ants, which all contain a single TET
gene, and, despite evolutionary divergence, their TET proteins are very highly
orthologous to the mammalian TET

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proteins in their previously mentioned

core domains. One notable exception of
the presence of a TET protein can be
found in the model organism Caenorhabditis elegans, which, like other nematodes,
contains no identifiable TET genes, but
also has no DNMT genes and no detectable genomic DNA methylation.
The alignments (Fig.2) show that the
evolutionary conservation of these nonvariable domains, the CXXC domain
and the dioxygenase domain, is remarkably high between humans, mouse,
Xenopus tropicalis, Apis mellifera, and
Drosophila melanogaster. An additional
~200 amino acid region just upstream of
the N-terminal half of the dioxygenase
domain is also highly conserved between
the Drosophila and the mammalian proteins. Three-dimensional modeling and

comparisons between human TET1,

TET2, and TET3 and Drosophila Tet
(dTet) also indicate a high degree of
similarity between all 4 proteins for the
catalytic domain including the conserved metal binding domain residues
(Fig.3AD). According to RSMD and
TM scores, Drosophila Tet is most closely
related to human TET3 (Fig.3E).
The presence of these proteins in
almost every multicellular organism
coupled with their very highly conserved
domains clearly implies that these proteins enzymatic activity must be equally
highly conserved having evolved at an
early stage in metazoan evolution, prior
to tissue differentiation. In mammals,
mainly humans and mice, the function
of 5hmC, the major product of TET protein activity, is becoming clearer with the

rapid pace of new studies with suggested

roles in gene regulation, mammalian
development, differentiation, and disease
states.44 In other organisms the function
of TET proteins has rarely been studied and, in many cases, only mentioned
in passing. Drosophila melanogaster, as a
model organism is well known as having a close ortholog of the mammalian
DNMT2 enzyme, but as mentioned earlier, no homologs of either DNMT1 or
DNMT3A and DNMT3B are present.
However, DNMT2 is now recognized as
a tRNA methyltransferase and is believed
not to be acting on DNA.12 The presence
of a Tet gene in Drosophila melanogaster is
therefore puzzling and leads to the interesting question of what is the function of a
5-methylcytosine oxidase in this organism
if there is no genomic 5mC?

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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Why Is There a 5mC Oxidase,

If There Is No 5mC?
Given the fact that there is a clearly
identifiable Drosophila Tet protein (Fig.2;
NM_001259652) the argument for the
presence of methylation in this species
takes on another dimension with the realization that, as dTet will very likely act
on 5mC and oxidize it, there is no reason
for dTet to be conserved in the Drosophila
genome, if there is no 5mC for it to act
upon. Since the substrate for the TET
proteins is genomic 5mC, it seems highly

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

structure, the complete lack of 5mC in

Drosophila, as now suggested by Raddatz
et al.,23 seems very unexpected.
Expression and Tissue
Distribution of dTet and Dnmt2
The bisulfite-sequencing method used
by Raddatz et al.23 would have detected
both 5hmC and 5mC due to its inability
to distinguish between the 2 base modifications.56,57 It may be possible that some
highly repetitive regions of the Drosophila
genome could contain 5mC/5hmC but

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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would have been excluded from the whole

genome analysis. In those cases where
5mC has been reported in Drosophila, it
has been at very low levels. It thus appears
that levels of 5mC, if present at all, are
either very low, limited to specific genomic
locations, limited to specific cell types or
developmental phases, or possibly a combination of all of these factors.
It is clear that there is a conserved Tet
gene in Drosophila, and it is also clear
that this gene is actively expressed in certain tissues. Examining available expression data for dTet mRNA obtained by in
situ hybridization reveals evidence that
expression of dTet does not appear until
after Drosophila developmental stage 6
(3 h post-fertilization). But, intriguingly,
by stage 16 (15 h post-fertilization) dTet
expression is strongly present in, and limited to, the central nervous system and the
brain (
ex/ CG2083)
(note: the old locus tag CG2083 is now
referred to as CG43444). Expression of
dTet (CG2083) is first detected in the ventral neuroectoderm, and transcripts are
also found in neuroblasts, ganglion mother
cells, and neurons of the central nervous
system. By stage 13, expression is observed
only in the nervous system.58 RNA-seq
data from early embryos reveals a similar pattern of dTet expression with a peak
during the development stages between
68 h (Fig.4).59 This tissue specificity

