 |
Introduction |
The character of a cell is defined by its constituent proteins,
which are the result of specific patterns of gene expression. Crucial
determinants of gene expression patterns are
DNA-binding transcription factors that choose genes for transcriptional
activation or repression by recognizing the sequence of DNA bases in
their promoter regions. Interaction of these factors with their cognate sequences triggers a chain of events, often involving changes in the
structure of chromatin, that leads to the assembly of an active
transcription complex (e.g., Cosma et al. 1999
). But the types of
transcription factors present in a cell are not alone sufficient to
define its spectrum of gene activity, as the transcriptional potential
of a genome can become restricted in a stable manner during
development. The constraints imposed by developmental history probably
account for the very low efficiency of cloning animals from the nuclei
of differentiated cells (Rideout et al. 2001; Wakayama and Yanagimachi
2001). A "transcription factors only" model would predict that the
gene expression pattern of a differentiated nucleus would be completely
reversible upon exposure to a new spectrum of factors. Although many
aspects of expression can be reprogrammed in this way (Gurdon 1999),
some marks of differentiation are evidently so stable that immersion in
an alien cytoplasm cannot erase the memory.
The genomic sequence of a differentiated cell is thought to be
identical in most cases to that of the zygote from which it is
descended (mammalian B and T cells being an obvious exception). This
means that the marks of developmental history are unlikely to be caused
by widespread somatic mutation. Processes less irrevocable than
mutation fall under the umbrella term "epigenetic" mechanisms. A
current definition of epigenetics is: "The study of mitotically and/or meiotically heritable changes in gene function that cannot be
explained by changes in DNA sequence" (Russo et al. 1996). There are
two epigenetic systems that affect animal development and fulfill the
criterion of heritability: DNA methylation and the Polycomb-trithorax
group (Pc-G/trx) protein complexes. (Histone modification has some
attributes of an epigenetic process, but the issue of heritability has
yet to be resolved.) This review concerns DNA methylation, focusing on
the generation, inheritance, and biological significance of genomic
methylation patterns in the development of mammals. Data will be
discussed favoring the notion that DNA methylation may only affect
genes that are already silenced by other mechanisms in the embryo.
Embryonic transcription, on the other hand, may cause the exclusion of
the DNA methylation machinery. The heritability of methylation states
and the secondary nature of the decision to invite or exclude
methylation support the idea that DNA methylation is adapted for a
specific cellular memory function in development. Indeed, the
possibility will be discussed that DNA methylation and Pc-G/trx may
represent alternative systems of epigenetic memory that have been
interchanged over evolutionary time. Animal DNA methylation has been
the subject of several recent reviews (Bird and Wolffe 1999; Bestor
2000; Hsieh 2000; Costello and Plass 2001; Jones and Takai 2001). For recent reviews of plant and fungal DNA methylation, see Finnegan et al.
(2000), Martienssen and Colot (2001), and Matzke et al. (2001).
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Variable patterns of DNA methylation in animals |
A prerequisite for understanding the function of DNA methylation is
knowledge of its distribution in the genome. In animals, the spectrum
of methylation levels and patterns is very broad. At the low extreme is
the nematode worm Caenorhabditis elegans, whose genome lacks
detectable m5C and does not encode a conventional DNA
methyltransferase. Another invertebrate, the insect Drosophila
melanogaster, long thought to be devoid of methylation, has a DNA
methyltransferase-like gene (Hung et al. 1999; Tweedie et al. 1999) and
is reported to contain very low m5C levels (Gowher et al.
2000; Lyko et al. 2000), although mostly in the CpT dinucleotide rather
than in CpG, which is the major target for methylation in animals. Most
other invertebrate genomes have moderately high levels of methyl-CpG
concentrated in large domains of methylated DNA separated by equivalent
domains of unmethylated DNA (Bird et al. 1979; Tweedie et al. 1997).
