1 Research Group Epigenetics, Deutsches Krebsforschungszentrum, Im Neuenheimer
Feld 580, 69120 Heidelberg, Germany
2 Division of Molecular Toxicology, Deutsches Krebsforschungszentrum, Im
Neuenheimer Feld 280, 69120 Heidelberg, Germany
* Author for correspondence (e-mail: f.lyko{at}dkfz.de)
Accepted 10 July 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: DNA methylation, Drosophila, DNA methyltransferase, Dnmt2, Su(var)3-9
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Based on sequence homology, animal DNA methyltransferases can be subdivided
into three families: Dnmt1, Dnmt2 and Dnmt3
(Colot and Rossignol, 1999).
Because of their substrate preference for hemimethylated CpG dinucleotides
(Bestor and Ingram, 1983
;
Gruenbaum et al., 1982
), Dnmt1
enzymes are generally regarded as maintenance methyltransferases. Accordingly,
it has been suggested that the primary function of these enzymes might be the
copying of cytosine methylation patterns from the parental DNA strand to the
newly synthesized strand during or shortly after replication. The function of
the second family of DNA methyltransferases, Dnmt2, has been enigmatic for a
long time. Direct evidence for a catalytic activity could not be provided yet
(Okano et al., 1998a
) and it
has been suggested that Dnmt2 proteins might not function as DNA
methyltransferases (Dong et al.,
2001
). However, more recent data indicated a weaker DNA
methyltransferase activity of Dnmt2 in mouse and human cells
(Liu et al., 2003
). The role
of the third family of animal DNA methyltransferases is defined by their
distinct preference for unmethylated DNA
(Okano et al., 1998b
). Dnmt3a
and Dnmt3b function as de novo DNA methyltransferases
(Hsieh, 1999
;
Lyko et al., 1999
;
Okano et al., 1999
) and are
considered to be important for the establishment of DNA methylation patterns
during embryogenesis.
The question of DNA methylation in Drosophila has been discussed
controversially for a long period of time. There are several reports that
demonstrate the absence of 5-methylcytosine from pupal and adult stages of fly
development (Patel and Gopinathan,
1987; Tweedie et al.,
1999
). In addition, it has also been shown that DNA from
Drosophila embryos is largely unmethylated at CpG dinucleotides
(Urieli-Shoval et al., 1982
).
This is an apparent contradiction to two more recent reports that provided
evidence for low levels of DNA methylation both in embryos and in adults
(Gowher et al., 2000
;
Lyko et al., 2000b
). The
contradiction could be partially resolved by analysing the sequence context of
5-methylcytosine in the fly genome (Lyko
et al., 2000b
). This confirmed the virtual absence of CpG
methylation in Drosophila, but also provided strong evidence for low
amounts of non-CpG methylation in embryos
(Lyko et al., 2000b
).
The enzyme that mediates DNA methylation in the fly has remained unknown.
The Drosophila genome contains a single candidate DNA
methyltransferase gene (Hung et al.,
1999; Lyko, 2001
;
Tweedie et al., 1999
). This
gene (Mt2 - FlyBase) belongs to the widely conserved Dnmt2 family of putative
DNA methyltransferases with known homologues in humans, mice, insects and
fungi (Dong et al., 2001
;
Okano et al., 1998a
;
Wilkinson et al., 1995
;
Yoder and Bestor, 1998
).
Drosophila Dnmt2 (like other Dnmt2 proteins) contains all the
catalytic signature motifs of active DNA methyltransferases. In addition, the
structure of human DNMT2 is highly similar to active bacterial DNA
methyltransferases (Dong et al.,
2001
). However, all attempts to demonstrate a catalytic activity
of Dnmt2-like proteins have failed so far, and targeting of the mouse
Dnmt2 locus showed no detectable effects on DNA methylation
(Okano et al., 1998a
).
Similarly, Drosophila Dnmt2 failed to reveal any activity in standard
in vitro methylation assays (Tweedie et
al., 1999
). Thus, the function of Dnmt2 proteins has remained
enigmatic (Bestor, 2000
).
