Methylated DNA in mammals is associated with transcriptional repression and nuclease resistant chromatin. In this review we discuss how these effects may be mediated by proteins that bind to methylated DNA.

In mammals approximately 70% of all CpGs are methylated 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 methylation 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 introducing 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 methylation cause embryonic lethality? There is much evidence that methylation near promoters leads to stable inactivation of the associated 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 methylation (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 methylation associated inactivation occurs to retroviral proviruses following infection of early mouse embryos (Jahner et al., 1982). In both of these cases there is some evidence that the genes become inactivated prior to or at the onset of methylation (Gautsch and Wilson, 1983; Lock et al., 1987; Singer-Sam et al., 1990). Although DNA methylation may not be directly responsible for the initial inactivation event, it is seen as integral to the maintenance of gene repression (Pfeifer et al., 1990b). There may well be other cases where the repressive effects of methylation have been harnessed for the benefit of controlling gene expression during development. If we assume that methylation is functioning as a transcriptional repressor 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 inactivated retroviruses (Jahner et al., 1982), retroposons and genes that are usually tissue-specific in their expression.

At this point it is also worth considering that DNA methylation 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 satellite sequence is undermethylated in the embryonal carcinoma-derived F9 cells and is shifted in its replication timing from late to early S-phase in comparison with its methylated counterpart (Selig et al., 1988). This may implicate methylation in the maintainance of satellite DNA sequences in a late replicating mode in differentiated cells. It has also recently been shown that CpG methylation inhibits recombination between V(D)J substrates maintained on minichro-mosomes (Hsieh and Lieber, 1992). Likewise transcriptionally active DNA promoter regions are preferentially repaired after exposure to DNA damaging agents in comparison with inactive DNA regions (Mellon et al., 1986). Thus a more liberal interpretation of the function of DNA methylation would be as a chromatin repressor rather than solely as a transcriptional repressor.

The observation that ES cells can survive in tissue culture with reduced amounts of m5C in their DNA implies that embryonic cells are less dependent on DNA methylation 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 methylation at the promoter regions of genes that are probably 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.

There is a variety of evidence in the literature which suggests that the presence of DNA methylation at gene promoters can inhibit transcription in two possible ways (Watt and Molloy, 1988; Boyes and Bird, 1991). One model proposes that CpG methylation can interfere with transcription directly by modifying the binding site (through methylation) of transcription factors so that they can no longer bind their cognate sequences. Alternatively there are factors in the nucleus which specifically bind methylated DNA and thereby deny transcription factors access to gene promotors.

In the first case we know of some transcription factors that are sensitive to methylation in this way (Kovesdi et al., 1987; Watt and Molloy,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 promoters (Kovesdi et al., 1987; Watt 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 (Harrington 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 containing 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 transcribed normally for about 8 hours until repressed. The timing of inhibition coincided with assembly of the methylated tk gene into chromatin. This suggested that the inhibition 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; Antequera 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-ÇpG 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 chromatin suppressor effects associated with DNA methylation such as late replication and inhibition of recombination.

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). MeCPl binds in vitro to DNA containing 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 significantly to DNA that contains m5C in a sequence other than CpG (Meehan et al., 1989, 1992). Furthermore, neither protein shows an affinity for TpG, which also carries a methyl group at the 5 position of the pyrimidine ring preceding 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. MeCPl 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 Hl binding to methylated DNA in vitro can inhibit the normally methylation-insensitive restriction enzyme Mspï (Higurashi and Cole, 1991).

Studies of MeCPl have implicated it in methylation-associated gene inactivation. The expression of the X-linked mouse PGK1 gene promoter can be inhibited by methylation in transcription extracts and in cell lines which contain MeCPl (Boyes and Bird, 1991). Expression can be restored by competition with methylated DNA substrates which are specific for MeCPl. 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 Hl 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 recognition 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 MeCPl is absent (Boyes and Bird, 1991, 1992; Levine et al., 1991).

Thus the predominant repression mechanism appears to work via MeCPl. 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 (Murray and Grosveld, 1987).