mirrors the expression pattern in mice,

where Tet3 expression has been reported
to be high in the developing brain,60 the
tissue which also has the highest reported
levels of 5hmC.61 Tissue expression data
from later stages of development, including
larvae and adults, show that there are still
high levels of dTet present in the brain and
central nervous system when compared
with other tissues such as salivary gland
and the digestive system (Fig.5). From
the expression data, it can be predicted
that dTet could be playing an important
role necessary for brain and central nervous system development. In developing
mouse embryos, Tet proteins and 5hmC
are functionally involved in neuronal differentiation.60 Interestingly, in Drosophila
embryos, expression of Dnmt2 follows
a similar expression profile, albeit levels
of Dnmt2 RNA are scored as lower than
those of dTet (Fig.4). From these data, it
appears that Dnmt2 and dTet expression
levels are close to zero at embryonic stages
02 h, the time point from which Drosophila embryos were collected by Raddatz
et al. for bisulfite sequencing analysis.23
Combination of these data clearly suggests that searches for modified cytosines
in Drosophila should include these later
developmental time points when the cytosine modifying enzymes are expressed,
especially focusing on the central nervous
system tissues that show the highest levels
of expression of dTet and Dnmt2.

Another recent report could help shed

light on where else DNA methylation
could be found as well as a potential role
for hydroxymethylation. Yadlapalli and
Yamashita provided evidence for the nonrandom segregation of Y and X chromosomes in male Drosophila germline stem
cells.62 They show that the non-random
chromosome segregation is heritable and is
dependent on a wild-type Dnmt2 protein.
This would imply that Dnmt2 could be
capable of leaving an epigenetic mark on
certain chromosomes. Taking a cue from
mammalian zygote differential methylation and hydroxymethylation immediately
following fertilization, in which paternal
DNA from the sperm is converted from
5mC to 5hmC and maternal DNA from
the egg is left untouched,48,49 it would
seem logical to propose that during this
non-random chromosome segregation in
Drosophila, there could also be 5mC and/
or 5hmC present on one set of chromosomes, which could similarly play a role in
Alternative Roles of Drosophila Tet?
As summarized earlier, dTet contains
several highly conserved TET protein
domains, including the CXXC domain,
the catalytic dioxygenase domain, and a
200 amino acid region just N-terminal to
the first half of the catalytic domain, suggesting that dTet is similar to mammalian

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.

TET proteins and may function as a 5mC

oxidase. However, current data do not
exclude the possibility that dTet would act
on 5mC in tRNA, produced by Dnmt2,
and would thus be an RNA 5mC oxidase.
So far, there have been no reports confirming the existence of 5hmC in RNA,
or specifically in tRNA, in any organism. However, there is widespread presence of 5mC not only in tRNA, but also
in mRNA and in other non-coding RNA
species.63 Since 5hmC in mammalian
DNA has remained unconfirmed until
2009,42,64 it would not be too surprising if
this base were also present in RNA, and
its detection has so far been hampered
by the lack of highly sensitive technology, including specific mass spectrometry-based approaches. The dioxygenase
domain present in dTet may be capable of
5mC oxidation in RNA. More puzzling
is the simultaneous presence of a CXXC
domain, a protein module generally
thought to interact with unmethylated
CpG dinucleotides in DNA.52 However,
this domain may also be capable of binding to CpG sites in RNA or has adopted

an alternative function, for example by

providing a specific structural feature
to the protein. Experimental tests with
recombinant or overexpressed dTet will
need to be conducted to study its ability
to oxidize 5mC in tRNA. While there is
evidence that 5mC in tRNA may have a
stabilizing function and protects it from
stress-induced cleavage,65 the biological
effect of converting 5mC in tRNA to
5hmC would remain to be determined.
Hypothetically, oxidation of 5mC in this
tRNA could lead to tRNA destabilization.
Finally, dTet may have evolved a function that is largely independent of its
catalytic activity altogether. Mammalian
TET proteins have large uncharacterized regions that could potentially interact with other nuclear proteins and may
facilitate 5mC-independent functions of
TET proteins. However, these regions
are poorly conserved between different
TET proteins, including dTet. What is
known already is that TET proteins can
bind to chromatin modifier complexes,
including Sin3A, NuRD, and O-linked

