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First published online 27 February 2008
doi: 10.1242/dev.016402
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1 Human Genetics Unit, MRC, Western General Hospital, Crewe Road, Edinburgh EH4
2XU, UK.
2 Genes and Development group, School of Biomedical Sciences, The University of
Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK.
3 Institute of Gene Biology, Russian Academy of Sciences, Vavilova 34/5, Moscow,
119334, Russian Federation.
* Author for correspondence (e-mail: richard.meehan{at}hgu.mrc.ac.uk)
Accepted 16 January 2008
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: DNA methylation, MBT, Xenopus
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The enzymatic function of DNMT1 is necessary to
maintain and perpetuate DNA methylation patterns at CpGs laid down by de novo
methyltransferases in response to developmental cues. DNA methylation in
mammals is essential for transcriptional silencing of transposons, regulation
of many imprinted genes and the maintenance of X-inactivation in female
somatic cells (Goll and Bestor,
2005). Methylated CpG pairs can repress transcription of adjacent
genes either by directly interfering with nuclear factor site recognition or,
indirectly by binding methyl-CpG specific binding proteins
(Klose and Bird, 2006).
Altered patterns of gene expression (including single copy genes, imprinted
genes and transposons) occurs in hypomethylated (dnmt1) mutant mouse
embryonic fibroblasts (Jackson-Grusby et
al., 2001). Nonetheless, many tissue-specific gene promoters are
hypo-methylated and are not expressed in early mouse or Xenopus
embryos (Walsh and Bestor,
1999; Stancheva et al.,
2002), implying that additional silencing mechanisms may be
operative. A screen of dnmt1n/n fibroblast cells also
noted that a high proportion of mis-expressed genes are transcribed from
CpG-island promoters that are constitutively unmethylated
(Lande-Diner et al.,
2007).
A decrease in DNMT1 levels results in early embryonic lethality in mouse,
frog and zebrafish, probably owing to multiple defects, including activation
of a cell death pathway (Li et al.,
1992; Stancheva and Meehan,
2000; Jackson-Grusby et al.,
2001; Stancheva et al.,
2001; Rai et al.,
2006). Recently a mutant mouse with a catalytically inactive form
of Dnmt1 has been generated that exhibits a developmental arrest
phenotype that is very similar to those observed for targeted deletion mutants
(Takebayashi et al., 2007).
This suggests that the catalytic function of DNMT1 is very important in early
mouse embryogenesis, although undifferentiated embryonic stem cells are
relatively unaffected by loss of DNMT1 activity
(Jackson-Grusby et al., 2001;
Tsumura et al., 2006). By
contrast, complete inactivation of DNMT1 in human cancer cells leads to
activation of a G2/M checkpoint and mitotic catastrophe with minimal changes
in DNA methylation levels (Chen et al.,
2007). Taken together these studies suggest the reported
phenotypes of DNMT1 depletion in early development and cell lines may reflect
the loss of both its enzymatic and non-enzymatic functions.
Although there is no evidence of global demethylation, imprinting, or
inactivation of sex-specific chromosomes in Xenopus laevis, we have
shown previously that xDnmt1 has an essential function in maintaining gene
silencing prior to zygotic gene activation at the mid-blastula transition
(MBT) in early amphibian development
(Stancheva and Meehan, 2000).
However, it was not clear from this study whether maintenance of gene
silencing prior to the MBT in Xenopus depended on the enzymatic or
non-enzymatic functions of xDnmt1. Here, we show that the silencing function
of xDnmt1 in early amphibian development is independent of its
methyltransferase activity. We report that a partial reduction in xDnmt1p
levels by morpholino (xDMO) injection into Xenopus laevis embryos
results in premature zygotic gene activation without a concomitant decrease in
DNA methylation levels, either globally or at specific loci. Rescue
experiments with an mRNA encoding a catalytically inactive form of human Dnmt1
(DNMT1) strongly suggest that DNA methylation is not used as a general
silencer of gene expression in Xenopus embryos. Our data support a
model in which xDnmt1 can regulate embryonic gene silencing directly and
independently of its catalytic function.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Morpholino oligonucleotides against xDnmt1 (xDMO) were designed and
synthesised by GeneTools. Human rescue mRNAs were synthesised from wild-type
or mutant (C1226Y) DNMT1 plasmids (gift from Michael Rountree) using the T3/T7
Capscribe kit (Boehringer).