Although purified MeCP2 has a preference for DNA symmetrically methylated at the dinucleotide CpG, it cannot selectively inhibit transcription from CpG-rich methylated DNA templates in vitro (Meehan et al., 1992). Thus the biological significance of MeCP2 is presently uncertain. In addition to its high specificity for methyl-CpG pairs, the protein contains domains that can interact with low affinity with non-methylated DNA sequences. The latter are readily competed out in the presence of excess non-methylated DNA, implying that they do not overlap the protein domain responsible for methyl-CpG binding (Meehan et al.,1992). It would be premature to conclude, however, that MeCP2 is not involved in transcriptional repression. Nuclease treatment of chromatin indicates that MeCP2 is bound to chromatin, but the transcription experiments were carried out in histone-free extracts in which chromatin cannot form (Meehan et al., 1992). If the natural ligand for MeCP2 is chromatin, as is the case for histone Hl (Wolffe, 1990), this could explain our failure to observe specific effects.

Evidence that MeCP2 is associated with methyl-CpGs in vivo relies upon in situ localisation of the protein using antibodies. A polyclonal antibody raised against purified, denatured rat MeCP2 cross reacts with the homologous protein from mouse (Fig. 1). The mobility of the reacting protein is identical to that of the mouse MeCP2-like activity (80 kD) detected by southwestern assay (Meehan et al., 1992). Antibodies against MeCP2 localise preferentially to centromeric heterochromatin regions, although euchromatic chromosome arms are also stained (Fig. 2; see also Lewis et al., 1992). Pre-immune serum did not stain the chromosomes significantly (Fig. 2). More than half of the methyl-CpGs in the mouse genome are located in the major satellite DNA which is concentrated in centromeric heterochromatin (Manuelides, 1981; Horz and Altenberger, 1981), as calculated from the known distribution of CpGs in satellite and bulk DNA. The asymmetrical distribution of methyl-CpG can also be visualised directly using antibodies against 5-methylcytosine, which preferentially stain regions of pericentromeric heterochromatin (Miller et al., 1974). Thus MeCP2 colocalizes with chromosomal regions that are known to be rich in methyl-CpG. We could also demonstrate that the methylated form of the 234-bp satellite monomer DNA was a good substrate for MeCP2 by southwestern analysis (Lewis et al., 1992) but did not bind MeCPl in the bandshift assay (S. Cross, unpublished data).

Fig. 1.

Detection of MeCP2 in rodent nuclear extracts by western blotting. Nuclear proteins equivalent to 20 (_lg of DNA were transferred to nitrocellulose membranes after separation by SDS/PAGE and incubated with antibody (Ab76) raised against purified rat MeCP2 (Lewis et al., 1992). The nuclei were from the following sources: lanel, mouse kidney; lane 2, mouse brain; lane 3, mouse spleen; lane 4, mouse liver; lane 5, rat testes; lane 6, rat brain; lane 7, PC13 a mouse 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 markers. Pre-immune serum failed to give any signal under these conditions (data not shown).

Fig. 1.

Detection of MeCP2 in rodent nuclear extracts by western blotting. Nuclear proteins equivalent to 20 (_lg of DNA were transferred to nitrocellulose membranes after separation by SDS/PAGE and incubated with antibody (Ab76) raised against purified rat MeCP2 (Lewis et al., 1992). The nuclei were from the following sources: lanel, mouse kidney; lane 2, mouse brain; lane 3, mouse spleen; lane 4, mouse liver; lane 5, rat testes; lane 6, rat brain; lane 7, PC13 a mouse 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 markers. Pre-immune serum failed to give any signal under these conditions (data not shown).

Fig. 2.

Distribution of MeCP2 in mouse nuclei and metaphase chromosomes. MeCP2 was detected by indirect immunofluorescence of nuclei and metaphase chromosomes 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 immunofluorescence (A) is confined to nuclei and metaphase chromosomes, where it is particularly enriched in the heterochromatic domains but there is also significant staining on the euchromatic arms. (B) The same field is observed using the DNA fluorochrome Hoechst 33258 and shows the brightly fluorescent heterochromatin 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 immunofluorescently labelled. (D.E) A similar preparation of mouse L929 cells was labelled under identical conditions to those described for A and B using pre-immune rabbit serum instead of AB76. Only background non-specific FITC fluorescence is evident (C). The same field observed by Hoechst 33258 fluorescence is shown in D.

Fig. 2.

