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Development 1990 Supplement, 55-62
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55
Preferential X-chromosome inactivation, DNA methylation and imprinting
MARILYN MONK and MARK GRANT
MRC Mammalian Development Unit, Wolfson House, 4 Stephenson Way, London NW1 2HE, UK
Summary
Non-random X-chromosome inactivation in mammals
was one of the first observed examples of differential
expression dependent on the gamete of origin of the
genetic material. The paternally-inherited X chromosome is preferentially inactive in all cells of female
marsupials and in the extra-embryonic tissues of
developing female rodents. Some form of parental
imprinting during male and female gametogenesis must
provide a recognition signal that determines the nonrandomness of X-inactivation but its nature is thus far
unknown. In the mouse, the imprint distinguishing the X
chromosomes in the extra-embryonic tissues must be
erased early in development since X-inactivation is
random in the embryonic cells. Random X-chromosome
inactivation leads to cellular mosaicism in expression
and differential methylation of active and inactive Xlinked genes. Transgene imprinting shares many features with X-inactivation, including differential DNA
methylation.
In this paper we consider when methylation differ-
ences in early development affecting X-chromosome
activity and imprinting are established. There are
processes of methylation and demethylation occurring in
early development, as well as changes in the activity of
the DNA methylase itself. Methylation of a specific CpG
site associated with activity of the X-linked PGK-1 gene
has been studied. This site is already methylated on the
inactive X chromosome by 6.5 days' gestation, close to
the time of X-inactivation. However, differential methylation of this site is not the primary imprint marking the
paternal X chromosome for preferential inactivation in
the extra-embryonic membranes.
A consideration of factors influencing both Xchromosome inactivation and imprinting suggests that a
process of communication and comparison between nonidentical alleles might by the basis for the differential
modification and expression patterns observed.
Introduction
VandeBurgh et al. 1987) and in the extra-embryonic
tissues of rodents (Takagi and Sasaki, 1975; West et al.
1977; Harper et al. 1982). In this paper we will review
changing patterns of X-chromosome activity and
methylation in development and consider the role of
methylation in the initiation and specificity of inactivation. This specificity (or choice) of which X chromosome will be active or inactive is affected by a number
of factors. These influencing factors may also result in
differential allele expression in general, i.e. whether the
region concerned is on the X chromosome or is on an
autosome (see Monk, 19906). In order to clarify these
influences, we will start by defining what we mean by
gamete-specific imprinting in the context of differential
gene expression in general.
In all female mammals one or other of the two X
chromosomes is inactivated in all somatic cells (Lyon,
1961; reviewed in Grant and Chapman, 1988). This
means that females are eqivalent to males with respect
to X-linked gene dosage. Once established, X-inactivation in somatic cells is clonally inherited and
extremely stable. The inactive X chromosome may be
distinguished from the active X chromosome by a
number of criteria. The inactive X chromosome is: (1)
heterochromatic: in certain interphase cells it can be
seen as a dark staining body, the sex chromatin or Barr
body, in the nucleus; (2) late replicating in the cell cycle
(Takagi and Oshimura, 1973); (3) transcriptionally
inactive (except for a short region at one end which is
homologous to the Y chromosome, and where pairing
and recombination between the X and the Y occurs
(Burgoyne, 1982); (4) differentially methylated
(reviewed in Monk, 1986).
One of the first examples of imprinting observed in
mammals was the preferential inactivation of the
paternally-inherited X chromosome in female marsupials (Cooper etal. 1971; Richardson etal. 1971;
Key words: X chromosome, DNA, methylation,
imprinting.
Classes of imprinting
We define imprinting in a general sense as the
differential modification and/or expression in the
offspring of the homologous alleles or chromosome
regions inherited from each parent.
56
M. Monk and M. Grant
Gamete-specific imprinting
Gamete-specific imprinting applies to differential modification or expression of an allele depending on whether
its inheritance is via the sperm or the egg. The term
'imprinting' originally applied to gamete-specific
imprinting (Crouse, 1960), and is most commonly used
in this context.
Gamete-specific imprinting may be seen only in a
particular tissue. For example, in rodents the preferential inactivation of the paternal X chromosome,
dependent on gamete of origin, is only seen in the extraembryonic tissues. Similarly, certain imprinted transgenes showing differential methylation of transgene
sequences which depend on the gamete of origin of the
transgene may show tissue differences, e.g. the methylation imprint may not occur in certain tissues of the
developing conceptus (Reik et al. 1987). In insects, the
behaviour of the paternal set of chromosomes may be
different in germ line and soma (see Chandra and
Nanjundiah, this volume). In maize, only the endosperm shows imprinting (Kermicle and Alleman, this
volume).