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.
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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understanding of any possible DNA-based

epigenetic mechanisms present in this
organism. Alternative possibilities, specifically that dTet and its mammalian
counterparts have functional properties as
tRNA 5mC oxidases producing 5hmC in
tRNA, should also strongly be considered.
Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were


Work of the authors was supported by

NIH grant CA160965 to GPP.
Supplemental Materials

Supplemental materials may be found










Suzuki MM, Bird A. DNA methylation landscapes:

provocative insights from epigenomics. Nat Rev
Genet 2008; 9:465-76; PMID:18463664; http://
Pfeifer GP. Mutagenesis at methylated CpG
sequences. Curr Top Microbiol Immunol 2006;
Goll MG, Bestor TH. Eukaryotic cytosine methyltransferases. Annu Rev Biochem 2005; 74:481514; PMID:15952895;
Jones PA, Liang G. Rethinking how DNA methylation patterns are maintained. Nat Rev Genet
2009; 10:805-11; PMID:19789556; http://dx.doi.
Chedin F, Lieber MR, Hsieh CL. The DNA methyltransferase-like protein DNMT3L stimulates de novo
methylation by Dnmt3a. Proc Natl Acad Sci U S A
2002; 99:16916-21; PMID:12481029; http://dx.doi.
Gowher H, Liebert K, Hermann A, Xu G, Jeltsch
A. Mechanism of stimulation of catalytic activity
of Dnmt3A and Dnmt3B DNA-(cytosine-C5)methyltransferases by Dnmt3L. J Biol Chem 2005;
Xie ZH, Huang YN, Chen ZX, Riggs AD, Ding
JP, Gowher H, Jeltsch A, Sasaki H, Hata K,
Xu GL. Mutations in DNA methyltransferase
DNMT3B in ICF syndrome affect its regulation
by DNMT3L. Hum Mol Genet 2006; 15:1375-85;
Okano M, Xie S, Li E. Dnmt2 is not required for
de novo and maintenance methylation of viral
DNA in embryonic stem cells. Nucleic Acids Res
1998; 26:2536-40; PMID:9592134; http://dx.doi.
Yoder JA, Bestor TH. A candidate mammalian DNA
methyltransferase related to pmt1p of fission yeast.
Hum Mol Genet 1998; 7:279-84; PMID:9425235;