In order to characterize Drosophila DNA methylation in higher detail we established a protocol for the immunodetection of 5-methylcytosine in embryos. Confocal analysis of stained embryos confirmed the presence of 5-methylcytosine in embryonic genomes. To analyse the function of Dnmt2, we depleted the protein by RNA interference. This demonstrated that Dnmt2 is essential for DNA methylation in Drosophila embryos. Additional experiments showed that overexpression of Dnmt2 resulted in significant genomic hypermethylation at CpT and CpA dinucleotides. Our results thus demonstrate that DNA methylation in Drosophila is mediated by Dnmt2.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Dnmt2 protein analysis
The Dnmt2-specific antiserum was raised by standard immunization of rabbits
with a mixture of three KLH-coupled peptides (YAHNYGSNLVKTRNC,
CQPHTRQGLQRDTEDK and CDTSNQDASKSEKILQ; Peptide Specialty Laboratories,
Heidelberg, Germany). For developmental western blots, protein extracts were
prepared from various developmental stages of wild-type flies. Equal amounts
of protein were separated on a 10% SDS-polyacrylamide gel and blotted using
standard procedures. A specific antibody against ribosomal protein P40 was
used for loading controls (Torok et al.,
1999). For the confocal analysis of subcellular Dnmt2
distribution, wild-type embryos were stained with our Dnmt2-specific antiserum
(1:200).
Depletion of Dnmt2 by RNAi
Double-stranded Dnmt2 RNA was prepared by in vitro transcription of a Dnmt2
cDNA clone (Lyko et al.,
2000a) using Megascript kits from Ambion. For controls, we
synthesized double-stranded RNA from an EST clone (CK00414)
(Kopczynski et al., 1998
) of
the CG11840 gene. Annealing of complementary RNA strands was verified by
agarose gel electrophoresis. For microinjection, wild-type embryos were
collected over 30 minutes at 18°C and directly injected under hydrocarbon
oil. Development was closely monitored through the adult stage. For DNA
methylation analysis, series of about 200 embryos were injected at 18°C
and subsequently aged for 3 hours at 25°C and then dechorionated, fixed,
permeabilized and stained as described above. All experiments were repeated
several times and the results were found to be strictly reproducible.
Overexpression of Dnmt2
We generated the transgenic construct for GAL4-inducible Dnmt2
overexpression by subcloning a Dnmt2 cDNA into the pUAST vector
(Brand and Perrimon, 1993).
Several independent UAS-Dnmt2 strains were generated by
P-element mediated transformation using standard procedures and
w1118 as host. For Dnmt2 overexpression,
UAS-Dnmt2 females were crossed to hs-GAL4
(Brand et al., 1994
) males, and
offspring were heat shocked daily for 1 hour at 37°C in a water bath. For
controls, we used w1118 instead of UAS-Dnmt2
females. Protein overexpression was confirmed by western blotting using our
Dnmt2 antiserum. For DNA methylation analysis, genomic DNA was isolated from
10-day-old adult flies. DNA samples were hydrolysed, derivatized and analyzed
by capillary electrophoresis as described previously
(Stach et al., 2003
). Samples
from independent DNA preparations were measured at least three times, and the
results were found to be strictly reproducible. To minimize the possibility of
bacterial contaminations, fly food was supplemented with tetracycline (0.25
mg/ml) (Holden et al., 1993
)
in all experiments.
Bisulfite genomic shotgun sequencing
Genomic DNA from adult flies overexpressing Dnmt2 and from control flies
(see above) was subjected to bisulfite sequencing as described previously
(Ramsahoye et al., 2000).
Sequences were aligned to the Drosophila genome sequence and only
sequences with an extensive alignment were used for analysis. To reduce the
number of potential experimental artefacts further, only 5-methylcytosine
residues with a matching cytosine residue in the Drosophila genome
sequence were considered methylated. The presence of a methylated cytosine
residue was always confirmed by sequencing from both strands.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The characterization of Drosophila DNA methylation patterns by
biochemical or molecular methods has been precluded by the low overall
methylation level and the small amount of DNA that can be extracted from early
embryonic stages. To confirm the methylation of Drosophila genomic
DNA by an independent method and to address the distribution of methylated DNA
in the fly genome, we established a protocol for the immunological detection
of 5-methylcytosine. Similar protocols have recently proved very useful for
the analysis of DNA methylation during early vertebrate development
(Dean et al., 2001;
Mayer et al., 2000
). Overnight
collections of wild-type embryos were obtained from population cages; they
were washed, fixed, treated with RNase and then extensively denatured in 2 M
HCl. This procedure leaves only DNA intact for immunostaining and eliminates
other potentially crossreacting epitopes. Subsequently, embryo preparations
were double stained with antibodies that specifically recognize
5-methylcytosine and DNA, respectively. The use of the DNA-specific antibody
also provided us with a valuable internal control for our staining procedure.
