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Journal of Cell Science, Supplement 19, 37-39 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
37
Studies of DNA méthylation in animals
Adrian Bird, Peri Tate, Xinsheng Nan, Javier Campoy, Richard Meehan, Sally Cross, Susan Tweedie,
Jillian Charlton and Donald Macleod
Institute of Cell and Molecular Biology, University of Edinburgh, Kings Buildings, Mayfield Road, Edinburgh EH9 3JR, UK
SUMMARY
We have been studying the evolution and function of DNA
méthylation in vertebrate animals using three related
approaches. The first is to further characterise proteins
that bind to methylated DNA. Such proteins can be viewed
as ‘receptors’ of the methyl-CpG ‘ligand’ that mediate
downstream consequences of DNA modification. The
second approach involves CpG islands. These patches of
non-methylated DNA coincide with most gene promoters,
but their origin and functional significance have only
recently become the subject of intensive study. The third
approach is to trace the evolution of DNA méthylation.
Genomic méthylation patterns of vertebrates are strikingly
different from those of invertebrates. By studying méthyl
ation in animals that diverged from common ancestors
near to the invertebrate/vertebrate boundary, we will
assess the possibility that changes in DNA méthylation con
tributed causally to the evolution of the complex vertebrate
lineage.
Key words: DNA méthylation, methyl-CpG binding protein, CpG
island, genome evolution
INTRODUCTION
The predominant methylated sequence in all animals is the
self-complementary dinucleotide CpG. In vertebrates, most
CpGs in the genome are methylated at the 5 position on the
cytosine ring. Several biological consequences of this post
synthetic modification are known. Best understood is the
methylation-associated mutagenesis that has caused the
under-representation of CpG in the genome and is responsi
ble for over one third of the point mutations that give rise to
human genetic diseases (Bird, 1980; Jones et al., 1992). It is
difficult to see this as a selected advantage of DNA méthyl
ation. More likely it is an unavoidable price to be paid for
some other benefit of methyl-CpG. Strikingly, the inverte
brates (which account for well over 95% of animal species)
may not pay this price, as few, if any of their genes are methy
lated (see below).
The need for DNA méthylation during normal mammalian
development has been shown by disruption of the gene for
cytosine methyltransferase (MTase) in mice (Li et al., 1992).
Mutant embryos have greatly reduced levels of DNA méthyl
ation, and die in mid-gestation. In seeking an explanation for
this embryonic lethal phenotype, it is tempting to focus on
the well-known effects of méthylation on transcription. DNA
méthylation has long been correlated with transcriptional
repression. That it causes repression has been shown by intro
duction of artificially methylated constructs into cells
(Vardimin et al., 1982; Stein et al., 1982), and by the use of
drugs that inhibit the MTase (Jones and Taylor, 1980). A rea
sonable hypothesis is that embryos lacking the MTase die
because the methylation-mediated repression mechanism
fails.
METHYLATION-MEDIATED REPRESSION OF
TRANSCRIPTION
Several parameters determine the influence of methyl-CpG on
transcription. The parameters are: the location of methyl-CpGs
relative to the promoter (they should be close-by; Murray and
Grosveld, 1987); the local density of methyl-CpGs (the
strength of repression is proportional to density of méthylation;
Boyes and Bird, 1992); the strength of the promoter (weak
promoters are repressed by lower méthylation densities than
strong ones; Boyes and Bird, 1992); and the dependence of
promoter function on transcription factors that are sensitive to
methyl-CpG (reviewed by Tate and Bird, 1993). We have iden
tified a protein that interacts with methylated DNA according
to the density of methyl-CpGs, and have implicated this protein
as a mediator of transcriptional repression (Meehan et al.,
1989; Boyes and Bird, 1991). The activity is known as methvl-
CpG binding protein 1 or MeCPl. Considerable effort has been
expended on purification of M eCPl. As might be expected
from its size (800 kDa by gel fitration), MeCPl comprises
several polypeptide chains, and dissociates upon affinity chro
matography with methylated DNA, leading to loss of activity.
Our belief that MeCPl may be of central importance in under
standing the mechanism of methylation-mediated repression
has sustained us through the trials of its purification.
Studies of the second methyl-CpG binding protein, MeCP2,
have recently advanced significantly. Following preliminary
characterisation of MeCP2 and its gene (Lewis et al., 1992),
we set out to assess the biological significance of the protein.
We know that it is very abundant (over 106 molecules per cell)
and is a tightly bound component of mammalian chromo
somes. Recent studies of its localisation made use of a fusion
38 A. Bird and others
A.
f3geo
MeCP2 Bgal neo
Fig. 1. Localisation of an MeCP2-lac Z fusion protein to
heterochramatic foci in mouse cells. (A) Diagram of the fusion
protein between MeCP2 (dotted and solid shading, left) and the lac
Z-neomycin resistance fusion gene fi-geo (Friedrich and Soriano,
1991). The solid box within the MeCP2 moiety represents the
‘methyl-CpG binding domain’, which is essential for correct
localisation (X. Nan, unpublished results). (B and C) Staining of a
mouse L cell nucleus with Hoescht 33258 (B) and anti-Pgal
antibodies (C). The cell that contained this nucleus had been
transfected with a gene expressing the fusion protein diagrammed in
A. The heterochromatic foci that are intensely stained by Hoechst are
the primary targets of the MeCP2 fusion protein.
between the cDNA for MeCP2 and lacZ-neoR gene as
reporter. When mouse cells are transfected with this construct,
the resulting fusion protein localises preferentially to hete
rochromatin, thereby mimicking endogenous MeCP2 (Fig. 1).