10. Okano M, Bell DW, Haber DA, Li E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de
novo methylation and mammalian development. Cell
1999; 99:247-57; PMID:10555141; http://dx.doi.
11. Li E, Bestor TH, Jaenisch R. Targeted mutation of
the DNA methyltransferase gene results in embryonic
lethality. Cell 1992; 69:915-26; PMID:1606615;
12. Goll MG, Kirpekar F, Maggert KA, Yoder JA, Hsieh
CL, Zhang X, Golic KG, Jacobsen SE, Bestor TH.
Methylation of tRNAAsp by the DNA methyltransferase homolog Dnmt2. Science 2006; 311:3958; PMID:16424344;
13. Klose RJ, Bird AP. Genomic DNA methylation:
the mark and its mediators. Trends Biochem Sci
2006; 31:89-97; PMID:16403636; http://dx.doi.
14. Urieli-Shoval S, Gruenbaum Y, Sedat J, Razin
A. The absence of detectable methylated bases in
Drosophila melanogaster DNA. FEBS Lett 1982;
15. Achwal CW, Ganguly P, Chandra HS. Estimation
of the amount of 5-methylcytosine in Drosophila
melanogaster DNA by amplified ELISA and photoacoustic spectroscopy. EMBO J 1984; 3:263-6;
16. Tweedie S, Ng HH, Barlow AL, Turner BM,
Hendrich B, Bird A. Vestiges of a DNA methylation system in Drosophila melanogaster? Nat Genet
1999; 23:389-90; PMID:10581020; http://dx.doi.
17. Roder K, Hung MS, Lee TL, Lin TY, Xiao H, Isobe
KI, Juang JL, Shen CJ. Transcriptional repression by
Drosophila methyl-CpG-binding proteins. Mol Cell
Biol 2000; 20:7401-9; PMID:10982856; http://
18. Lyko F, Ramsahoye BH, Jaenisch R. DNA methylation in Drosophila melanogaster. Nature 2000;
19. Kunert N, Marhold J, Stanke J, Stach D, Lyko FA.
A Dnmt2-like protein mediates DNA methylation in Drosophila. Development 2003; 130:508390; PMID:12944428;
20. Phalke S, Nickel O, Walluscheck D, Hortig F, Onorati
MC, Reuter G. Retrotransposon silencing and telomere integrity in somatic cells of Drosophila depends
on the cytosine-5 methyltransferase DNMT2. Nat
Genet 2009; 41:696-702; PMID:19412177; http://
21. Schaefer M, Lyko F. Lack of evidence for DNA
methylation of Invader4 retroelements in Drosophila
and implications for Dnmt2-mediated epigenetic
regulation. Nat Genet 2010; 42:920-1, author reply
921; PMID:20980983;
22. Phalke S, Nickel O, Reuter G. Reply to Lack of
evidence for DNA methylation of Invader4 retroelements in Drosophila and implications for Dnmt2mediated epigenetic regulation. Nat Genet 2010;
23. Raddatz G, Guzzardo PM, Olova N, Fantappi MR,
Rampp M, Schaefer M, Reik W, Hannon GJ, Lyko
F. Dnmt2-dependent methylomes lack defined DNA
methylation patterns. Proc Natl Acad Sci U S A
2013; 110:8627-31; PMID:23641003; http://dx.doi.
24. Bonasio R, Li Q, Lian J, Mutti NS, Jin L, Zhao H,
Zhang P, Wen P, Xiang H, Ding Y, et al. Genomewide and caste-specific DNA methylomes of the ants
Camponotus floridanus and Harpegnathos saltator.
Curr Biol 2012; 22:1755-64; PMID:22885060;

Cell Cycle

25. Elango N, Hunt BG, Goodisman MA, Yi SV. DNA

methylation is widespread and associated with differential gene expression in castes of the honeybee, Apis
mellifera. Proc Natl Acad Sci U S A 2009; 106:1120611; PMID:19556545;
26. Feliciello I, Parazajder J, Akrap I, Ugarkovi D. First
evidence of DNA methylation in insect Tribolium
castaneum: Environmental regulation of DNA
methylation within heterochromatin. Epigenetics
2013; 8:534-41; PMID:23644818; http://dx.doi.
27. Fneich S, Dheilly N, Adema C, Rognon A, Reichelt
M, Bulla J, Grunau C, Cosseau C. 5-methylcytosine and 5-hydroxy-methyl-cytosine in the
genome of Biomphalaria glabrata, a snail intermediate host of Schistosoma mansoni. Parasit Vectors
2013; 6:167; PMID:23742053; http://dx.doi.
28. Lyko F, Foret S, Kucharski R, Wolf S, Falckenhayn
C, Maleszka R. The honey bee epigenomes: differential methylation of brain DNA in queens and workers. PLoS Biol 2010; 8:e1000506; PMID:21072239;
29. Walsh TK, Brisson JA, Robertson HM, Gordon
K, Jaubert-Possamai S, Tagu D, Edwards OR. A
functional DNA methylation system in the pea
aphid, Acyrthosiphon pisum. Insect Mol Biol 2010;
19(Suppl 2):215-28; PMID:20482652; http://
30. Ye YH, Woolfit M, Huttley GA, Rances E, Caragata
EP, Popovici J, et al. Infection with a Virulent
Strain of Disrupts Genome Wide-Patterns of
Cytosine Methylation in the Mosquito. PLoS ONE
2013; 8:e66482; PMID:23840485; http://dx.doi.
31. Zwier MV, Verhulst EC, Zwahlen RD, Beukeboom
LW, van de Zande L. DNA methylation plays a crucial role during early Nasonia development. Insect
Mol Biol 2012; 21:129-38; PMID:22122805; http://
32. Simola DF, Wissler L, Donahue G, Waterhouse RM,
Helmkampf M, Roux J, Nygaard S, Glastad KM,
Hagen DE, Viljakainen L, et al. Social insect genomes
exhibit dramatic evolution in gene composition
and regulation while preserving regulatory features
linked to sociality. Genome Res 2013; 23:123547; PMID:23636946;
33. Lenhard B, Sandelin A, Carninci P. Metazoan promoters: emerging characteristics and insights into
transcriptional regulation. Nat Rev Genet 2012;
13:233-45; PMID:22392219
34. Hoskins RA, Landolin JM, Brown JB, Sandler JE,
Takahashi H, Lassmann T, Yu C, Booth BW, Zhang
D, Wan KH, et al. Genome-wide analysis of promoter
architecture in Drosophila melanogaster. Genome Res
2011; 21:182-92; PMID:21177961; http://dx.doi.
35. Jeltsch A, Nellen W, Lyko F. Two substrates are
better than one: dual specificities for Dnmt2 methyltransferases. Trends Biochem Sci 2006; 31:3068; PMID:16679017;
36. Schaefer M, Steringer JP, Lyko F. The Drosophila
cytosine-5 methyltransferase Dnmt2 is associated
with the nuclear matrix and can access DNA during
mitosis. PLoS One 2008; 3:e1414; PMID:18183295;
37. Schaefer M, Lyko F. Solving the Dnmt2 enigma.
Chromosoma 2010; 119:35-40; PMID:19730874;
38. Jurkowski TP, Jeltsch A. On the evolutionary origin
of eukaryotic DNA methyltransferases and Dnmt2.
PLoS One 2011; 6:e28104; PMID:22140515; http://