Strain- and species-specific imprinting
Apart from classical imprinting dependent on the
gamete of origin of the X chromosome, strain
(Cattanach, 1975) and species (Zakien et al. 1987)
differences between the parents also influence the
randomness of X-chromosome inactivation. There may
be a bias towards inactivation of a particular X
chromosome depending upon its strain of origin in the
mouse cross (Cattanach, 1975; this volume). There are
also similar effects, depending on the combination of
mouse strains in a given cross, on the degree of
methylation and expression of a transgene in the
progeny (Sapienza et al. 1989; Allen et al. 1990; Reik
et al., this volume).
Species differences may have even more pronounced
effects: there may be preferential inactivation of a
particular X chromosome from one of the species in an
interspecific cross (e.g. in interspecific hybrid voles,
Zakian et al. 1987). However, this is not a rule of
interspecific crosses - mules and hybrids between foxes
do not show preferential X-inactivation (Serov et al.
I978a,b).
In these latter examples of strain- and species-specific
X chromosome imprinting, non-random X chromosome expression is not concerned with whether the X
chromosome comes from the mother or the father but
with a 'dominance" of one X over the other. Nevertheless, interspecific crosses can show parental source
effects, i.e. phenotypic differences depending on
gamete of origin. The classical example given is the
horse/donkey hybrid (Fig. 1; Chandley, 1989) although
it has not been clearly demonstrated that the differences are due to gamete of origin of either the horse or
donkey genome or to the different uterine environments when the horse or the donkey is the mother.
Allele-specific imprinting
Mention should also be made of allele-specific imprint-
Fig. 1. (A) shows a hinny (donkey mother and horse
father) and (B) shows a mule (horse mother and donkey
father). The hinny and the mule clearly demonstrate the
differential roles of paternal and maternal genomes in this
interspecific cross. These pictures were kindly provided by
Dr Ann Chandley, MRC, Edinburgh, Scotland and
Lorraine Travis. Hope Mount Farm. Alstonfield,
Derbyshire. England. See Chandley (1989).
ing. Silva and White (1988) demonstrated that in the
human a number of loci, distinguished by differences in
size of tandem repeat sequences (VNTR), may be
differently methylated. The methylation pattern
specific for a particular allele is heritable through
several generations. The allele appears to be irreversibly modified and imprinted. If such a modification
were to result in the silencing of expression of an allele,
it would appear to be a mutation. Such a heritable
modification of gene expression has been termed an
epimutation by Holliday (1987).
X-chromosome activity in development
X-chromosome inactivation occurs in early development. Therefore to understand the mechanisms which
X chromosome, methylation and imprinting
PGK in 6.5 day embryo tissues
Male
57
Fig. 2. The embryonic
lineage, epiblast (epi),
and two extra-embryonic
lineages, extrae.e.e.
embryonic ectoderm
(e.e.e.) and primary
PGK-2
endoderm (end), were
PGK-1 A dissected apart from 6.5
day embryos and extracts
end
PGK-1 B
electrophoresed and
stained from PGK-1
activity. The embryos
were derived from a
T epi end e.e.e.
PGK-IB mother and a
PGK-JA father. Male embryos inherit only PGK-IB on the X chromosome from the mother. Female embryos show
random inactivation in epiblast (both PGK-IA and PGK-IB are expressed) and non-random inactivation in the extraembryonic lineages (only PGK-IB of the maternal X chromosome is expressed). A, PGK-IA control; T, testis control from
a PGK-IA male, showing the position of the testis-speeific autosome-coded PGK-2. Data from Harper etal. (1982).
affect inactivation and imprinting in general, we must
investigate the early developing embryo. Changes in Xchromosome activity may be monitored by the use of
highly sensitive microassays for X-coded enzymes. For
example, the two X chromosome from either parent
can be marked with different alleles for the electrophoretically distinguishable forms of phosphoglycerate
kinase (PGK-1, EC 2.7.2.3). In this way we can see the
proportions of cells in a particular tissue with one or the
other X chromosome active. Fig. 2 shows evidence for
preferential inactivation of the paternal X chromosome
in the extra-embryonic tissues of 6.5 day embryos.
Using such highly sensitive assays, we and others
(Epstein et al. 1978; Kratzer and Gartler, 1978; Monk
and Harper, 1979; Kratzer and Chapman, 1981; Monk
and McLaren, 1981) have established the timing and
tissue specific patterns of X-activation, X-inactivation
and X-reactivation in female mouse embryo development. Fig. 3 summarises these changes. Two X chromosomes are active during oocyte growth and maturation,
whilst in the later stages of spermatogenesis the single X
chromosome is inactive, heterochromatic and sequestered away from the events of meiosis in the sex vesicle.