Distribution of MeCP2 in mouse nuclei and metaphase chromosomes. MeCP2 was detected by indirect immunofluorescence of nuclei and metaphase chromosomes 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 immunofluorescence (A) is confined to nuclei and metaphase chromosomes, where it is particularly enriched in the heterochromatic domains but there is also significant staining on the euchromatic arms. (B) The same field is observed using the DNA fluorochrome Hoechst 33258 and shows the brightly fluorescent heterochromatin 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 immunofluorescently labelled. (D.E) A similar preparation of mouse L929 cells was labelled under identical conditions to those described for A and B using pre-immune rabbit serum instead of AB76. Only background non-specific FITC fluorescence is evident (C). The same field observed by Hoechst 33258 fluorescence is shown in D.

One speculation for MeCP2 function may be in the genome-wide protection of methyl-CpGs against nucleases, as it is much more abundant and more tightly bound in the nucleus than MeCPl. Brain nuclei, which have the highest levels of MeCP2, show particularly striking protection of methyl-CpGs against nucleases (Antequera et al., 1989). Conversely PC 13 cells, which have very reduced levels of MeCP 1 and MeCP2, show markedly reduced levels of protection (Antequera et al., 1989; Levine et al., 1991). As stated earlier, it has been reported that histone Hl binding to methylated DNA in vitro can inhibit the normally methylation-insensitive restriction enzyme MspI, implying that Hl could be responsible for nuclease protection in nuclei (Higurashi and Cole, 1991). However, this observation does not explain the different levels of protection seen between mouse brain and PC 13 nuclei, which appear to have comparable levels of histone Hl, unless histone Hl is very different between PC 13 cells and brain. As yet, we have been unable to detect a preference for binding to methylated DNA by histone Hl in either the bandshift or southwestern assays.

Genomic sequencing studies on the human PGK1 promoter have not provided any evidence for 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 (Xa) and the inactive X chromosome (Xi). The unmethylated Xa promoter was free of nucleosomes but did show several footprints indicative of transcription factors (including Spl), whereas the methylated DNA (60 out of a possible 61 CpG sites) of the Xi promoter was wrapped around positioned particles, which are presumed to be nucleosomes. Interestingly the phased nucleosomes on the Xi promoter covered most of the Mspl sites tested for nuclease sensitivity.

The lack of footprints attributable to MeCPs over the methylated PGK1 promoter does not prove their absence. If MeCPs bind randomly and weakly with methyl-CpGs then it may be difficult to detect their interaction with methylated DNA by present methods (Pfeifer and Riggs, 1991). We know that MeCP2 binding to chromatin is very sensitive to nuclease treatment (Meehan et al., 1992). This may account for the failure to detect MeCPs interacting with methyl-CpGs of PGK1 on the Xi using DNase I. There is no evidence that methylation of DNA favours nucleosome formation or determines positioning (Felsenfeld et al., 1982; Drew and McCall. 1987). Without MeCPs it is difficult to account for the reduced MspI resistance in MeCP deficient cells (Antequera et al., 1989; Levine et al., 1991). This problem may be overcome if MeCPs guide nucleosome formation so that methyl-CpGs are rendered nuclease resistant (see Riggs and Pfeifer. 1992, and below).

Mechanisms of methylation-mediated repression

The similarities and differences between MeCPl and MeCP2 suggest a working model for their roles in methylation-mediated repression. MeCPl 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. MeCPl may thus compete with transcription factors in the nucleoplasm for binding to methylated DNA, the outcome depending on the density of methylation and the affinity of the factors (Boyes and Bird. 1992; Bird, 1992). We hypothesise that binding to MeCPl then guides the DNA into a heterochromatic structure involving stable association with MeCP2. How this might happen is unknown, but it probably occurs at DNA replication, since the resistance of methylated DNA to nucleases is known to increase dramatically 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 following replication, consistent with the idea that methylation guides the replicating DNA into a ‘heterochromatic’ structure.

Although direct interference of methyl-CpG with transcription 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 methylation 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 promoter strength may weaken certain promoters, and may therefore bias the competition between MeCPl binding and transcription factor binding in favour of MeCPl. In this hypothetical case, direct and indirect inhibition mechanisms, which were thought to be mutually exclusive alternatives, would work together to bring about repression.