This mosaic methylation pattern has been confirmed at higher resolution
in the sea squirt, Ciona intestinalis (Simmen et al. 1999). At
the opposite extreme from C. elegans are the vertebrate
genomes, which have the highest levels of m5C found in the
animal kingdom. Vertebrate methylation is dispersed over much of the
genome, a pattern referred to as global methylation. The variety of
animal DNA methylation patterns highlights the possibility that
different distributions reflect different functions for the DNA
methylation system (Colot and Rossignol 1999).
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Mammalian DNA methylation patterns vary in time and space |
In human somatic cells, m5C accounts for ~1% of total
DNA bases and therefore affects 70%-80% of all CpG dinucleotides in
the genome (Ehrlich 1982). This average pattern conceals intriguing temporal and spatial variation. During a discrete phase of early mouse
development, methylation levels in the mouse decline sharply to ~30%
of the typical somatic level (Monk et al. 1987; Kafri et al. 1992). De
novo methylation restores normal levels by the time of implantation. A
much more limited drop in methylation occurs in the frog Xenopus
laevis (Stancheva and Meehan 2000), and no drop is seen in the
zebrafish, Danio rerio (MacLeod et al. 1999). Even within
vertebrates, therefore, interspecies variation is seen that could
reflect differences in the precise role played by methylation in these
organisms. For mice and probably other mammals, however, the cycle of
early embryonic demethylation followed by de novo methylation is
critical in determining somatic DNA methylation patterns. A genome-wide
reduction in methylation is also seen in primordial germ cells (Tada et
al. 1997; Reik et al. 2001) during the proliferative oogonial and
spermatogonial stages.
The most striking feature of vertebrate DNA methylation patterns is the
presence of CpG islands, that is, unmethylated GC-rich regions that
possess high relative densities of CpG and are positioned at the 5'
ends of many human genes (for review, see Bird 1987). Computational
analysis of the human genome sequence predicts 29,000 CpG islands
(Lander et al. 2001; Venter et al. 2001). Earlier studies estimated
that ~60% of human genes are associated with CpG islands, of which
the great majority are unmethylated at all stages of development and in
all tissue types (Antequera and Bird 1993). Because many CpG islands
are located at genes that have a tissue-restricted expression pattern,
it follows that CpG islands can remain methylation-free even when their
associated gene is silent. For example, the tissue-specifically
expressed human 
-globin (Bird et al. 1987) and 2(1) collagen
(McKeon et al. 1982) genes have CpG islands that remain unmethylated in
all tested tissues, regardless of expression.
A small but significant proportion of all CpG islands become methylated
during development, and when this happens the associated promoter is
stably silent. Developmentally programmed CpG-island methylation of
this kind is involved in genomic imprinting and X chromosome
inactivation (see below). The de novo methylation events occur in germ
cells or the early embryo (Jaenisch et al. 1982), suggesting that de
novo methylation is particularly active at these stages. There is
evidence, however, that de novo methylation can also occur in adult
somatic cells. A significant fraction of all human CpG islands are
prone to progressive methylation in certain tissues during aging (for
review, see Issa 2000), or in abnormal cells such as cancers (for
review, see Baylin and Herman 2000) and permanent cell lines (Harris
1982; Antequera et al. 1990; Jones et al. 1990). The rate of
accumulation of methylated CpGs in somatic cells appears to be very
slow. For example, de novo methylation of a provirus in murine
erythroleukemia cells took many weeks to complete (Lorincz et al.
2000). Similarly, the recovery of global DNA methylation levels
following chronic treatment of mouse cells with the DNA methylation
inhibitor 5-azacytidine required months (Flatau et al. 1984).
How do patterns of methylated and unmethylated mammalian DNA arise in
development and how are they maintained? Why are CpG islands usually,
but not always, methylation-free? What causes methylation of bulk
non-CpG-island DNA? These burning questions cannot be answered
definitively at present, but there are distinct hypotheses that have
been addressed experimentally. The available data will be conveniently
considered in three parts: (1) mechanisms for maintaining DNA
methylation patterns; (2) mechanisms and consequences of methylation
gain; and (3) mechanisms and consequences of methylation loss.