Volume 12 Issue 21

39. Jurkowski TP, Meusburger M, Phalke S, Helm M,

Nellen W, Reuter G, Jeltsch A. Human DNMT2
methylates tRNA(Asp) molecules using a DNA
methyltransferase-like catalytic mechanism. RNA
2008; 14:1663-70; PMID:18567810; http://dx.doi.
40. Ito S, DAlessio AC, Taranova OV, Hong K, Sowers
LC, Zhang Y. Role of Tet proteins in 5mC to
5hmC conversion, ES-cell self-renewal and inner
cell mass specification. Nature 2010; 466:112933; PMID:20639862;
41. Ito S, Shen L, Dai Q, Wu SC, Collins LB, Swenberg
JA, He C, Zhang Y. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 2011; 333:1300-3; PMID:21778364;
42. Tahiliani M, Koh KP, Shen Y, Pastor WA,
Bandukwala H, Brudno Y, Agarwal S, Iyer LM, Liu
DR, Aravind L, et al. Conversion of 5-methylcytosine
to 5-hydroxymethylcytosine in mammalian DNA
by MLL partner TET1. Science 2009; 324:9305; PMID:19372391;
43. He YF, Li BZ, Li Z, Liu P, Wang Y, Tang Q, Ding
J, Jia Y, Chen Z, Li L, et al. Tet-mediated formation of 5-carboxylcytosine and its excision by TDG
in mammalian DNA. Science 2011; 333:13037; PMID:21817016;
44. Pastor WA, Aravind L, Rao A. TETonic shift: biological roles of TET proteins in DNA demethylation and
transcription. Nat Rev Mol Cell Biol 2013; 14:34156; PMID:23698584;
45. Pfeifer GP, Kadam S, Jin SG. 5-hydroxymethylcytosine and its potential roles in development and cancer.
Epigenetics Chromatin 2013; 6:10; PMID:23634848;
46. Shen L, Zhang Y. 5-Hydroxymethylcytosine: generation, fate, and genomic distribution. Curr Opin
Cell Biol 2013; 25:289-96; PMID:23498661; http://
47. Hackett JA, Sengupta R, Zylicz JJ, Murakami K,
Lee C, Down TA, Surani MA. Germline DNA
demethylation dynamics and imprint erasure through
5-hydroxymethylcytosine. Science 2013; 339:44852; PMID:23223451;
48. Iqbal K, Jin SG, Pfeifer GP, Szab PE.
Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine. Proc Natl Acad Sci U S A 2011; 108:3642-7;
49. Wossidlo M, Nakamura T, Lepikhov K, Marques CJ,
Zakhartchenko V, Boiani M, Arand J, Nakano T, Reik
W, Walter J. 5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming. Nat Commun 2011; 2:241; PMID:21407207;
50. Iyer LM, Tahiliani M, Rao A, Aravind L. Prediction
of novel families of enzymes involved in oxidative and
other complex modifications of bases in nucleic acids.
Cell Cycle 2009; 8:1698-710; PMID:19411852;
51. Ko M, An J, Bandukwala HS, Chavez L, Aij T,
Pastor WA, Segal MF, Li H, Koh KP, Lhdesmki H,
et al. Modulation of TET2 expression and 5-methylcytosine oxidation by the CXXC domain protein
IDAX. Nature 2013; 497:122-6; PMID:23563267;
52. Long HK, Blackledge NP, Klose RJ. ZF-CxxC
domain-containing proteins, CpG islands and
the chromatin connection. Biochem Soc Trans
2013; 41:727-40; PMID:23697932; http://dx.doi.

