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Jo urna l of Cell S cie nce, S upp lem ent 16, 9-1 4 (199 2)
Pr inted in Grea t Brit ain © T he Co mpa ny o f B iolo gists L imi ted 1992 9
Transcriptional repression by méthylation of CpG
RICHARD MEEH AN1, JOE LEWIS1, SALLY CROSS1, XINSHENG NA N1, PETER JEPPESEN2
and ADRIAN BIRD1
1Institute o f C ell and Molecula r Biology, U niversity of Edin burgh, Kin gs B uildin gs, Edinb urgh EH 9 3JR, UK
2MRC, Hum an Genetics Unit, Western Genera l Hospital, Cre we Road, Edin burg h EH4 2XU, UK
Summary
Methylated DNA in mammals is associated with tran
scriptional repression and nuclease resistant chromatin.
In this review we discuss how these effects may be medi
ated by proteins that bind to methylated DNA.
Méthylation of DNA is essential in mammals
In mammals approximately 70% of all CpGs are m ethy
lated at the 5 position of cytosine. It is not clear what the
functional role of this modification is, but it has been
recently shown by gene targeting experiments that méthyl
ation of DNA is necessary for mouse development (Li et
al., 1992). Mutant mouse embryos that have reduced levels
(1/3 of wild type) of genomic m5C were generated by intro
ducing a mutation into the DNA methyltransferase (MTase)
gene. Homozygous mutant embryos were stunted and died
at midgestation. A similar reduction of m5C in the DNA of
embryonic stem (ES) cells had no effect on their ability to
survive in tissue culture. This suggests that reduced levels
of MTase cause abnormal development.
How might decreased levels of DNA méthylation cause
embryonic lethality? There is much evidence that méthyl
ation near promoters leads to stable inactivation of the asso
ciated gene, and in one case (X-inactivation in placental
mammals) we know that the stability of repression in the
organism is due in part to méthylation (see Riggs and
Pfeifer, 1992). For example the CpG island-containing
genes on the X chromosome become methylated following
X chromosome inactivation in eutherian mammals (Grant
and Chapman, 1988). The equivalent CpG island genes
remain non-methylated on the active X chromosome. A
similar process of méthylation associated inactivation
occurs to retroviral proviruses following infection of early
mouse embryos (Jâhner et al., 1982). In both of these cases
there is some evidence that the genes becom e inactivated
prior to or at the onset of méthylation (Gautsch and Wilson,
1983; Lock et al., 1987; Singer-Sam et al., 1990). Although
DNA méthylation may not be directly responsible for the
initial inactivation event, it is seen as integral to the main
tenance of gene repression (Pfeifer et al., 1990b). There
may well be other cases where the repressive effects of
méthylation have been harnessed for the benefit of con-
Key words: DNA méthylation, transcriptional repression, methyl-
CpG binding proteins.
trolling gene expression during development. If we assume
that méthylation is functioning as a transcriptional repres
sor in the developing embryo then one possibility is that
high levels of MTase are required to repress genes stably
whose inappropiate expression could cause embryonic
lethality. These ‘genes’ may correspond to normally inac
tivated retroviruses (Jâhner et al., 1982), retroposons and
genes that are usually tissue-specific in their expression.
At this point it is also worth considering that DNA
méthylation has been proposed to have numerous other
roles in the regulation of chromatin activity with respect to
DNA repair, recombination and replication. Perturbation of
these processes could also have lethal consequences for the
developing embryo. For example, the mouse major satel
lite sequence is undermethylated in the embryonal carci
noma-derived F9 cells and is shifted in its replication timing
from late to early S-phase in comparison with its methy
lated counterpart (Selig et al., 1988). This may implicate
méthylation in the maintainance of satellite DNA sequences
in a late replicating mode in differentiated cells. It has also
recently been shown that CpG méthylation inhibits recom
bination between V(D)J substrates maintained on minichro
mosomes (Hsieh and Lieber, 1992). Likewise transcrip
tionally active DNA promoter regions are preferentially
repaired after exposure to DNA damaging agents in com
parison with inactive DNA regions (Mellon et al., 1986).
Thus a more liberal interpretation of the function of DNA
méthylation would be as a chromatin repressor rather than
solely as a transcriptional repressor.