MeCPs have been detected in organisms which maintain a large fraction of their genomes in a methylated state, including mammals, birds and plants (Meehan et al., 1992; Zhang et al., 1989). Thus the interaction of MeCPs with methylated DNA may be a common strategy in regulating chromatin activity in these types of organisms. This idea is reinforced 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 reconstruct 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 Antequera for critical reading of the manuscript through many drafts and Christine Struthers for help with the manuscript. This work was supported by the Imperial Cancer Research Fund and the Wellcome trust. R. M. and J. L. are members of the ICRF epigenetics laboratory; S. C. and X. N. are supported by the Wellcome trust.

Antequera
,
F.
,
MacLeod
,
D.
and
Bird
,
A. P.
(
1989
).
Specific protection of methylated CpGs in mammalian nuclei
.
Cell
58
,
509
517
.
Antequera
,
F.
,
Boyes
,
J.
and
Bird
,
A.
(
1990
).
High levels of de novo methylation and altered chromatin structure at CpG islands in cell lines
.
Cell
62
,
503
514
.
Bird
,
A. P.
(
1992
).
The essentials of DNA methylation
.
Cell
70
,
5
8
.
Boyes
,
J.
and
Bird
,
A.
(
1991
).
DNA methylation inhibits transcription indirectly via a methyl-CpG binding protein
.
Cell
64
,
1123
1134
.
Boyes
,
J.
and
Bird
,
A.
(
1992
).
Repression of genes by DNA methylation depends on CpG density and promoter strength: evidence for involvement of a methyl-CpG binding protein
.
EMBO J
.
11
,
327
333
.
Buschhausen
,
G.
,
Wittig
,
B.
,
Graessman
,
M.
and
Graessman
,
H.
(
1987
).
Chromatin structure is required to block transcription of the methylated herpes simplex virus thymidine kinase gene
.
Proc. Nat. Acad. Sci. USA
84
,
1177
1181
.
Comb
,
M.
and
Goodman
,
H. M.
(
1990
).
CpG methylation inhibits proenkephalin gene expression and binding of the transcription factor AP-2
.
Nucl. Acids Res
.
18
,
3975
3982
.
Drew
,
M. R.
and
McCall
,
M. J.
(
1987
).
Structural analysis of a reconstituted DNA containing three histone octamers and histone H5
.
J. Mol. Biol
.
197
,
485
511
.
Felsenfeld
,
G.
,
Nickol
,
J.
,
Behe
,
M.
,
McGhee
,
J. D.
and
Jackson
,
D.
(
1982
).
Methylation and chromatin structure
.
Cold Spring Harbor Symp. Quant. Biol
.
47
,
577
584
.
Gautsch
,
J. W.
and
Wilson
,
M. C.
(
1983
).
Delayed de novo methylation in tetracarcinoma cells suggests additional tissue-specific mechanisms for controlling gene expression
.
Nature
301
,
32
37
.
Grant
,
S. G.
and
Chapman
,
V.M.
(
1988
).
Mechanisms of X-chromosome regulation
.
Annu. Rev. Genet
.
22
,
199
233
.
Hansen
,
R. S.
,
Ellis
,
N. A.
and
Gartler
,
S. M.
(
1988
).
Demethylation of specific sites in the 5’ region of the inactive X-linked human phosphoglycerate kinase gene correlates with appearance of nuclease sensitivity and gene expression
.
Mol. Cell Biol
.
8
,
4692
4699
.
Harrington
,
M. A.
,
Jones
,
P. A.
,
Masayoshi
,
I.
and
Karin
,
M.
(
1988
).
Cytosine methylation does not effect binding of transcription factor Spl
.
Proc. Nat. Acad. Sci. USA
85
,
2066
2070
.
Higurashi
,
M.
and
Cole
,
R. D.
(
1991
).
The combination of DNA methylation and Hl histone binding inhibits the action of a restriction nuclease on plasmid DNA