TNT
cDNA encoding full-length xDnmt1 was used as a template in coupled in
vitro transcription-translation (TNT, Promega) reactions performed in the
absence or presence of xDMO followed by PAGE.
RT-PCR
RT-PCR was performed as published
(Ruzov et al., 2004).
In situ analysis
In situ protocols were performed using standard methods.
Southern blotting
Southern blotting was performed as described
(Stancheva and Meehan, 2000).
xSatellite I (xSatI) probe was generated by PCR.
Bisulfite sequencing
The bisulfite sequencing protocol has been described previously
(Stancheva et al., 2002).
Promoter cloning
xOct91 and xOct25 promoter regions were cloned from
Xenopus laevis genomic DNA using the DNA Walking Kit (SeeGene,
Korea). xOct60 promoter was cloned by synteny PCR and the xOct91
promoter region (-463 to -12) was cloned into SacI/BglII
sites of pGL3-Luc basic.
Western blotting
Embryonic extracts were isolated using RIPA buffer and xDnmt1 levels were
detected by immunoblotting with 
-xDnmt1 antibody 3C6
(Shi et al., 2001). Mouse cell
extracts were prepared and the following antibodies were used: -T7
(T7-xSp1) (Novagen); -human DNMT1 (NEB); and PCNA (Abcam).
Embryonic histone extracts were prepared by acid extraction and blotted with
the following antisera: panAcH4 (Cell Signalling, 9441S), H3K9Ac (Abcam,
AB4441), H4K5Ac (Upstate, 06-759), PanMethKH3 (Abcam, AB7315), H3K4me3 (Abcam,
AB8580), H4K20me3 (Abcam, AB9053), H3K9me3 (Abcam, AB8898 and H3 (Abcam,
AB1791).
GST pull down
Binding reactions for DNA GST pull downs were prepared as for EMSA
(Ruzov et al., 2004). CpGpos
oligonucleotide probes were used (Voo et
al., 2000). Reactions were incubated for 10 minutes on a shaker at
30°C, washed four times with PBS, treated with Proteinase K, extracted and
analysed using PAGE.
Transient transfections and reporter assays
Human 293T cells and mouse N2A cells were cultured using standard methods.
Constructs for reporter assays were transfected into Neuro2A cells using
established methods (Invitrogen). Assays were carried out independently in
quadruplicate.
ChIP analysis
Xenopus A6 cells were transfected with xDnmt1-GFP, pCMVxDnmt1 or
without plasmid DNA. CHIP was performed with a GFP antibody (Abcam).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Here, we use highly stable (xDMO) morpholinos,
which inhibit xDnmt1 mRNA translation in vitro and in vivo
(Fig. 1A), and result in a
phenotype that is indistinguishable from antisense RNA depletion. The xDMO
morphants develop normally up to MBT but at gastrulation exhibit an extended
open blastopore, which by neurulation results in the appearance of dead
shedding white cells on the surface of a high proportion (80%) of embryos
(arrows in Fig. 1B and insert)
and a failure to form a neural tube. By tadpole stage, only 2% of the xDMO
morphants are phenotypically normal compared with 91% for controls (see
Fig. 3). This is intriguing as
antisense injection results in almost complete depletion of xDnmt1 in stage 8
pre-MBT embryos (Stancheva and Meehan,
2000), whereas xDMO injection results in a 40-50% reduction of
xDnmt1 protein (Fig. 1A).