The observation that ES cells can survive in tissue cul
ture with reduced amounts of m5C in their DNA implies
that embryonic cells are less dependent on DNA méthyl
ation than somatic cells. It would of be interest to know
whether somatically derived cell lines carrying the same
MTase mutation could also maintain viability in culture. In
fact many established cell lines have high levels of de novo
méthylation at the promoter regions of genes that are prob
10 R. M eehan and others
ably not required by the cell for growth on a plastic dish
(Jones et al., 1990; Antequera et al., 1990). A somatically
derived cell line may find it selectively more advantageous
to utilize the inhibitory effects of DNA methylation on
genes that are dispensable in culture.
How does methylation inhibit transcription?
There is a variety of evidence in the literature which
suggests that the presence of DNA methylation at gene pro
moters can inhibit transcription in two possible ways (Watt
and Molloy, 1988; Boyes and Bird, 1991). One model pro
poses that CpG methylation can interfere with transcription
directly by modifying the binding site (through methyl
ation) of transcription factors so that they can no longer
bind their cognate sequences. Alternatively there are fac
tors in the nucleus which specifically bind methylated DNA
and thereby deny transcription factors access to gene pro
motors.
In the first case we know of some transcription factors
that are sensitive to methylation in this way (Kovesdi et al.,
1987; Watt and M olloy,1988; Iguchi-Ariga and Schaffner,
1989; Shen and Whitlock, 1989; Comb and Goodman,
1990). Two factors detected in HeLa cells are unable to
bind to sites containing methyl-CpG and are thus unable to
stimulate transcription from the adenovirus late and E2 pro
moters (Kovesdi et al., 1987; W att and Molloy, 1988). On
the other hand the general transcription factor Spl binds
equally well to methylated and non-methylated sites and
stimulates transcription from both kinds of template (Har
rington et al., 1988; Hoeller et al., 1988). It should also be
recognised that many transcription factors do not contain
the dinucleotide CpG in their recognition sequence, so it is
difficult to envisage how methylation could directly inhibit
the binding of such factors.
Current evidence strongly favours the second idea that
repression is mediated by proteins that bind to DNA con
taining methyl-CpG (Boyes and Bird, 1991; Levine et al.,
1991). Early evidence supporting such a mechanism came
from microinjection studies with the methylated Herpes
simplex virus thymidine kinase (tk) gene (Buschausen et
al., 1987). In this case the methylated tk gene was tran
scribed normally for about 8 hours until repressed. The
timing of inhibition coincided with assembly of the methy
lated tk gene into chromatin. This suggested that the inhi
bition was indirect and may involve proteins that bind to
methylated DNA. Additional evidence for the existence of
such factors in mammalian nuclei was that m5C (but not
thymidine) in chromatin is refractory to digestion by
microccoccal nuclease (Solage and Cedar, 1978) and to
nucleases that can cleave at CpG (Hansen et al., 1988; Ante
quera et al., 1989). Sequences artificially methylated in vitro
are transcriptionally repressed when transfected into cells
and also adopt a nuclease-insensitive chromatin structure
(Keshet et al., 1986). A refinement of the indirect model
which can accommodate both gene repression and altered
chromatin would be that methyl-CpG binding proteins, or
MeCPs, bind to a methylated gene leading to an altered
chromatin structure which would in turn deny access to the
transcription machinery (Lewis and Bird, 1991). Binding of
MeCPs in this model could also account for the other chro
matin suppressor effects associated with DNA methylation
such as late replication and inhibition of recombination.
Methyl-CpG binding proteins (MeCPs)
We have detected two such proteins (MeCPs 1 and 2) by
conventional biochemical techniques in mammalian and
avian nuclei. We have purified, cloned and sequenced one
of these (MeCP2). MeC Pl binds in vitro to DNA contain
ing at least 12 symmetrically methylated CpGs (Meehan et
al., 1989), while MeCP2 can bind to a single methylated
CpG pair (Lewis et al., 1992). Neither protein will bind sig
nificantly to DNA that contains m5C in a sequence other
than CpG (Meehan et al., 1989, 1992). Furthermore, nei
ther protein shows an affinity for TpG, which also carries
a methyl group at the 5 position of the pyrimidine ring pre
ceding a G (but is not self complementary). The relaxed
sequence specificity and widespread tissue distribution of
these proteins makes them likely candidates for mediators
of the effects of methylation. M eCPl is of relatively low
abundance (about 5000 molecules per nucleus), and is
loosely bound, whereas MeCP2 is comparatively abundant
(about 200,000 molecules per nucleus) and is only released
from chromatin by high salt concentrations. Both proteins
are deficient in embryonal carcinoma (EC) and stem (ES)
cells.