.
J. Biol. Chem
.
266
,
8619
8625
.
Hoeller
,
M.
,
Westin
,
G.
,
Jiricny
,
J.
and
Schaffner
,
W.
(
1988
).
Spl transcription factor binds DNA and activates transcription even when the binding site is CpG methylated
.
Genes Dev
.
2
,
1127
1135
.
Horz
,
W.
and
Altenberger
,
W.
(
1981
).
Nucleotide sequence of mouse satellite DNA
.
Nucl. Acids Res
.
9
,
683
676
.
Hsieh
,
C-L.
and
Lieber
,
M. R.
(
1992
).
CpG methylated minichromosomes become inaccessible for V(D)J recombination after undergoing replication
.
EMBO J
.
11
,
315
325
.
Huang
,
L-H.
,
Wang
,
R.
,
Gama-Sosa
,
M. A.
,
Shenoy
,
S.
and
Ehrlich
,
M.
(
1984
).
A protein from human placental nuclei binds preferentially to 5-methylcytosine rich DNA
.
Nature
308
,
293
295
.
Iguchi-Ariga
,
S. M. M.
and
Schaffner
,
W.
(
1989
).
CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation
.
Genes Dev
.
3
,
612
619
.
Jahner
,
D.
,
Stihlmann
,
H.
,
Stewart
,
L. L.
,
Harkens
,
K.
,
Liihlen
,
J.
,
Simon
,
I.
and
Jaenisch
,
R.
(
1982
).
De novo methylation and expression of retroviral genomes during mouse embryogenesis
.
Nature
298
,
632628
.
Jeppesen
,
P. N.
,
Mitchell
,
A.
,
Turner
,
B.
and
Perry
,
P.
(
1991
).
Antibodies to defined histone epitopes reveal variations in chromatin conformation and underacetylation of centric heterochromatin in human metaphase chromosomes
.
Chromosoma
101
,
322
332
.
Jones
,
P.
,
Woikowicz
,
M.
,
Rideout
,
W.
,
Gonzales
,
F.
,
Marziasz
,
C.
,
Coetzee
,
G.
and
Tapscott
,
S.
(
1990
).
De novo methylation of the MyoDl CpG island during the establishment of immortal cell lines
.
Proc. Nat. Acad. Sci. USA
87
,
6117
6121
.
Keshet
,
I.
,
Lieman-Hurwitz
,
J.
and
Cedar
,
H.
(
1986
).
DNA methylation affects the formation of active chromatin
.
Cell
44
,
535
543
.
Kovesdi
,
I.
,
Reichel
,
R.
and
Nevins
,
J. R.
(
1987
).
Role of adenovirus E2 promoter binding factor in ElA-mediated coordinate gene control
.
Proc. Nat. Acad. Sci. USA
84
,
2180
2184
.
Levine
,
A.
,
Cantoni
,
G-L.
and
Razin
,
A.
(
1991
).
Inhibition of promoter activity by methylation: possible involvement of protein mediators
.
Proc. Nat. Acad. Sci. USA
88
,
6515
6518
.
Lewis
,
J. D.
and
Bird
,
A. P.
(
1991
).
DNA methylation and chromatin structure
.
FEBSLett
.
285
,
155
159
.
Lewis
,
J. D.
,
Meehan
,
R. R.
,
Henzel
,
W. J.
,
Maurer-Fogy
,
I.
,
Jeppesen
,
P.
,
Klein
,
F.
and
Bird
,
A.
(
1992
).
Purification, sequence and cellular localization of a novel chromosomal protein that binds to methylated DNA
.
Cell
69
,
905
914
.
Li
,
E.
,
Bestor
,
T. H.
and
Jaenisch
,
R.
(
1992
).
Targeted mutation of the DNA methyltransferase gene results in embryonic lethality
.
Cell
61
,
915926
.
Lock
,
L. F.
,
Takagi
,
N.
and
Martin
,
G. R.
(
1987
).
Methylation of the HPRT gene on the inactive X chromosome occurs after chromosome inactivation
.
Cell
48
,
39
46
.
Manuelides
,
L.
(
1981
).
Consensus sequence of mouse satellite DNA indicates it is derived from tandem 116 base pair repeats
.
FEBS Lett
.
129
,
25
28
.
Meehan
,
R. R.
,
Lewis
,
J. D.
,
McKay
,
S.
,
Kleiner
,
E. L.
and
Bird
,
A. P.
(
1989
).
Identification of a mammalian protein that binds specifically to DNA containing methylated CpGs
.
Cell
58
,
499