Truncations and deletions of the MeCP2 moiety have estab
lished that the 80 amino acid methyl binding domain (MBD;
Nan et al., 1993) is both necessary and sufficient for locali
sation. More directly, it was found that the association of
MeCP2 with chromosomes is dependent on méthylation, as
cells lacking DNA méthylation cannot localise the protein
efficiently (X. Nan et al., unpublished results). Thus MeCP2
is a methyl-CpG binding protein in vivo as well as in vitro,
and as such may be a major mediator of the effects of DNA
méthylation on cells. If MeCP2 is a mediator of the effects of
méthylation, it should, like the MTase itself, be essential for
mouse development. By disrupting the X-linked gene in
embryonic stem (ES) cells, we have shown that it is indeed
essential (P. H. Tate et al., unpublished results). Chimaeric
embryos show developmental abnormalities whose severity
depends on the proportion of mutant cells. ES cells lacking
the MeCP2 gene grow normally, as do ES cells that lack the
MTase. Taken together, the results tell us that our interest in
MeCP2 is justified, but they do not reveal its biological
function. Future work will address this problem.
HISTONE H1 DOES NOT HAVE A HIGH AFFINITY
FOR METHYLATED DNA
Several laboratories have proposed that the linker histone H 1
binds preferentially to methylated DNA, and may therefore be
involved in methylation-mediated transcriptional repression
(Levine et al., 1993; Johnson et al., 1995). We have spent some
time testing this idea using a variety of assays (Campoy et al.,
unpublished results). In our hands, no preferential affinity of
HI for methylated DNA could be detected. This was true for
naked DNA and also for DNA that had been assembled into
poly-nucleosomal chromatin using a Xenopus oocyte extract.
Thus it is unlikely that histone HI is involved in mediating the
biological consequences of CpG methylation.
ORIGIN OF CpG ISLANDS
Islands of non-methylated CpG-rich DNA (CpG or HTF
islands) occur at the majority of human genes. They usually
cover the promoter and extend downstream into the gene for
1,000 base pairs (bp) on average (Bird, 1986). We and others
have used a transgenic mouse assay to find out which parts
of a CpG island determine its methylation-free status. In the
case of the adenine phosphoribosyltransferase gene, retention
of the island depended on the presence of sites for the tran
scription factor Spl (Macleod et al., 1994; Brandeis et al.,
1994). These sites, which are required for transcription of the
gene, are occupied by protein (presumably Spl) in vivo, and
surprisingly are located at the extreme 5' edge of the island
rather than in its centre (Fig. 2; Macleod et al., 1994). Two
questions are raised by these findings. Firstly, how do
occupied Spl sites at the edge of a CpG island keep 1,000 bp
1000 2000 3000
MOUSEAPRT I -
Fig. 2. Peripheral Spl sites are essential for maintaining the methylation-free status of the CpG island at the mouse adenine
phosphoribosyltranferase gene (Macleod et al., 1994). The CpG island is denoted by the bracket. Spl sites are shown by the three vertical bars.
Vertical crosslines on the map represent CpGs. Open boxes are exons. The two transcription starts are joined to an arrow below the diagram.
Studies of DNA méthylation in animals 39
downstream free of méthylation? Secondly, if transcription is
necessary for the creation of CpG islands, why do many
tissue-specific genes (e.g. human alpha-globin) have non-
methylated islands in tissues where they are not expressed
(Bird et al., 1987)? These questions will be important themes
for the future.
EVOLUTION OF DNA METHYLATION PATTERNS
It has been known for some time that the extensive genomic
DNA methylation seen in vertebrates is exceptional (Bird et
al., 1979; Bird and Taggart, 1980). Methylation of invertebrate
genomes is confined to a small fraction of the genome, and in
some cases (e.g. Drosophila melanogaster and Caenorhabdi-
tis elegans) may be absent altogether. Although the data are
incomplete, there is reason to believe that methylated DNA in
invertebrates comprises transposable elements and other poten
tially damaging DNA sequences that have been detected and
silenced by a mechanism involving methylation. No methy
lated gene has yet been reliably reported in an invertebrate, and
the primary function of DNA methylation in these organisms
may be to protect the genome by neutralising disruptive
elements. In vertebrates, on the other hand, the genome as a
whole is heavily methylated, and most genes are methylated to
some extent.
The transition from the predominantly non-methylated
genome of invertebrates to the predominantly methylated
genome of vertebrates appears to occur within the chordates
(A. Bird, S. Tweedie and J. Charlton, unpublished results).
Could this dramatic change have facilitated the evolutionary
development of the complex vertebrate lineage? It has been
suggested that the total number of genes in vertebrates is con
siderably higher than in invertebrates (50,000-100,000 versus
10,000-25,000; Bird, 1995). On the strength of this and other
data, it was proposed that the increased gene number (and
therefore complexity) of vertebrates is due to improved
methods of reducing transcriptional noise (that is, transcription
of non-genic DNA or of genes that are inappropriate for the
cell type concerned). The theory has the virtue that it makes
some testable predictions and that it might explain a major
macroevolutionary change. Its disadvantage is that it is rather
speculative, going some way beyond the available data.
Whether or not the noise reduction idea is relevant to the evo
lutionary origin of vertebrates, the transition in methylation
patterns deserves further study for the light that it may shed on
the biology of DNA methylation generally.
We thank Joan Davidson and Aileen Greig for technical assistance.
This work was funded by grants from The Wellcome Trust, Imperial
Cancer Research Fund and The Howard Hughes Medical Institute.
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