53. Xu C, Bian C, Lam R, Dong A, Min J. The structural

basis for selective binding of non-methylated CpG
islands by the CFP1 CXXC domain. Nat Commun
2011; 2:227; PMID:21407193; http://dx.doi.
54. Xu Y, Xu C, Kato A, Tempel W, Abreu JG, Bian C,
Hu Y, Hu D, Zhao B, Cerovina T, et al. Tet3 CXXC
domain and dioxygenase activity cooperatively regulate key genes for Xenopus eye and neural development. Cell 2012; 151:1200-13; PMID:23217707;
55. Cierpicki T, Risner LE, Grembecka J, Lukasik
SM, Popovic R, Omonkowska M, Shultis DD,
Zeleznik-Le NJ, Bushweller JH. Structure of the
MLL CXXC domain-DNA complex and its functional role in MLL-AF9 leukemia. Nat Struct Mol
Biol 2010; 17:62-8; PMID:20010842; http://dx.doi.
56. Jin SG, Kadam S, Pfeifer GP. Examination of the
specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine. Nucleic Acids Res 2010; 38:e125;
57. Huang Y, Pastor WA, Shen Y, Tahiliani M, Liu DR,
Rao A. The behaviour of 5-hydroxymethylcytosine
in bisulfite sequencing. PLoS One 2010; 5:e8888;
58. Brody T, Stivers C, Nagle J, Odenwald WF.
Identification of novel Drosophila neural precursor
genes using a differential embryonic head cDNA
screen. Mech Dev 2002; 113:41-59; PMID:11900973;
59. Graveley BR, Brooks AN, Carlson JW, Duff MO,
Landolin JM, Yang L, Artieri CG, van Baren MJ,
Boley N, Booth BW, et al. The developmental
transcriptome of Drosophila melanogaster. Nature
2011; 471:473-9; PMID:21179090; http://dx.doi.
60. Hahn MA, Qiu R, Wu X, Li AX, Zhang H, Wang
J, Jui J, Jin SG, Jiang Y, Pfeifer GP, et al. Dynamics
of 5-hydroxymethylcytosine and chromatin marks
in Mammalian neurogenesis. Cell Rep 2013; 3:291300; PMID:23403289;
61. Mnzel M, Globisch D, Brckl T, Wagner M,
Welzmiller V, Michalakis S, Mller M, Biel M, Carell
T. Quantification of the sixth DNA base hydroxymethylcytosine in the brain. Angew Chem Int Ed
Engl 2010; 49:5375-7; PMID:20583021; http://
62. Yadlapalli S, Yamashita YM. Chromosome-specific
nonrandom sister chromatid segregation during stem-cell division. Nature 2013; 498:251-4;
63. Squires JE, Patel HR, Nousch M, Sibbritt T,
Humphreys DT, Parker BJ, Suter CM, Preiss T.
Widespread occurrence of 5-methylcytosine in
human coding and non-coding RNA. Nucleic Acids
Res 2012; 40:5023-33; PMID:22344696; http://
64. Kriaucionis S, Heintz N. The nuclear DNA base
5-hydroxymethylcytosine is present in Purkinje
neurons and the brain. Science 2009; 324:92930; PMID:19372393;
65. Schaefer M, Pollex T, Hanna K, Tuorto F, Meusburger
M, Helm M, Lyko F. RNA methylation by Dnmt2
protects transfer RNAs against stress-induced cleavage. Genes Dev 2010; 24:1590-5; PMID:20679393;
66. Shi FT, Kim H, Lu W, He Q, Liu D, Goodell MA,
Wan M, Songyang Z. Ten-eleven translocation 1
(Tet1) is regulated by O-linked N-acetylglucosamine
transferase (Ogt) for target gene repression in mouse
embryonic stem cells. J Biol Chem 2013; 288:2077684; PMID:23729667;