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Maintenance methylation not so simple |
Maintenance methylation describes the processes that reproduce DNA
methylation patterns between cell generations. The simplest conceivable
mechanism for maintenance depends on semiconservative copying of the
parental-strand methylation pattern onto the progeny DNA strand
(Holliday and Pugh 1975; Riggs 1975). In keeping with the model, the
methylating enzyme DNMT1 prefers to methylate those new CpGs whose
partners on the parental strand already carry a methyl group (Bestor
1992; Pradhan et al. 1999). Thus a pattern of methylated and
nonmethylated CpGs along a DNA strand tends to be copied, and this
provides a way of passing epigenetic information between cell
generations. The idea that mammalian DNA methylation patterns are
established in early development by de novo methyltransferases DNMT3A
and DNMT3B (Okano et al. 1998a, 1999; Hsieh 1999b) and then copied to
somatic cells by the maintenance DNA methyltransferase DNMT1 is elegant
and simple, but, as discussed below, may not fully explain persistence
of methylation patterns during cell proliferation.
Experiments that first showed replication of methylation patterns on
artificially methylated DNA also revealed a relatively low fidelity for
the process (Pollack et al. 1980; Wigler et al. 1981). After many cell
generations, methylation of the introduced DNA was retained, but at a
much lower level than in the starting plasmid. The failure of
maintenance was estimated to occur with a frequency of ~5% per CpG
site per cell division. Quantitative studies of an endogenous CpG site
broadly agreed with this figure (Riggs et al. 1998). Cell clones in
which this site was initially unmethylated acquired methylation and
clones where it was methylated lost methylation. The rate of change was
estimated at ~4% per cell generation. Error rates of this magnitude
mean that a detailed methylation pattern would eventually become
indistinct as cells proliferate. Indeed, dynamic changes in detailed
methylation patterns have been observed in monoclonal lyomyomas (Silva
et al. 1993) and at the methylated FMR1 gene (Stöger et al.
1997). These studies established that clonal populations of cells do
not have the homogeneous methylation patterns that would be predicted
by the replication model of maintenance methylation. Not only does DNA
methyltransferase fail to complete half-methylated sites at a
significant rate, but also significant de novo methylation occurs at
unmethylated sites.
At first sight, these findings appear to undermine the concept of
maintenance methylation, but this does not follow. Although detailed
methylation patterns may not be maintained at the level of a single CpG
nucleotide, the methylation status of DNA domains appears to be
faithfully propagated during development (Pfeifer et al. 1990). CpG
islands, for example, keep their overall unmethylated state (or
methylated state) extremely stably through multiple cell generations.
DNMT1 is partly responsible for this stability, but there is likely to
be another as yet unknown component to the maintenance process.
Dramatic evidence for this alternative maintenance mechanism comes from
the finding that CpG-island methylation is stably maintained even in
the apparent absence of the only known maintenance DNA
methyltransferase, DNMT1 (Rhee et al. 2000). A similar phenomenon may
account for the maintenance of allele-specific DNA methylation imprints
under conditions where the concentration of DNMT1 is severely limiting
(Jaenisch 1997).
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De novo DNA methylation by default? |
The origin of DNA methylation patterns is a long-standing mystery in
the field. The de novo methyltransferases DNMT3A and DNMT3B (Okano et
al. 1998a, 1999) are highly expressed in early embryonic cells, and it
is at this stage that most programmed de novo methylation events occur.
What determines which regions of the genome should be methylated? An
extreme possibility is that de novo DNA methylation in early mammalian
development is an indiscriminate process potentially affecting all
CpGs. Compatible with the default model is the apparent absence of
intrinsically unmethylatable DNA sequences in mammalian genomes. Even
CpG islands, most of which are unmethylated at all times in normal
cells, can acquire methylation under special developmental
circumstances or in abnormal cells (permanent cell lines or cancer
cells). It is clear, however, that not all regions of the genome are
equally accessible to DNA methyltransferases. DNMT3B in particular is known to be required for de novo methylation of specific genomic regions, as mice or human patients with DNMT3B mutations are deficient in methylation of pericentromeric repetitive DNA sequences and at CpG
islands on the inactive X chromosome (Miniou et al. 1994; Okano et al.