Analysis of the developmental 5-methylcytosine pattern by confocal microscopy
revealed a weak signal in early cleavage embryos
(Fig. 1A). During syncytial
blastoderm, the distribution of 5-methylcytosine became more inhomogeneous
(data not shown). The intensity of the 5-methylcytosine signal appeared
strongest in cellular blastoderm embryos
(Fig. 1B). Later stages of
embryonic development still showed detectable DNA methylation, but the
intensity of the signal appeared to be reduced
(Fig. 1C). Interestingly, pole
cells also revealed clear 5-methylcytosine staining
(Fig. 1D), which indicated the
presence of DNA methylation in the germline. Our results thus confirm previous
observations that DNA methylation is most prevalent during earlier stages of
Drosophila embryogenesis (Lyko et
al., 2000b
). In addition, they suggest a fractional distribution
of 5-methylcytosine during the blastoderm stages.
|
The primary candidate for a Drosophila DNA methyltransferase is
the Dnmt2 protein. As an initial step towards the characterization of Dnmt2,
we raised an antibody against the protein. We obtained a rabbit polyclonal
antiserum that specifically recognizes a single band at 45 kDa in western
blots (Fig. 2A). This band was
not observed with preimmune serum (data not shown) and the signal was strongly
reduced upon depletion of Dnmt2 by RNA interference (see below). In order to
analyse the developmental expression pattern of Dnmt2, protein extracts were
prepared from all stages of Drosophila development and analyzed by
western blotting. This demonstrated low but detectable levels of Dnmt2 in
embryos (Fig. 2A). By contrast,
larvae, pupae or adults contained only background levels of protein
(Fig. 2A). The expression of
Dnmt2 is therefore developmentally regulated. Our results are consistent with
previous findings that described the presence of Dnmt2 mRNA in ovaries and in
early embryos (Lyko et al.,
2000a
). Most of the mRNA from ovaries is probably not translated
but rather deposited in embryos as a maternal component. The specificity of
the antibody was further confirmed by analysis of protein extracts derived
from adult flies overexpressing Dnmt2. Western blots revealed a strong ectopic
band that co-migrated with the weaker, embryo-specific bands at
45 kDa
(Fig. 2A, right lane).
|
Because fly strains with a mutation in the Dnmt2 gene are not
available, we used RNAi to analyse the function of Dnmt2. After injection of
Dnmt2 dsRNA at the syncytial blastoderm stage, embryos were aged for 3 hours.
This procedure efficiently removed most of the Dnmt2 protein from embryos
(Fig. 2C). Double staining of
dsRNA-injected embryos with antibodies against 5-methylcytosine and DNA
revealed a strong reduction of 5-methylcytosine signals
(Fig. 3A). The effect was
specific for the 5-methylcytosine signal and the staining pattern for DNA was
not affected (Fig. 3A). For
controls, we injected embryos with dsRNA from an independent gene. CG11840
encodes a Drosophila signal peptide peptidase homologue
(Weihofen et al., 2002) and
has no conceivable function in epigenetic regulation. Embryos injected with
CG11840 dsRNA showed the characteristic nuclear 5-methylcytosine staining
pattern also observed in wild-type embryos
(Fig. 3B). From these results,
we concluded that Dnmt2 is required for embryonic DNA methylation.
Interestingly, RNA interference of Dnmt2 appeared not to have a detectable
effect on embryonic viability (Fig.
3C). Similarly, injection of a 5 µM solution of 5-azacytidine
caused pronounced demethylation but showed only minimal effects on the
survival rates of embryos (data not shown). By contrast, control injection
with CG11840 dsRNA caused detectable embryonic lethality
(Fig. 3C).