A third methylated DNA binding protein has been
detected in human placenta but this protein is highly
sequence-specific (Huang et al., 1984; Wang et al., 1986)
and may actually correspond to a methylation-insensitive
transcription factor. This protein is therefore unlikely to be
involved in general methylation-mediated inhibition. It has
also been reported that histone HI binding to methylated
DNA in vitro can inhibit the normally methylation-insensi
tive restriction enzyme Mspl (Higurashi and Cole, 1991).
Biological significance of MeCPs
Studies of M eCPl have implicated it in methylation-asso-
ciated gene inactivation. The expression of the X-linked
mouse PGK1 gene promoter can be inhibited by methyl
ation in transcription extracts and in cell lines which con
tain MeCPl (Boyes and Bird, 1991). Expression can be
restored by competition with methylated DNA substrates
which are specific for M eCPl. In addition, methylated
genes are not efficiently repressed in ES and EC cell lines
or extracts which lack MeCPs (Boyes and Bird, 1991;
Levine et al., 1991). Histone HI is present in ES and EC
cell lines and so can be ruled out as a methylation-specific
transcriptional repressor.
Methylation can also inhibit transcription directly by
interference of site-specific methylation within a recogni
tion site for a transcription factor with the binding of that
factor. Although we know of some transcription factors that
are blocked in this way (see earlier), it is striking that out
of seven fully methylated promoters studied so far, none
are inhibited strongly under conditions where M eCPl is
absent (Boyes and Bird, 1991, 1992; Levine et al., 1991).
Transcriptional repression by meth yla tion o f CpG 11
12 3 4 5 6 7 8
180 -
116 —
58 —
48 —
Fig. 1. Detection o f MeCP2 in rodent nuclear extracts by western
blotting. Nuclear proteins equivalent to 20 (Xg of DNA were
transferred to nitrocellulose m embranes after separation by
SDS/PA GE and incubated with antibody (Ab76) raised against
purified rat MeCP2 (Lewis et al., 1992). The nuclei were from the
follow ing sources: lanel, mouse kidney; lane 2, m ouse brain; lane
3, mouse spleen; lane 4, mouse liver; lane 5, rat testes; lane 6, rat
brain; lane 7, PC 13 a m ouse embryonal carcinoma cell line; lane
8, D2 an SV40 transformed mouse embryo stem cell line. The
numbers on the left correspond to the molecular mass (in kDa) of
protein size m arkers. Pre-immune serum failed to give any signal
under these conditions (data not shown).
Thus the predom inant repressio n m echanism appears to
work via M e CP l. In keeping with this, several studies have
shown that transcription is sensitive to any methylation near
the promoter, not just to specific methyl groups at key sites
(M urray and Grosveld, 1987).
Although purified M eCP 2 has a preferenc e for D NA
symm etrically m ethylated at the dinucleotide CpG , it cannot
selectively inhibit transcription from CpG-rich m ethylated
DNA templates in vitro (M eehan et al., 1992). Thu s the
biological significance of M eCP2 is presen tly uncertain. In
addition to its high specificity for m ethyl-CpG pairs, the
protein contains d omains that can interact with low affin
ity with non-m ethylated DNA sequences. T he latter are
readily competed out in the presence of excess non-m ethy
lated DNA, im plying tha t they do not overlap the protein
domain responsible for m ethyl-CpG bind ing (Meehan et al.,
1992). It would be prematu re to conc lude, however, that
MeC P2 is not involved in transcriptional repression. Nucle
ase treatment of chromatin indicates that MeCP2 is bound
to chromatin, but the transcription experimen ts w ere car
ried out in histone-free extracts in wh ich chromatin cannot
form (Meehan et al., 1992). If the natural ligand for M eCP2
is chrom atin, as is the case for histone HI (W olffe, 1990),
this could explain our failure to observe specific effects.