1998b; Hansen et al. 2000; Kondo et al. 2000). DNMT3B may therefore be
adapted to methylate regions of silent chromatin.
Evidence that accessory factors are also needed to ensure appropriate
methylation came initially from plants, where the SNF2-like protein
DDM1 was shown to be essential for full methylation of the
Arabidopsis thaliana genome (Jeddeloh et al. 1999). An
equivalent dependence is seen in animals, as mutations in human
ATRX (Gibbons et al. 2000) and mouse Lsh2 genes
(Dennis et al. 2001), both of which encode relatives of the
chromatin-remodeling protein SNF2, have significant effects on global
DNA methylation patterns. Loss of LSH2 protein, in particular, matches
the phenotype of the DDM1 mutation in Arabidopsis,
for both mutants lose methylation of highly repetitive DNA sequences,
but retain some methylation elsewhere in the genome. Perhaps efficient
global methylation of the genome requires perturbation of chromatin
structure by these chromatin-remodeling proteins so that DNMTs can gain
access to the DNA. Collaboration between DNMTs and factors that allow
them access to specialized chromosomal regions may be particularly
important in regions that are heterochromatic and inaccessible.
Although the net result of these processes is apparently global genomic
methylation, the evidence for selectivity means that the word
"default" is probably not appropriate.
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Targeting de novo methylation to preferred DNA sequences |
Another hypothesis to explain global methylation is that the DNA
methylation machinery is preferentially attracted by certain DNA
sequences in the mammalian genome (Turker 1999). The presence of high
levels of methylation in DNA outside such a DNA methylation center
could be explained by spreading into the surrounding DNA. Barriers to
spreading would lead to the formation of CpG islands. A hypothetical
trigger for DNA methylation is DNA sequence repetition, which can
promote de novo methylation in filamentous fungi and plants under
certain circumstances (Selker 1999; Martienssen and Colot 2001). The
most suggestive evidence in mammals concerns manipulation of transgene
copy number at a single locus in the mouse genome using cre-lox
technology (Garrick et al. 1998). High levels of transgene repetition
were found to cause significant transgene silencing and concomitant
methylation. The efficiency of expression increased as copy number was
reduced at the locus, and the level of methylation decreased. Whether
repetition caused methylation directly, or indirectly as a consequence of some
other event (e.g., transcriptional silencing; see below), is not known.
The clearest definition of a DNA methylation center comes from the
fungus Neurospora, where short TpA-rich segments of DNA were
found to induce methylation (Miao et al. 2000). Identification of a
mammalian DNA methylation center located upstream of the mouse adenine
phophoribosyltransferase (APRT) gene has been reported (Mummaneni et
al. 1993; Yates et al. 1999). The region contains B1 repetitive
elements and attracts high levels of de novo methylation upon
transfection into embryonic cells, although the effect is relative,
because many DNA sequences are subject to de novo methylation in these
cells. The APRT methylation center becomes methylated in
DNMT1-deficient ES cells, supporting the idea that it corresponds to a
region that is a favorable substrate for de novo methylation (Yates et
al. 1999).
Because the evidence suggests that replication of methylation patterns
by DNMT1 is only partly responsible for maintenance methylation (see
above), an attractive possibility is that the features of a DNA domain
that help maintain its methylated status are the same features that
promote its de novo methylation. Imprinting boxes, for example, whose
differential methylation is associated with genomic imprinting
(Tremblay et al. 1997; Birger et al. 1999; Shemer et al. 2000), tend to
retain their methylation levels tenaciously even when the amount of the
maintenance enzyme DNMT1 is reduced (Beard et al. 1995). The de novo
methylases DNMT3A and DNMT3B (Okano et al. 1998a, 1999) may be
attracted disproportionately to these sequences, and this attraction
may also underlie the decision to methylate the box in the first place.
In other words, de novo methylation may not occur once at a discrete
and perhaps rather inaccessible stage of germ-cell development, but may
happen repeatedly (assisted by DNMT1) as embryonic cells divide.