67. Williams K, Christensen J, Pedersen MT, Johansen

JV, Cloos PA, Rappsilber J, Helin K. TET1 and
hydroxymethylcytosine in transcription and DNA
methylation fidelity. Nature 2011; 473:343-8;
68. Chen Q, Chen Y, Bian C, Fujiki R, Yu X. TET2 promotes histone O-GlcNAcylation during gene transcription. Nature 2013; 493:561-4; PMID:23222540;
69. Vella P, Scelfo A, Jammula S, Chiacchiera F, Williams
K, Cuomo A, Roberto A, Christensen J, Bonaldi T,
Helin K, et al. Tet proteins connect the O-linked
N-acetylglucosamine transferase Ogt to chromatin
in embryonic stem cells. Mol Cell 2013; 49:64556; PMID:23352454;
70. Deplus R, Delatte B, Schwinn MK, Defrance M,
Mndez J, Murphy N, Dawson MA, Volkmar M,
Putmans P, Calonne E, et al. TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through
OGT and SET1/COMPASS. EMBO J 2013; 32:64555; PMID:23353889;
71. Yu M, Hon GC, Szulwach KE, Song CX, Jin P,
Ren B, He C. Tet-assisted bisulfite sequencing of
5-hydroxymethylcytosine. Nat Protoc 2012; 7:215970; PMID:23196972;
72. Sun Z, Terragni J, Borgaro JG, Liu Y, Yu L, Guan
S, Wang H, Sun D, Cheng X, Zhu Z, et al. Highresolution enzymatic mapping of genomic 5-hydroxymethylcytosine in mouse embryonic stem cells. Cell
Rep 2013; 3:567-76; PMID:23352666; http://
73. McGuffin LJ, Buenavista MT, Roche DB. The
ModFOLD4 server for the quality assessment of 3D
protein models. Nucleic Acids Res 2013; 41(Web
Server issue):W368-72; PMID:23620298; http://
74. Roche DB, Buenavista MT, Tetchner SJ, McGuffin
LJ. The IntFOLD server: an integrated web resource
for protein fold recognition, 3D model quality assessment, intrinsic disorder prediction, domain prediction and ligand binding site prediction. Nucleic
Acids Res 2011; 39(Web Server issue):W171-6;
75. Yang Y, Faraggi E, Zhao H, Zhou Y. Improving protein fold recognition and template-based modeling by
employing probabilistic-based matching between predicted one-dimensional structural properties of query
and corresponding native properties of templates.
Bioinformatics 2011; 27:2076-82; PMID:21666270;
76. Zhang Y. I-TASSER server for protein 3D
2008; 9:40; PMID:18215316; http://dx.doi.
77. Zhang Y, Skolnick J. TM-align: a protein structure
alignment algorithm based on the TM-score. Nucleic
Acids Res 2005; 33:2302-9; PMID:15849316;
78. Xu J, Zhang Y. How significant is a protein structure similarity with TM-score = 0.5? Bioinformatics
2010; 26:889-95; PMID:20164152; http://dx.doi.
79. Graveley BR, May G, Brooks AN, Carlson JW,
Cherbas L, Davis CA, et al. he D. melanogaster
transcriptome: modENCODE RNA-Seq data for
dissected tissues. FlyBase, personal communication

Cell Cycle 3365