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
(1) Our experiments revealed a strong dependency on the Su(var)3-9 histone
H3-K9 methyltransferase. A similar dependency has been recently described to
involve the Su(var)3-9 homologues dim-5 in Neurospora
(Tamaru and Selker, 2001) and
KRYPTONITE in Arabidopsis
(Jackson et al., 2002
) and has
been interpreted to reflect a role of DNA methylation in heterochromatin
stability. Our observations are also consistent with our own previous results
that demonstrated a strong effect of Su(var)3-9 mutations on
ectopically induced hypermethylation in the fly
(Weissmann et al., 2003
).
(2) DNA methyltransferase activity in Drosophila embryos could be
effectively inhibited by 5-azacytidine. This compound functions as a suicide
substrate for cytosine-5 DNA methyltransferases by covalently trapping the
enzymes (Santi et al., 1984).
It has been shown recently that 5-azacytidine also forms covalent bonds with
the human and mouse Dnmt2 homologues (Liu
et al., 2003
). This result had provided the first indication for
an active DNA methyltransferase function of Dnmt2 proteins.
The primary candidate DNA methyltransferase gene for Drosophila
has been Dnmt2. Based on antibody stainings, the presence of a second
Dnmt1-like methyltransferase protein has also been suggested
(Hung et al., 1999). However,
there is no corresponding open reading frame in the Drosophila genome
sequence (Adams et al., 2000
).
The complete loss of DNA methylation in embryos injected with double-stranded
Dnmt2 RNA also argues against the presence of a second DNA methyltransferase.
While these results provided unambiguous evidence for a DNA methyltransferase
activity of Dnmt2, they did not reveal a function of DNA methylation in the
fly. Depletion of Dnmt2 by RNA interference appeared not to have any
detectable consequences for embryonic development. We also monitored
development during later stages and observed only minor phenotypic aberrations
that could not be unambiguously linked to the demethylation of the genome
(data not shown). The apparent lack of phenotypic effects is consistent with
the absence of major phenotypes in null mutant Su(var)3-9 fly strains
(Tschiersch et al., 1994
) that
we have shown to lack most, if not all, DNA methylation. In this context it is
noteworthy that experimental demethylation of the Neurospora genome
also has no major phenotypic consequences
(Kouzminova and Selker,
2001
).
Dnmt2-like genes are widely conserved during evolution, but their function
has been elusive for a considerable period of time
(Dong et al., 2001). No
catalytic activity could be demonstrated by standard in vitro methylation
assays and by gene targeting of the mouse Dnmt2 gene
(Okano et al., 1998a
;
Tweedie et al., 1999
;
Wilkinson et al., 1995
;
Yoder and Bestor, 1998
). Our
results revealed a comparatively low activity of Dnmt2 that was specific for
CpT and CpA dinucleotides. Both characteristics could have contributed to the
apparent lack of DNA methyltransferase activity in previous assays. The
specificity for CpT and CpA distinguishes Dnmt2 from all other known animal
DNA methyltransferases and confirms our previous suggestion of predominant
non-CpG methylation in Drosophila
(Lyko et al., 2000b
). CpT/A
methylation has also been reported in various mammalian test systems
(Clark et al., 1995
;
Lorincz et al., 2000
;
Toth et al., 1990
;
Woodcock et al., 1997
). Most
notably, the B29 gene promoter in human B cells has been found to be
methylated in the context of CCTGG or CCAGG
(Malone et al., 2001
), and it
has been hypothesized that Dnmt2 might be the responsible enzyme
(Lorincz and Groudine, 2001
).
Our data did not reveal a consensus sequence that extended beyond the CpT/A
dinucleotide. However, mammalian CpT/A methylation is not restricted to CCTGG
or CCAGG pentanucleotides (Ramsahoye et
al., 2000
). Thus, it will be interesting to analyse the
methylation of CpT/A sites in other animal systems for their dependency on
Dnmt2-like proteins.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams, M. D., Celniker, S. E., Holt, R. A., Evans, C.A.,
Gocayne, J. D., Amanatides, P. G., Scherer, S. E., Li, P. W., Hoskins, R. A.,
Galle, R. F. et al. (2000). The genome sequence of
Drosophila melanogaster. Science
287,2185
-2195.
Bestor, T. H. (2000). The DNA
methyltransferases of mammals. Hum. Mol. Genet.
9,2395
-2402.