Despite this difference, the phenotypes of the AS and xDMO embryos are
virtually indistinguishable. Array screens in our laboratory (D.S.D. and R.R.M., unpublished) indicate that up to 25% of genes in these experiments are mis-expressed in stage 8 xDMO morphants. We used RT-PCR to verify the expression of these putative methyl-CpG dependent target genes in pre-MBT (stage 7-8) embryos (wild type and xDMO morphants). All tested genes were mis-expressed in xDMO morphants (xCycD1, xSox17β, xMix1, xp68, xDep, xOct91 and xID2) relative to histone H4 and xOct60 expression (Fig. 1C; see Fig. S1 in the supplementary material). Whole-mount RNA in situ hybridisation revealed that ectopic transcripts are present throughout the animal pole of xDMO stage 8 morphants (Fig. 1D). A control shows equal expression of the maternal oocyte-specific gene xOct60 between the two embryo sets. We conclude that the xDnmt1p reduction in xDMO morphants is sufficient for premature gene activation before MBT, the induction of apoptosis and phenotypic defects that result in reduced survival rates. More importantly, premature gene activation is a general feature of xDMO embryos, implying an essential global role for xDnmt1p in embryonic gene repression.
xDMO morphants retain normal DNA methylation at repeat and unique sequences
To determine whether global methylation levels were altered in xDMO
morphants, we tested the dispersed satellite I repeat (xSatI) by Southern
blotting, which is methylated at its two HpaII (CCGG)
sites through development (Stancheva et
al., 2002). Genomic DNA from both wild-type and xDMO siblings
showed a comparable resistance to HpaII digestion either by itself or
in double digestion with HindIII
(Fig. 2A). We
used bisulphite sequencing analysis to precisely map CpG methylation at xSatI
sequences in wild-type and xDMO genomic DNA. No hypomethylated CpGs were
observed in xDMO DNA relative to the wild type
(Fig. 2B; boxed numbers
indicate % methylation at each CpG). As xSatI is distributed through the
Xenopus genome, this suggests there are no genome-wide changes in DNA
methylation in xDMO morphants.
Subsequent to finding normal methylation patterns in xDMO repeat DNA, the next issue was to evaluate the methylation profile of xDMO target genes (xOct91 and xCycD1, Fig. 2C). This analysis showed that the pattern of methylation at xOct91 and xCycD1 promoters and upstream regions in stage 8 xDMO morphants was identical to stage 8 wild type (Fig. 2D). xOct91 and xCycD1 are zygotically activated during normal development after MBT (see Fig. S2A in the supplementary material) so we compared bisulfite maps when they are either transcriptionally silent (stage 8) or active (stage 10). There was no significant difference in CpG methylation at the xOct91 and xCycD1 loci between the inactive and active stages (data not shown), indicating that DNA methylation does not play a direct role in regulating their expression during normal development. We made similar observations for the xOct25 and xSox17β promoter regions (data not shown). Together with the xSatI methylation analysis, this led us to conclude that DNA methylation is not directly regulating the expression of the xOct91 and xCycD1 loci in xDMO morphants and by extension other genes that are misexpressed in stage 8 xDMO embryos. Our data imply that premature gene activation during Xenopus embryogenesis is governed by a mechanism independent of DNA modification.
Several studies have identified crosstalk between the DNMT1 proteins and
histone modifying enzymes (Fuks,
2005). One possibility is that premature transcription in xDMO
morphants may be due to global alterations in histone modification states as a
consequence of xDnmt1p depletion. We tested this possibility by direct
comparisons of histone mark abundance levels by immunoblotting. These
experiments revealed no significant differences for various histone
acetylation and histone methylation marks globally in early (stage 8) or later
(stage 15) xDMO morphants (Fig.
2E). In stage 8 embryos, most histone marks are low to
undetectable and only accrue as development proceeds, particularly in the case
of H3K4me3 (see Fig. S2 in the supplementary material) and H4K20me3. However,
these experiments cannot completely rule out subtle histone mark changes at
specific gene promoters. Our data imply that neither global changes in DNA
methylation or specific histone modifications are associated with activation
of xDMO target genes in stage 8 embryos, but rather that high levels of
xDnmt1p that are present in early Xenopus embryos are essential for
their repression (Shi et al.,
2001).