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Unusual DNA structures and RNAi as triggers for de novo methylation |
Studies of purified DNMT1 revealed that the enzyme prefers to
methylate unusual DNA structures in vitro (Smith et al. 1991; Laayoun
and Smith 1995). This led to the idea that such structures might be
generated during recombination between repetitive elements or during
transposition events and directly trigger de novo methylation (Bestor
and Tycko 1996). Subsequent evidence, however, does not support a role
for DNMT1 in de novo methylation in vivo (Lyko et al. 1999; Howell et
al. 2001), and therefore the biological significance of its
predilection for deformed DNA is uncertain. There is evidence for
transfer of methylation from one copy of a sequence to a second
previously unmethylated copy of the same sequence in the fungus
Ascobolus (Colot et al. 1996). The process might use
mechanisms involved in homologous DNA recombination and may therefore
involve deformation of DNA. How identical sequences sense one another
and transfer epigenetic information remains unknown, however.
Exciting recent developments in the DNA methylation field have arisen
through molecular genetic studies of posttranscriptional gene silencing
in plants. Double-stranded RNA directs the destruction of transcripts
containing the same sequence, but there is compelling evidence that it
can also direct de novo methylation of homologous genomic DNA
(Wassenegger et al. 1994; Bender 2001; Matzke et al. 2001).
Posttranscriptional gene silencing by double-stranded RNA is probably
an ancient genome defence system because it occurs in fungi, plants,
and animals; but DNA methylation is not an obligatory accompaniment, as
silencing is efficient in C. elegans in the complete absence
of genomic m5C. Even in the fungus Neurospora, where
transgene arrays are often methylated, DNA methylation is not required
for posttranscriptional gene silencing (or quelling; Cogoni et al.
1996). There are also specific features of RNA-directed DNA methylation
that may not occur in animals; notably the occurrence of methylation at
multiple non-CpG cytosines in an affected DNA sequence tract. Although there is evidence for non-CpG methylation in ES cells, most probably owing to DNMT3A, which strongly methylates CpA as well as CpG (Ramsahoye et al. 2000; Gowher and Jeltsch 2001), non-CpG methylation is barely detectable in adult cells (Ramsahoye et al. 2000). Plants have a CpG methylation system, but it does not appear to be essential for RNA-directed gene silencing (for reviews, see Wassenegger et al.
1994; Bender 2001; Matzke et al. 2001). Optimism that RNA-directed de
novo methylation will also apply in mammals is tempered by this
sequence disparity, and by the absence so far of a clear demonstration
that mammalian double-stranded RNA leads to DNA methylation-mediated
gene silencing.
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Transcriptionally silent chromatin as a de novo methylation target |
Several lines of evidence suggest that DNA methylation does not
intervene to silence active promoters, but affects genes that are
already silent. It was reported many years ago that retroviral transcription is repressed in embryonic cells at ~2 d after
infection, whereas de novo methylation is delayed until ~15 d
(Gautsch and Wilson 1983; Niwa et al. 1983). De novo methylation of
proviral sequences in embryo cells depends on DNMT3A and DNMT3B (Okano et al. 1999), but initial retroviral shutdown occurs as usual even when
both these de novo methyltransferases are absent (Pannell et al. 2000).
Clearly, de novo methylation is not required for silencing in the first
instance, reinforcing the view that methylation is a secondary event.
Methylation of genes that are already silent is also observed during X
chromosome inactivation in the mammalian embryo. Kinetic studies showed
that the phosphoglycerate kinase gene is silent on the mammalian
inactive X chromosome before methylation of its CpG-island promoters
occurs (Lock et al. 1987). Subsequent studies of the mouse, in which
the process is best understood, have established that expression of a
noncoding chromosomal RNA from the Xist gene on the inactive X
chromosome triggers the inactivation process in cis.