Bestor, T. H. and Ingram, V. M. (1983). Two DNA methyltransferases from murine erythroleukemia cells: purification, sequence specificity, and mode of interaction with DNA. Proc. Natl. Acad. Sci. USA 80,5559 -5563.[Abstract]
Bird, A. (2002). DNA methylation patterns and
epigenetic memory. Genes Dev.
16, 6-21.
Brand, A. H. and Perrimon, N. (1993). Targeted
gene expression as a means of altering cell fates and generating dominant
phenotypes. Development
118,401
-415.
Brand, A. H., Manoukian, A. S. and Perrimon, N. (1994). Ectopic expression in Drosophila. Methods Cell Biol. 44,635 -654.[Medline]
Clark, S. J., Harrison, J. and Frommer, M. (1995). CpNpG methylation in mammalian cells. Nat. Genet. 10,20 -27.[Medline]
Colot, V. and Rossignol, J. L. (1999). Eukaryotic DNA methylation as an evolutionary device. BioEssays 21,402 -411.[CrossRef][Medline]
Dean, W., Santos, F., Stojkovic, M., Zakhartchenko, V., Walter,
J., Wolf, E. and Reik, W. (2001). Conservation of methylation
reprogramming in mammalian development: aberrant reprogramming in cloned
embryos. Proc. Natl. Acad. Sci. USA
98,13734
-13738.
Dong, A., Yoder, J. A., Zhang, X., Zhou, L., Bestor, T. H. and
Cheng, X. (2001). Structure of human DNMT2, an enigmatic DNA
methyltransferase homolog that displays denaturant-resistant binding to DNA.
Nucleic Acids Res. 29,439
-448.
Gowher, H., Leismann, O. and Jeltsch, A.
(2000). DNA of Drosophila melanogaster contains
5-methylcytosine. EMBO J.
19,6918
-6923.
Gruenbaum, Y., Cedar, H. and Razin, A. (1982). Substrate and sequence specificity of a eukaryotic DNA methylase. Nature 295,620 -622.[Medline]
Holden, P. R., Jones, P. and Brookfield, J. F. (1993). Evidence for a Wolbachia symbiont in Drosophila melanogaster. Genet. Res. 62, 23-29.[Medline]
Hsieh, C. L. (1999). In vivo activity of murine
de novo methyltransferases, Dnmt3a and Dnmt3b. Mol. Cell.
Biol. 19,8211
-8218.
Hung, M. S., Karthikeyan, N., Huang, B., Koo, H. C., Kiger, J.
and Shen, C. J. (1999). Drosophila proteins related
to vertebrate DNA (5-cytosine) methyltransferases. Proc. Natl.
Acad. Sci. USA 96,11940
-11945.
Jackson, J. P., Lindroth, A. M., Cao, X. and Jacobsen, S. E. (2002). Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase. Nature 416,556 -560.[CrossRef][Medline]
Jackson-Grusby, L., Beard, C., Possemato, R., Tudor, M., Fambrough, D., Csankovszki, G., Dausman, J., Lee, P., Wilson, C., Lander, E. et al. (2001). Loss of genomic methylation causes p53-dependent apoptosis and epigenetic deregulation. Nat. Genet. 27,31 -39.[CrossRef][Medline]
Jones, P. A. and Baylin, S. B. (2002). The fundamental role of epigenetic events in cancer. Nat. Rev. Genet. 3,415 -428.[Medline]
Kennerdell, J. R. and Carthew, R. W. (1998). Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway. Cell 95,1017 -1026.[Medline]
Kopczynski, C. C., Noordermeer, J. N., Serano, T. L., Chen, W.
Y., Pendleton, J. D., Lewis, S., Goodman, C. S. and Rubin, G. M.
(1998). A high throughput screen to identify secreted and
transmembrane proteins involved in Drosophila embryogenesis.
Proc. Natl. Acad. Sci. USA
95,9973
-9978.
Kouzminova, E. and Selker, E. U. (2001). dim-2
encodes a DNA methyltransferase responsible for all known cytosine methylation
in Neurospora. EMBO J.
20,4309
-4323.
Li, E., Bestor, T. H. and Jaenisch, R. (1992). Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69,915 -926.[Medline]
Li, E. (2002). Chromatin modification and epigenetic reprogramming in mammalian development. Nat. Rev. Genet. 3,662 -673.[CrossRef][Medline]
Liu, K., Wang, Y. F., Cantemir, C. and Muller, M. T.