Evidence that MeCP2 is associated w ith methyl-C pGs in
vivo relies u pon in situ localisation o f the protein usin g
antibodies. A polyclonal antibody raised against purified,
denatured rat MeCP2 cross reacts with the h omologous pro
tein from mouse (Fig. 1). The mobility of the reacting pro
tein is identical to that of the mouse M eCP2-like activity
(80 kD) detected by sou thwestern assay (Meehan et al.,
1992). An tibodies against MeCP2 localise preferentially to
centromeric heterochrom atin regions, although euchro matic
chromoso me arm s are also stained (Fig. 2; see also Lewis
et al., 1992). Pre-im mune serum did no t stain the chrom o
somes significantly (Fig. 2). More than half of the methyl-
CpG s in the mouse genome are located in the major satel
lite DNA which is co ncentrated in centromeric
heterochromatin (M anuelides, 1981; Horz and Altenberger,
1981), as calculated from the known distribution of CpGs
in satellite and bulk D NA. The asym metrical distribution
of m ethyl-C pG can also be visualised directly using anti
bodies against 5-m ethylcytosine, which preferentially stain
regions of pericentrom eric hetero chromatin (Miller et al.,
1974). Thus M eCP 2 colocalizes with ch rom osom al regions
that are known to be rich in m ethyl-CpG. W e could also
dem onstrate that the m ethylated form of the 234 -bp satel
lite m onomer D NA was a good substrate for M eCP2 by
southw estern analysis (Lewis et al., 1992) but did not bind
M eCP l in the bandsh ift assay (S. Cross, unpublished data).
One speculation for MeCP2 function may be in the
genome-w ide protection o f methyl-C pGs against nucleases,
as it is much more abundant and more tightly bound in the
nucleus than MeC Pl. Brain nuclei, which have the highest
levels of MeC P2, show particularly striking protection of
methyl-CpGs against nucleases (Antequera et al., 1989).
Conversely PC 13 cells, which have very reduced levels of
M eCPl and MeCP2, show mark edly reduced levels of pro
tection (A ntequera et al., 1989; L evine et al., 1991). As
stated earlier, it has been reported th at histone HI binding
to m ethylated D NA in vitro can inhibit the norm ally
methylation-insensitive restriction enzym e M sp l, implying
that H I could be respo nsible fo r nuclease pro tection in
nuclei (H igurashi and Cole, 1991). How ever, this observa
tion does not ex plain the different levels of p rotec tion seen
between mouse brain and PC 13 nuclei, w hich appear to
have comparable levels of histone H I, unless histone HI is
very d ifferent between PC 13 cells an d brain. As yet, we
have been unable to detect a preference for binding to
methylated DNA by histone HI in either the bandshift or
southwestern assays.
Genom ic sequencing studies on the human PGK1 pro
moter have not provided any evid ence fo r binding by
MeCPs in vivo (Pfeifer et al., 1990a; Pfeifer and Riggs,
1991). However differences were observed between the
PGK1 promoters on the active X chromosome (X a) and the
inactiv e X chro mosom e (Xi). The unm ethylated X a pro
moter was free of nucleo som es but did show several foot
prints indicative o f transcription factors (including S pl),
whereas the m ethylated DNA (60 out of a po ssible 61 CpG
sites) of the Xi promote r was w rapped around positioned
particles, w hich are presum ed to be nucleosom es. Interest
ingly the phased nucleosom es on the Xi promoter covered
most of the M s pl sites tested for nuclease sensitivity.
The lack of footprints attributable to M eCPs over the
methylated PGK1 p rom oter does not prove the ir absence.
If M eCPs bind randomly and w eakly with methyl-C pGs
then it may be difficult to detect their in teraction with
methylated D NA by present m ethods (Pfeifer and Riggs,
1991). We know that MeCP2 binding to chro matin is very
sensitive to nuclease treatm ent (M eehan et al., 1992). This
may ac cou nt for the failure to detec t MeCPs interacting
with m ethyl-C pGs of PGK1 on the Xi using DN ase I. There
12 R. Meeh an a n d others
Fig. 2. Distribution o f MeC P2 in mouse
nuclei and metaphase chromosomes.
MeCP2 was detected by indirect
immunofluorescence o f nuclei and
metaphase chrom osomes from mouse
fibroblasts (cell line L929) using Ab76.
Antibody labelling was carried out as
described by Jeppesen et al. (1992) and
modified by Lewis et al. (1992). Anti-
MeCP2 im munofluorescence (A) is
confined to nuclei and metaphase
chrom osom es, where it is particularly
enriched in the heterochromatic domains
but there is also significant staining on the
euchrom atic arms. (B) The same field is
observed using the DNA fluorochrome
Hoechst 33258 and shows the brightly
fluorescent heterochrom atin in both nuclei
and chromosomes. The fluorescent
heterochromatin corresponds with the
concentration of anti-MeCP2
immunofluorescence. This is especially
striking in the case of a marker multicentric
chromosome (arrowhead) where each block
of residual pericentromeric heterochromatin
is imm unofluorescently labelled. (D,E) A
sim ilar preparation of m ouse L929 cells
was labelled under identical conditions to
those described for A and B using pre
immune rabbit serum instead o f AB76.