Specifically, activation of the Xist gene and onset of its
late replication precede CpG-island methylation by several days
(Keohane et al. 1996; Wutz and Jaenisch 2000). In other words, methylation affects the X chromosome on which genes are already shut
down by other mechanisms. Is transcriptional inertia during embryogenesis the trigger for de novo methylation? Studies of the
origin of methylation-free CpG islands offer some support for this
idea. The coincidence between CpG islands and promoters is striking
(Bird 1987), and footprinting shows that the 5' extremity of CpG
islands often corresponds to the region occupied by transcription factors in vivo (Cuadrado et al. 2001). Even when CpG islands are
identified in unusual locations, they have turned out to correspond to
promoters. For example, a CpG island located in intron 2 of the
Igf2r gene is an active promoter (Wutz et al. 1997; Lyle et al. 2000), as is a CpG island that covers exon 2 of the class II major
histocompatibility gene (MacLeod et al. 1998). The potential importance
of promoter function in the genesis of CpG islands is highlighted by
studies in transgenic mice. CpG-island-containing transgenes normally
faithful reproduce their methylation-free character, but their immunity
to methylation is lost if promoter function is impaired (Brandeis et
al. 1994; MacLeod et al. 1994). Similarly, viral DNA integrated into ES
cell genomes by homologous recombination becomes methylated when the
promoter is weakened by absence of an enhancer, but excludes
methylation when an enhancer is present (Hertz et al. 1999). A
parsimonious interpretation of the results is that failure to
transcribe invites de novo methylation (see Fig. 2 below), although
other potential explanations (Brandeis et al. 1994; Mummaneni et al.
1998) cannot be discounted.
The signal for this putative gene silence-related de novo methylation
is unknown, but the possibility that chromatin states inform the DNA
methylation machinery is attractive (Selker 1990). The acetylation
and methylation state of nucleosomal histones is tightly correlated
with transcriptional activity (Jenuwein and Allis 2001) and could be
read by the methylation machinery, leading it to either methylate or
fail to methylate a particular domain. Indeed, recent work on
Neurospora (Tamaru and Selker 2001) has shown an
intimate link between histone methylation and DNA methylation in that
fungus, as mutation of a histone methyltransferase that methylates Lys
9 of histone H3 abolished genomic methylation. In mammalian and yeast
systems, histone H3 Lys 9 methylation is associated with
transcriptionally repressed heterochromatin (Bannister et al. 2001;
Nakayama et al. 2001; Noma et al. 2001; Zhang and Reinberg 2001). If
the dependence of DNA methylation on prior histone methylation turns
out to be applicable to mammals, this would further strengthen the
argument that DNA methylation is targeted to genes that are already
silent. The nature of the molecular cues that trigger transfer of
methyl groups to unmethylated DNA should be illuminated by ongoing
studies of multiprotein complexes that contain DNA methyltransferases
(Fuks et al. 2000, 2001; Robertson et al. 2000; Bachman et al. 2001)
and the identification of genes that modify DNA methylation patterns
(Weng et al. 1995).
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Consequences of methylation gain: stable transcriptional silencing
of genes |
Why methylate genes that are already silent? A plausible answer is:
to silence them irrevocably. Methylation clearly contributes to the
stability of inactivation, because both X inactivation (Mohandas et al.
1981a; Graves 1982; Venolia et al. 1982) and retroviral silencing
(Stewart et al. 1982; Jaenisch et al. 1985) can be relieved by
treatment of somatic cells with demethylating agents. Individuals who
lack DNMT3B show reduced methylation of some CpG islands on the
inactive X chromosome and also silence X-linked genes imperfectly
(Miniou et al. 1994; Hansen et al. 2000). The implication that
irreversibility involves DNA methylation is supported by the frequent
reactivation of an X-linked transgene in mouse embryo cells and in
cultured somatic cells when DNMT1 is absent or inhibited (Sado et al.
2000). This view is sustained by differences in the stability of
inactivity states pre- and postmethylation. For example, X inactivation
caused by expression of an Xist transgene in embryonic stem
cells is initially reversed when the Xist gene is shut down,
but after 3 d, inactivation becomes irreversible and independent of
Xist (Wutz and Jaenisch 2000). Irreversibility may reflect the
arrival of promoter methylation.