(2003). Endogenous assays of DNA methyltransferases: Evidence for
differential activities of DNMT1, DNMT2, and DNMT3 in mammalian cells In vivo.
Mol. Cell. Biol. 23,2709
-2719.
Lorincz, M. C., Schubeler, D., Goeke, S. C., Walters, M.,
Groudine, M. and Martin, D. I. (2000). Dynamic analysis of
proviral induction and de novo methylation: implications for a histone
deacetylase-independent, methylation density-dependent mechanism of
transcriptional repression. Mol. Cell. Biol.
20,842
-850.
Lorincz, M. C. and Groudine, M. (2001).
C(m)C(a/t)GG methylation: a new epigenetic mark in mammalian DNA?
Proc. Natl. Acad. Sci. USA
98,10034
-10036.
Lyko, F. (2001). DNA methylation learns to fly. Trends Genet. 17,169 -172.[CrossRef][Medline]
Lyko, F., Ramsahoye, B. H., Kashevsky, H., Tudor, M., Mastrangelo, M. A., Orr-Weaver, T. L. and Jaenisch, R. (1999). Mammalian (cytosine-5) methyltransferases cause genomic DNA methylation and lethality in Drosophila. Nat. Genet. 23,363 -366.[CrossRef][Medline]
Lyko, F., Whittaker, A. J., Orr-Weaver, T. L. and Jaenisch, R. (2000a). The putative Drosophila methyltransferase gene dDnmt2 is contained in a transposon-like element and is expressed specifically in ovaries. Mech. Dev. 95,215 -217.[CrossRef][Medline]
Lyko, F., Ramsahoye, B. H. and Jaenisch, R. (2000b). DNA methylation in Drosophila melanogaster. Nature 408,538 -540.[CrossRef][Medline]
Malone, C. S., Miner, M. D., Doerr, J. R., Jackson, J. P.,
Jacobsen, S. E., Wall, R. and Teitell, M. (2001). CmC(A/T)GG
DNA methylation in mature B cell lymphoma gene silencing. Proc.
Natl. Acad. Sci. USA 98,10404
-10409.
Marhold, J., Zbylut, M., Lankenau, D. H., Li, M., Gerlich, D., Ballestar, E., Mechler, B. M. and Lyko, F. (2002). Stage-specific chromosomal association of Drosophila dMBD2/3 during genome activation. Chromosoma 111, 13-21.[CrossRef][Medline]
Mayer, W., Niveleau, A., Walter, J., Fundele, R. and Haaf, T. (2000). Demethylation of the zygotic paternal genome. Nature 403,501 -502.[CrossRef][Medline]
Miura, A., Yonebayashi, S., Watanabe, K., Toyama, T., Shimada, H. and Kakutani, T. (2001). Mobilization of transposons by a mutation abolishing full DNA methylation in Arabidopsis. Nature 411,212 -214.[CrossRef][Medline]
Okano, M., Xie, S. and Li, E. (1998a). Dnmt2 is
not required for de novo and maintenance methylation of viral DNA in
embryonic stem cells. Nucleic Acids Res.
26,2536
-2540.
Okano, M., Xie, S. and Li, E. (1998b). Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases. Nat. Genet. 19,219 -220.[CrossRef][Medline]
Okano, M., Bell, D. W., Haber, D. A. and Li, E. (1999). DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99,247 -257.[Medline]
Patel, C. V. and Gopinathan, K. P. (1987). Determination of trace amounts of 5-methylcytosine in DNA by reverse-phase high-performance liquid chromatography. Anal. Biochem. 164,164 -169.[Medline]
Ramsahoye, B. H., Biniszkiewicz, D., Lyko, F., Clark, V., Bird,
A. P. and Jaenisch, R. (2000). Non-CpG methylation is
prevalent in embryonic stem cells and may be mediated by DNA methyltransferase
3a. Proc. Natl. Acad. Sci. USA
97,5237
-5242.
Robertson, K. D. and Wolffe, A. P. (2000). DNA methylation in health and disease. Nat. Rev. Genet. 1, 11-19.[CrossRef][Medline]
Santi, D. V., Norment, A. and Garrett, C. E. (1984). Covalent bond formation between a DNA-cytosine methyltransferase and DNA containing 5-azacytosine. Proc. Natl. Acad. Sci. USA 81,6993 -6997.[Abstract]
Schotta, G., Ebert, A., Krauss, V., Fischer, A., Hoffmann, J.,
Rea, S., Jenuwein, T., Dorn, R. and Reuter, G. (2002).