After fertilisation, the paternally-inherited X chromosome becomes active but then it is inactivated again in
the trophectoderm and in the primary endoderm as
Female
these extra-embryonic tissues are formed. Clearly,
there is some memory mechanism (imprint) distinguishing the paternal from the maternal X chromosome
when inactivation occurs in these tissues. However, in
the fetal precursor cells inactivation is random. Either
the imprint has been erased by this stage or it is not seen
in these cells. Reactivation of the inactive X chromosome occurs in the germ line around the time of
meiosis.
We are interested in the possible role of methylation
in these changes - activation, inactivation, reactivation
- and in particular whether methylation is associated
with the gamete-specific imprinting of the X chromosome observed in extra-embryonic membranes. We
know that differential methylation is correlated with the
active and inactive state of X chromosomes in somatic
tissues (reviewed in Monk, 1986) and that it is also
observed for imprinted transgenes (reviewed by Surani
etal. 1988).
Changes in X-chromosome activity and
methylation
Several lines of evidence suggest that DNA methylation
plays a role in the maintenance of dosage compensation
oocyte Xm
morula
trophectoderm
XmXp
foetus
XmXp orXmXp
primary
endoderm
Fig. 3. Diagrammatic representation
of the changes in X-chromosome
activity in female mouse embryo
development, m, maternal;
p, paternal; +, active; —, inactive.
Reproduced with permission from
Monk (1990a).
58
M. Monk and M. Grant
of X-linked genes. The observed methylation differences may be summarised as follows (reviewed in
Monk, 1986): (1) CpG islands at the 5' end of X-linked
genes are methylated on the inactive X chromosome. In
contrast, other CpG sequences in the body of X-linked
genes may be more methylated on the active X
chromosome (Wolf et al. 1984; Yen et al. 1984; Toniolo
etal. 1984; Keith etal. 1986; Lindsay etal. 1985); (2)
certain tissues are hypomethylated on the inactive X
chromosome suggesting that there are other modifying
mechanisms apart from methylation which maintain the
inactive state (Monk, 1986). For instance, CpG sites on
the inactive X chromosome are undermethylated in
extra-embryonic tissues in mouse and man and in
somatic tissues of marsupials. This undermethylation
may explain a greater instability of the inactive state in
these tissues (Migeon et al. 1985; Kaslow and Migeon,
1987); (3) Msp\ and HpaU restriction digest studies
only look at a proportion of CpG sites and, in addition,
it has been shown recently that not all of these sites
show methylation differences which are critically
correlated with active and inactive status (Yen et al.
1986; Hansen etal. 1988).
The important questions are when do these methylation changes occur in development and how do they
relate to changes in X-chromosome activity and
imprinting? Do the methylation differences imprint the
X chromosomes in the gametes? Do they precede or
follow inactivation or reactivation of the X-linked
genes? Similar questions could be asked about the role
of methylation in imprinted transgenes, i.e. are certain
transgenes differentially methylated in the sperm and
egg and does the observed pattern correspond to the
methylation imprinting seen in the offspring?
Methylation in early development
Very little is known about methylation in early
development at the time when methylation may be
playing a role in imprinting, i.e. determining preferential modification and/or expression of the X chromosome, transgenes and endogenous genes. Until recently
it has been impossible to look at CpG methylation of
specific single copy genes in development because of
the shortage of biological meterial. However, attempts
have been made to look at total methylation (Monk
et al. 1987) as well as methylation of repetitive and low
copy number sequences (Sanford et al. 1987; Monk
et al. 1987) at various stages of development. The data
is summarised in Table 1 and gives the following
picture.
The sperm genome is more methylated than the egg
genome, although both are globally undemethylated
compared to somatic tissue (Monk etal. 1987). Dispersed repetitive LI sequences are methylated in sperm
but undermethylated in fetal oocytes (Sanford et al.
1984). In the preimplantation embryo, there is a loss of
overall methylation by the blastocyst stage, occurring
between the 8-cell and blastocyst stages, but this could
be due to a lack of methylation in the trophectoderm
Table 1. Methylation in development
Overall
Specialised
and low
copy
Repetitive
Satellite
Sperm
Oocyte
Blastocyst
(ICM)
Post implantation
7.5d
Primordial germ
cells
Extra-embryonic
cells
cells (which are increasing in number) rather than an
absolute decrease (Monk etal. 1987). [n the rabbit
blastocyst the trophectoderm is also markedly undermethylated compared with the ICM (Manes and
Menzel, 1981).
In the preimplantation stages there is not much sign
of de novo methylation, either overall or for LI
sequences and satellite sequences (Sanford et al. 1987).