In artificial systems, DNA methylation represses transcription in a
manner that depends on the location and density of the methyl-CpGs
relative to the promoter (Boyes and Bird 1992; Hsieh 1994; Kass et al.
1997a,b). But what genes are affected by DNA methylation-mediated gene
silencing? Early studies relied on the use of the demethylating drug
5-azacytidine (Jones and Taylor 1980), which was shown to activate
genes on the inactive X in rodent-human cell hybrids (Mohandas et al.
1981b; Graves 1982). More recently, mice and murine cell lines lacking
DNMT1 (Li et al. 1992) have clarified the effects of DNA methylation on
gene expression. In placental mammals, repression of X-linked genes follows expression of Xist, which sets in train the
inactivation process, culminating in widespread methylation of CpG
islands. The active X chromosome, on the other hand, must be protected from silencing, and this requires repression of Xist and again depends on methylation (Panning and Jaenisch 1996). An intact DNA
methylation system is also essential for genomic imprinting, because
deletion of Dnmt1 leads to disruption of the monoallelic expression of several imprinted genes (Li et al. 1993).
Both X inactivation and genomic imprinting involve silencing of one
allele only, leaving the other unaffected. An unusual set of genes that
are active in the germ line, most of which are X-linked, appears to use
methylation for complete silencing in somatic cells (De Smet et al.
1996, 1999). Several of the human and murine MAGE genes, for
example, have CpG-island promoters that are methylation-free in germ
cells, but are methylated in somatic cells of the adult. The genes were
discovered as novel antigens in tumors, where genomic methylation
levels are often low and MAGE-gene CpG islands are
undermethylated. MAGE expression can be induced by treating
nonexpressing cells with demethylating agents, supporting the idea that
methylation is an important component of the repression of these genes
in somatic cells.
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Transposable element silencing as a consequence of DNA methylation |
Another well-documented consequence of DNA methylation deficiency is
the activation of transposable element-derived promoters. Like much of
the mammalian genome, transposable element-related sequences are
heavily methylated and transcriptionally silent in somatic cells. Mouse
cells, for example, normally repress transcription of intracisternal A
particle (IAP) elements, which constitute a homogeneous and
transpositionally active family of elements. In embryos lacking DNMT1,
transcription of IAP elements is massively induced, arguing that
methylation is normally responsible for their repression (Walsh et al.
1998). Derepression of LINE (Woodcock et al. 1997) and SINE (Liu et al.
1994) promoters in the human genome also occurs when DNA methylation is
reduced. The most abundant SINE in the human genome is the Alu family,
which consists of several hundred thousand elements (Smit 1999). Only a
tiny minority of elements are capable of transposition (<1%), but
many carry functional promoters. Interestingly, these promoters can be
activated by stress of various kinds without altering DNA methylation
(Liu et al. 1995; Chu et al. 1998), although artificial demethylation also stimulates expression.
The biological significance of transposable-element repression is
uncertain. Two kinds of explanation have been discussed: either that
repression is required to prevent DNA damage due to unconstrained
transposition (the genome defence model; Yoder et al. 1997); or that
transcription of a large excess of irrelevant promoters would
constitute an unacceptable level of transcriptional noise that would
interfere with gene expression programs (Bird 1995). Increased
transcription of elements in human and mouse cells has not so far been
found to lead to increased transposition. In undermethylated cancer
cells that show transposon promoter activity, for example, mutations
caused by transposition are exceedingly rare. It has, however, been
claimed that rampant transposition and reduced methylation are linked
in the case of an interspecific hybrid marsupial (Waugh O'Neill et al.
1998). The hybrid wallaby concerned was found to contain an abundant
transposable element near the centromeres of one parental chromosome
set, but not the other. Surprisingly, this element could not be
detected in either of the presumed parent species, and was therefore
hypothesized to have been assembled from related fragments in the
parental genomes following fertilization. It was suggested that,
because of perceived depression of methylation levels in the hybrid
embryo, the emergent element became transpositionally hyperactive,
being targeted exclusively to one parental genome. The parents of the hybrid were not available to verify this unprecedented scenario.