Only background non-specific FITC
fluorescence is evident (C). The same field
observed by Hoechst 33258 fluorescence is
shown in D.
is no evidenc e that m éthylation of DNA favours nucleo-
some formation or determ ines positioning (Felsenfeld et al.,
1982; Drew and M cCall, 1987). W ithout M eCPs it is dif
ficult to account for the reduced Ms pi resistance in M eCP
deficient cells (Antequera et al., 1989; Levine et al., 1991).
This problem m ay be overcome if MeCPs guide nucleo-
some formation so that methy l-CpG s are ren dered nuclease
resistant (see Riggs and Pfeifer, 1992, and below ).
Mechanisms of methylation-mediated repression
The sim ilarities and differences betw een MeC Pl and
MeC P2 suggest a workin g model for th eir roles in methyl-
ation-mediated repression. M eC Pl is of relatively low
abundance (about 5000 m olecules per nucleus), and is
loosely bound, whereas MeCP2 is com paratively abund ant
(about 200,000 molecules per nucleus) and is only released
from chromatin by high salt concentrations. M eC Pl may
thus compete with transcription factors in the nucleoplasm
for binding to methylated DN A, the outcom e depending on
the density of methylation and the affinity o f the factors
(Boyes and Bird. 1992; B ird, 1992). W e hypothesise that
binding to MeC Pl then guides the DNA into a hete
rochromatic structure involving stable association with
MeC P2. How this migh t hap pen is unknown, but it proba
bly occurs at DNA replication, since the resistance o f
methylated D NA to n ucleases is known to increase dra
Transcrip tiona l rep ression by m éthylation ofC pG 13
matically at this time. Hsieh and Lieber (1992) showed that
high-density CpG methylation prevents the V(D)J joining
reaction in lymphocytes, but only after DNA replication has
taken place. Resistance to Mspl was much higher follow
ing replication, consistent with the idea that methylation
guides the replicating DNA into a ‘heterochrom atic’ struc
ture.
Although direct interference of methyl-CpG with tran
scription factor binding may not be the primary mechanism
of methylation-mediated repression, it may contribute to the
MeCP-mediated repression described above. The three
parameters which determine the effects of DNA methyl
ation on gene expression are: (1) location of the methylated
sites relative to the promoter; (2) density of methylated
sites; (3) promoter strength (Boyes and Bird, 1992; Bird,
1992). Direct blockage of a factor which contributes to pro
moter strength may weaken certain promoters, and may
therefore bias the competition between M eCPl binding and
transcription factor binding in favour of MeCPl. In this
hypothetical case, direct and indirect inhibition mecha
nisms, which were thought to be mutually exclusive alter
natives, would work together to bring about repression.
Conclusions
MeCPs have been detected in organisms which maintain a
large fraction of their genomes in a methylated state, includ
ing mammals, birds and plants (Meehan et al., 1992; Zhang
et al., 1989). Thus the interaction of MeCPs with methy
lated DNA may be a common strategy in regulating chro
matin activity in these types o f organisms. This idea is rein
forced by the fact that we have not detected MeCPs in
organisms with genomes which have either very reduced
methylated DNA fractions or no detectable methylated
DNA (eg. Drosophila) (Meehan et al., 1992). Our ability
to isolate and clone these proteins should allow us to recon
struct in vitro the interaction of chromatin and MeCPs, and
investigate how this could influence chromatin structure and
activity. Of prime importance will be the generation of
mutations in the MeCP loci of mice and the comparison of
the phenotype of such mutants with the MTase mutation
(Li et al., 1992). We might expect that loss of MeCPs and
loss of MTase should give similar phenotypes.
We thank Jillian Charlton for expert technical assistance, Peri
Tate and Paco A ntequera for critical reading of the manuscript
through many drafts and C hristine Struthers for help with the m an
uscript. This work was supported by the Imperial Cancer Research
Fund and the Wellcom e trust. R. M. and J. L. are members of the
ICRF epigenetics laboratory; S. C. and X. N. are supported by the
Wellcome trust.
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