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xDnmt1p is a transcriptional repressor and can be localised to target gene promoters
In light of the above data, we sought to explore potential mechanisms for
DNMT1-mediated repression. The N-terminal non-catalytic region of mammalian
DNMT1 is an effective transcription repressor when artificially recruited to a
promoter (Fuks, 2005), and
knockdown of DNMT1 in transformed cells specifically activates expression of
two genes, independently of DNA methylation
(Milutinovic et al., 2004). We
hypothesised that if xDnmt1, like its mammalian counterparts, contained
regions that can directly bind non-methylated DNA, this would enable it to act
as a general repressor of transcription during early Xenopus
development (Chuang et al.,
1996; Suetake et al.,
2006). We tested three candidate regions
(Fig. 4A, black bars G1-G3) of
xDnmt1 as GST fusion proteins for DNA binding activity in vitro using a pull
down assay. Under the stringent conditions employed, all three GST fusion
proteins bound the CpGpos oligonucleotide
(Voo et al., 2000) as shown in
Fig. 4A. Additional experiments
suggest that xDnmt1 binding has relaxed sequence specificity (data not
shown).
We tested whether xDnmt1 can repress the activation of a minimum promoter
that has four copies of the xSp1-binding site driving luciferase expression
(p4xSp1-Lucif) (Kockar et al.,
2001). Relative induction by xSp1 is reduced by 55% in the
presence of xDnmt1 and both the catalytically active and inactive forms of
human DNMT1 (Fig. 4B), which
are expressed equally in transient transfection assays
(Fig. 4C, left panel). Analysis
of the raw luciferase data suggests that as the Dnmt1 dose is increased,
p4xSp1 activation is repressed up to 10-fold without affecting cell numbers
(data not shown). However, the expression of the co-transfected luciferase
(Renilla) reporter is concomitantly repressed by fivefold and results
in a normalised value of a twofold (50%) reduction. Western blot analysis
shows that there is no observable difference in xSp1 levels between cells
transfected with a 10-fold difference of the xDnmt1 expression plasmid
(Fig. 4C, right panel). Our
data suggest that untethered DNMT1 can act as a general repressor of promoters
when it is abundant, and that its catalytic activity is dispensable for this
function.
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50% or more relative to the empty vector control
(Fig. 4D). The repressive
capacity of hDNMT1C1226Y strongly indicates that the mechanism of
inhibition of these transgenes is independent of DNA methylation. In addition,
repression by DNMT1s is not relieved by an HDAC inhibitor (TSA) and requires
full-length forms of the protein for efficient silencing and xDMO rescue
(J.A.H., A.R. and R.R.M., unpublished). We conclude that DNMT1 can act as a
general repressor of non-methylated promoters and that its catalytic activity
is not required for this function.
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-Tubulin gene. Enrichment of GFP-xDnmt1 at
non-methylated, non-expressed loci (xDMO target genes) is consistent with a
model in which xDnmt1p can bind and repress gene expression independently of
DNA methylation during development. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Mutant mice expressing a DNMT1 point-mutant protein lacking catalytic activity
(DNMT1-C1229S) fail to develop, arrest after gastrulation (E9.5) with a
near-complete loss of DNA methylation and mis-express normally methylated
genes with phenotypes very similar to those of the
dnmt1c/c mutant (Lei
et al., 1996; Takebayashi et
al., 2007). This indicates that the catalytic function of
Dnmt1 is required to support early mouse embryogenesis, probably
owing to the many developmental processes, including X-chromosome
inactivation, suppression of retrotransposon activity, imprinting and
regulation of germ-cell specific gene expression, that are dependent on DNA
methylation (Goll and Bestor,
2005; Maatouk et al.,
2006). By contrast, Xenopus laevis lacks imprinting and
processes equivalent to X-inactivation, which may underlie its non-dependence
on DNA methylation in early development. Additionally primordial germ cell
development is specified by different mechanisms in amphibians and mammals
(Crother et al., 2007). Our
work suggests that the intrinsic repression function of xDnmt1p has been used
to maintain gene silencing in pre-MBT embryos until co-ordinated and precise
zygotic activation occurs at multiple gene loci. This period of gene silencing
over 11 cell divisions is not observed during mouse embryogenesis
(Meehan et al., 2005) and
implies distinct regulatory mechanisms for zygotic gene activation are used
between mice and frogs.