Phylogenetic studies of genomic methylation patterns in animals have
not yet offered support for the genome defence model. Effective
silencing due to sequence repetition has been observed in
Drosophila and C. elegans, but it is associated with
the polycomb group of proteins or posttranscriptional gene silencing
(Birchler et al. 2000). The possibility that the low level of
m5C in Drosophila (Lyko et al. 2000) is relevant to
silencing has not yet been addressed. Studies of the sea squirt C. intestinalis, a chordate belonging to the same phylum as
vertebrates, but which does not exhibit global methylation of the
genome, revealed that genes were often present in domains of methylated
DNA, whereas transposable element families, some of which appeared to
be mobile in the population, were unmethylated (Simmen et al. 1999).
This is the opposite of expectation, but may represent a frequent
situation in invertebrates, which account for >95% of animal species
(Tweedie et al. 1997).
Colonization of the genome by transposable elements can only occur in
the germ-cell lineage because somatic transposition events leave no
heritable trace. Paradoxically, transposable elements are often
transcriptionally active and unmethylated in germ cells and totipotent
ES cells (for review, see Bird 1997). IAP elements, for example, become
unmethylated during the gonial proliferation phase, when primordial
germ cell number increases from ~75 to ~25,000 (Walsh et al. 1998).
The frequent absence of DNA methylation in germ cells, when
transposition can do long-term damage (Malik et al. 1999), contrasts
with its repressive presence in somatic cells, where transposition
would be an evolutionary dead end. It is too early to discount the
possibility that transposon promoters, most of which belong to
degenerate elements that are incapable of transposition, must be
silenced to suppress transcriptional noise.
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Mechanisms of DNA methylation-mediated transcriptional repression |
Why does DNA methylation interfere with transcription? Two modes of
repression can be envisaged, and it is likely that both are
biologically relevant. The first mode involves direct interference of
the methyl group in binding of a protein to its cognate DNA sequence
(Fig. 1). Many factors are known to bind
CpG-containing sequences, and some of these fail to bind when the CpG
is methylated. Strong evidence for involvement of this mechanism in
gene regulation comes from studies of the role of the CTCF protein in
imprinting at the H19/Igf2 locus in mice (Bell and
Felsenfeld 2000; Hark et al. 2000; Szabo et al. 2000; Holmgren et al.
2001). CTCF is associated with transcriptional domain boundaries (Bell
et al. 1999) and can insulate a promoter from the influence of remote enhancers. The maternally derived copy of the Igf2 gene is
silent owing to the binding of CTCF between its promoter and a
downstream enhancer. At the paternal locus, however, these CpG-rich
binding sites are methylated, preventing CTCF binding and thereby
allowing the downstream enhancer to activate Igf2 expression.
Although there is evidence that H19/Igf2 imprinting
involves additional processes (Ferguson-Smith and Surani 2001), the
role of CTCF represents one of the clearest examples of transcriptional
regulation by DNA methylation.
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Figure 1.
Mechanisms of transcriptional repression by DNA
methylation. A stretch of nucleosomal DNA is shown with all CpGs
methylated (red circles). Below the diagram is a transcription factor
that is unable to bind its recognition site when a methylated CpG is
within it. Many transcription factors are repelled by methylation,
including the boundary element protein CTCF (see text). Above the line
are protein complexes that can be attracted by methylation, including
the methyl-CpG-binding protein MeCP2 (plus the Sin3A histone
deacetylase complex), the MeCP1 complex comprising MBD2 plus the NuRD
corepressor complex, and the uncharacterized MBD1 and Kaiso complexes.
MeCP2 and MBD1 are chromosome-bound proteins, whereas MeCP1 may be less
tightly bound. Kaiso has not yet been shown to associate with
methylated sites in vivo.
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MBD1,
MBD2, MBD3, and MeCP2
-globin) are not detected (Daniels et al.
1997
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Introduction
Variable patterns of DNA...
not so...