507
.
Meehan
,
R. R.
,
Lewis
,
J. D.
and
Bird
,
A. P.
(
1992
).
Characterization of MeCP2, a vertebrate DNA binding protein with affinity for methylated DNA
.
Nucl. Acids Res
.
20
,
5085
5092
.
Mellon
,
L
,
Bohr
,
V. A.
,
Smith
,
L. A.
and
Hanawalt
,
P. C.
(
1986
).
Preferential DNA repair of an active gene in human cells
.
Proc. Nat. Acad. Sci. USA
83
,
8878
8882
.
Miller
,
L. L.
,
Schnoedl
,
W
,,
Allen
,
J
, and
Erlanger
,
B. F.
(
1974
).
5-methylcytosine localized in mammalian constitutive heterochromatin
.
Nature
251
,
636
637
.
Murray
,
E. J.
and
Grosveld
,
F.
(
1987
).
Site specific demethylation in the promoter of human gamma globin gene does not alleviate methylation mediated repression
.
EMBO J
.
6
,
2329
2335
.
Pfeifer
,
G. P.
,
Tanguay
,
P. L.
Steigerwald
,
S. D.
and
Riggs
,
A. D.
(
1990a
).
In vivo footprint and methylation analysis by PCR-aided genomic sequencing: comparison of active and inactive X-chromosomal DNA at the CpG island and promoter of PGK-1
.
Genes Dev
4
,
1277
1287
.
Pfeifer
,
G. P.
,
Steigerwald
,
B. S. D.
,
Hansen
,
R. S.
,
Gartler
,
S. M.
and
Riggs
,
A. D.
(
1990b
).
Polymerase chain reaction aided genomic sequencing of an X-chromosome linked CpG island: methylation patterns suggest clonal inheritance, CpG site autonomy and an explanation of activity state stability
.
Proc. Nat. Acad. Sci. USA
87
,
8252
8256
.
Pfeifer
,
G. P.
and
Riggs
,
A. D.
(
1991
).
Chromatin differences between active and inactive X chromosomes revealed by genomic footprinting of permeabilized cells using DNasel and ligation mediated PCR
.
Genes Dev
5
,
1102
1113
.
Riggs
,
A. D.
and
Pfeifer
,
G. P.
(
1992
).
X-chromosome inactivation and cell memory
.
Trends Genet
.
8
,
169
174
.
Selig
,
S.
,
Ariel
,
M.
,
Goitein
,
R.
,
Marcus
,
M.
and
Cedar
,
H.
(
1988
).
Regulation of mouse satellite DNA replication time
.
EMBO J
.
7
,
419426
.
Shen
,
E. S.
and
Whitlock
,
J. P.
Jr
. (
1989
).
The potential role of DNA methylation in the response to 2,3,7,8-Tetrachlorodibenzo-p-dioxin
.
J. Biol. Chem
.
264
,
17754
17758
.
Singer-Sam
,
J.
,
Grant
,
M.
,
LeBon
,
J. M.
,
Okuyama
,
K.
,
Chapman
,
V.
,
Monk
,
M.
and
Riggs
,
A. D.
(
1990
).
Use of 77/MlI-polymerase chain reaction assay to study DNA methylation in the Pgk-1 CpG island of mouse embryos at the time of X-chromosome inactivation
.
Mol. Cell Biol
.
10
,
4987
4989
.
Solage
,
H.
and
Cedar
,
H.
(
1978
).
Organization of 5-methylcytosine in chromosomal DNA
.
Biochemistry
17
,
2934
2938
.
Wang
,
R.
,
Zhang
,
X. Y.
,
Khan
,
R.
,
Zhou
,
Y.
,
Huang
,
L. H.
and
Ehrlich
,
M.
(
1986
).
Methylated DNA-binding protein from human placenta recognizes specific methylated sites on several prokaryotic DNAs
.
Nucl. Acids Res
.
14
,
9843
9859
.
Watt
,
F.
and
Molley
,
P. L.
(
1988
).
Cytosine methylation prevents binding to DNA of a HeLa cell transcription factor required for optimal expression of the adenovirus major late promoter
.
Genes Dev
.
2
,
11361143
.
Wolffe
,
A. P.
(
1990
).
New approaches to chromatin function
.
New Biologist
2
,
211
218
.
Zhang
,
D.
,
Ehrlich
,
K. C.
,
Supakar
,
P. C.
and
Ehrlich
,
M.
(
1989
).
A plant DNA-binding protein that recognizes 5-methylcytosine residues
.
Mol. Cell. Biol
.
9
,
1351
1356
.