Zygotic activation of gene expression during early Xenopus
development is mainly dictated by maternal inheritance of repressor and
activator components (Veenstra,
2002). A two-cell Xenopus embryo contains up to 100 ng of
histones and 10 ng of xDnmt1 (Shi et al.,
2001; Veenstra,
2002), which together impose dominant repression of transcription.
At MBT, virtually all of the free histone pool has been depleted and we
hypothesize that transcription repression is now sensitive to xDnmt1 protein
levels. Radioactive labelling experiments and successful depletion by
antisense RNA and morpholinos suggest that continuous translation is required
to maintain high xDnmt1p levels in oocytes and embryos
(Kimura et al., 1999). The
rate of transcription per cell increases 200-fold when xDnmt1p levels are
reduced to approximately 2 pg/cell in normal embryos
(Newport and Kirschner, 1982a;
Hashimoto et al., 2003). In
xDMO morphants, a 40-50% reduction in xDnmt1p before the MBT is sufficient to
allow the low level of general transcription machinery components that are
present, such as RNA Pol I, Pol II and TBP, to dramatically activate a
generalised pattern of gene expression approximately two cell cycles earlier
than normal. However, this pattern of gene activation does not equate with a
normal transcriptional activation profile as the spectrum of genes that are
mis-expressed in xDMO morphants (and antisense-depleted embryos) is much
greater, as measured by cDNA library screens and array analysis (D.S.D. and
R.R.M., unpublished). This suggests that the transcriptional competence of
normal late blastula/early gastrula embryos is crucially dependent on the
non-catalytic and catalytic silencing function of xDnmt1. In general,
transcription at MBT occurs before histone modifications, such as H3K4M3
(Fig. 2E) or H4 acetylation,
accumulate (Almouzni et al.,
1994). This may explain why we do not observe an accumulation of
histone modifications in pre-MBT xDMO morphants. It is possible that changes
in histone modifications during Xenopus development are linked with a
dynamic alteration in the organization of different chromatin domains that
occurs after the MBT when gene-specific subdomains are set up
(Vassetzky et al., 2000).
|
).
It is possible that mammalian DNMT1 also has multiple silencing functions
because screens of mis-expressed genes in Dnmt1-/-,
Trp53-/- MEFs indicate that a high proportion (up to 80%) of
mis-expressed genes have CpG island promoters, which would be predicted to be
methylation free at all times
(Jackson-Grusby et al., 2001;
Lande-Diner et al., 2007).
Similar to our xDMO targets, it is highly possible that these genes are
inhibited through direct action of the Dnmt1 protein at promoters, hinting at
conservation of non-methyl dependent functions. Recent work demonstrates that
complete inactivation of DNMT1 function in human cancer cells results in cell
death (Chen et al., 2007), but
this decrease in viability occurs with minimal changes in global DNA
methylation. This observation supports the hypothesis that DNMT1 possesses
essential functions independent of its role as a maintenance
methyltransferase, and links its absence with activation of a cellular
checkpoint response. Unlike the situation in tumour cells
(Spada et al., 2007), limited
reduction in xDnmt1p levels appears to be sufficient to activate a cell death
program in Xenopus embryos. The pathway that mediates activation of
apoptosis in DNMT1-deficient cells is dependent on TRP53 function, but the
activating signal has yet to be identified
(Jackson-Grusby et al., 2001;
Stancheva et al., 2001). It
has also been observed that undifferentiated Dnmt1c/c ES
cells have a growth advantage compared with wild-type controls, but this
advantage was lost in the presence of a normal or mutant (C1229S) DNMT1
mini-genes (Damelin and Bestor,
2007). These observations support multifunctional non-enzymatic
roles for DNMT1 in development, cellular differentiation and cancer.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/7/1295/DC1
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ACKNOWLEDGMENTS |
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