Central role of Drosophila SU(VAR)3-9 in histone H3-K9 methylation
and heterochromatic gene silencing. EMBO J.
21,1121
-1131.
Stach, D., Schmitz, O. J., Stilgenbauer, S., Benner, A., Dohner,
H., Wiessler, M. and Lyko, F. (2003). Capillary
electrophoretic analysis of genomic DNA methylation levels. Nucleic
Acids Res. 31,e2
.
Stancheva, I. and Meehan, R. R. (2000).
Transient depletion of xDnmt1 leads to premature gene activation in
Xenopus embryos. Genes Dev.
14,313
-327.
Tamaru, H. and Selker, E. U. (2001). A histone H3 methyltransferase controls DNA methylation in Neurospora crassa. Nature 414,277 -283.[CrossRef][Medline]
Torok, I., Herrmann-Horle, D., Kiss, I., Tick, G., Speer, G.,
Schmitt, R. and Mechler, B. M. (1999). Down-regulation of
RpS21, a putative translation initiation factor interacting with P40, produces
viable minute imagos and larval lethality with overgrown hematopoietic organs
and imaginal discs. Mol. Cell. Biol.
19,2308
-2321.
Toth, M., Muller, U. and Doerfler, W. (1990). Establishment of de novo DNA methylation patterns. Transcription factor binding and deoxycytidine methylation at CpG and non-CpG sequences in an integrated adenovirus promoter. J. Mol. Biol. 214,673 -683.[Medline]
Tschiersch, B., Hofmann, A., Krauss, V., Dorn, R., Korge, G. and Reuter, G. (1994). The protein encoded by the Drosophila position-effect variegation suppressor gene Su(var)3-9 combines domains of antagonistic regulators of homeotic gene complexes. EMBO J. 13,3822 -3831.[Abstract]
Tweedie, S., Charlton, J., Clark, V. and Bird, A. (1997). Methylation of genomes and genes at the invertebrate-vertebrate boundary. Mol. Cell. Biol. 17,1469 -1475.[Abstract]
Tweedie, S., Ng, H. H., Barlow, A. L., Turner, B. M., Hendrich, B. and Bird, A. (1999). Vestiges of a DNA methylation system in Drosophila melanogaster? Nat. Genet. 23,389 -390.[CrossRef][Medline]
Urieli-Shoval, S., Gruenbaum, Y., Sedat, J. and Razin, A. (1982). The absence of detectable methylated bases in Drosophila melanogaster DNA. FEBS Lett. 146,148 -152.[CrossRef][Medline]
Walsh, C. P., Chaillet, J. R. and Bestor, T. H. (1998). Transcription of IAP endogenous retroviruses is constrained by cytosine methylation. Nat. Genet. 20,116 -117.[CrossRef][Medline]
Weihofen, A., Binns, K., Lemberg, M. K., Ashman, K. and
Martoglio, B. (2002). Identification of signal peptide
peptidase, a presenilin-type aspartic protease.
Science 296,2215
-2218.
Weissmann, F., Muyrers-Chen, I., Musch, T., Stach, D., Wiessler,
M., Paro, R. and Lyko, F. (2003). DNA hypermethylation in
Drosophila melanogaster causes irregular chromosome condensation and
dysregulation of epigenetic histone modifications. Mol. Cell.
Biol. 23,2577
-2586.
Wilkinson, C. R., Bartlett, R., Nurse, P. and Bird, A. P. (1995). The fission yeast gene pmt1+ encodes a DNA methyltransferase homologue. Nucleic Acids Res. 23,203 -210.[Abstract]
Woodcock, D. M., Lawler, C. B., Linsenmeyer, M. E., Doherty, J.
P. and Warren, W. D. (1997). Asymmetric methylation in the
hypermethylated CpG promoter region of the human L1 retrotransposon.
J. Biol. Chem. 272,7810
-7816.
Yoder, J. A. and Bestor, T. H. (1998). A
candidate mammalian DNA methyltransferase related to pmt1p of fission yeast.
Hum. Mol. Genet. 7,279
-284.