Detectable de novo methylation commences around the
time of implantation in the ICM of the late blastocyst
and increases throughout gastrulation. (Since it is
occurring independently in different lineages, it is
potentially occurring differently in these lineages and
may therefore be implicated in differential programming.) We, and others (Lock et al. 1987; Kaslow and
Migeon, 1987), proposed that methylation may serve to
lock in, or reinforce, patterns of potential gene
transcription established by other means.
It is clear that demethylation is also occurring in early
development. Dispersed repetitive sequences inherited
from the sperm are demethylated in primordial germ
cells of the progeny at some time before 11.5 days'
gestation (Monk etal. 1987). Sperm-derived LI sequences are also demethylated either before, or soon
after, the delineation of extra-embryonic tissues. Either
demethylation occurs in the preimplantation embryo
and the extra-embryonic and germ line lineages
segregate from demethylated precursor cells, or
demethylation may occur independently in the extraembryonic tissues and germ line after their segregation.
Methylation is certainly very low in the germ line; this
may be a prerequisite for reprogramming of the germ
line to developmental totipotency.
Although the data for methylation changes in
development is fairly meagre because of the tiny
amounts of tissue available, there are clearly processes
of both demethylation and de novo methylation
occurring in early development.
Are we able to obtain any clues as to what is
happening to DNA methylation by looking at the
activity of methylase, DNA(cytosine-5)methyltransferase, EC 2.1.1.37? Establishment and maintenance of
methylation patterns in development will depend upon
X chromosome, methylation and imprinting
the availability and activity of the methylase. This in
turn will depend on the amount of enzyme inherited in
the egg cytoplasm, the stability of the maternallyinherited enzyme and the time of onset of activity of the
embryonic gene coding for the methylase. In the egg the
low level of methylation observed suggests that
methylase activity might be low. The opposite turns out
to be true.
Using a highly sensitive microassay for the methylase
developed by Roger Adams (Department of Biochemistry, University of Glasgow, U.K.), we showed that the
level of maternally-inherited enzyme is extremely high
in the egg, and that the activity is stable for the first
three cleavage divisions but then is degraded between
the 8-cell and the blastocyst stage (M. Monk, R. Adams
and A. Rinaldi, unpublished data). There is a marked
absolute decrease (10-fold) of methylase activity per
embryo. On a per-cell basis, the fall in methylase
activity between the egg and the blastocyst is 1000-fold.
However, the activity in the egg is so high that, despite
the absolute decrease and dilution of enzyme, the final
level of enzyme activity at the blastocyst stage reaches a
similar level to that observed in cultured cells. The
reason for the high level of methylase activity in the egg
is open to speculation at this stage. One possibility is
that de novo methylation occurs at trie onset of
development of the fertilised egg although its efficiency
may be low.
Methylation of specific CpG sites in development
Is differential methylation the primary signal which
distinguishes the sperm and egg X chromosomes for
preferential paternal X inactivation? If not, when does
differential methylation occur - at the time of inactivation of the expression of the X chromosome or later?
When is the differential methylation erased - at the
time of X-reactivation or earlier, or later? Alternatively, do the X chromosomes in the female germ line
escape methylation modification? To attempt to answer
some of these questions we need to look at specific CpG
sites associated with X-linked genes.
The development of the polymerase chain reaction
(PCR) for amplification of specific DNA sequences
allows us to look at methylation in development more
closely. This procedure is so sensitive that we can now
look at changes in methylation of informative single
CpG sites whose methylation is correlated with activity
or inactivity of a gene when it is on the active or inactive
X chromosome (Singer-Sam etal. 1989; 1990a,/?).
Methylation of CpG of X-linked PGK
This work is being done in collaboration with Judy
Singer-Sam, Jeanne LeBon and Art Riggs (Beckman
Research Institute, LA, U.S.A.), and Kazuhika
Okuyama and Verne Chapman (Roswell Park, Buffalo,
U.S.A.) (Singer-Sam etal. 1990a). The methylation of
a specific CpG site in the 5' region of PGK is followed
by PCR amplification of a sequence containing that site,
before and after Hpall digestion. When the site
59
Table 2. Methylation of a critical Hpa// site
(designated HI) of PGK-1 during mouse
development*
Gestatior i
(dpc)
Tissue
%
methylationf
Female
Oocyte
Whole embryo
Epiblast
Extraembryonic
Ectoplacental cone
Embryonic
Chorion
Ectoplacental cone
Mesonephros
Kidney
Spleen
0.5
5.5
6.5
7.5
13.5
16.5
Adult
Male
all stages
Sperm, embryonic or adult
=£10
18±4
43±8
40±7
40±9
50±ll
41±6
57±6
58
52
51±3
=£10
•Data from Singer-Sam etal. (1990a).
PCR amplification after Hpall digestion
-thv ntinn —
PCR amplification control
becomes methylated on the inactive X in the female, at
some point in development, amplification will be
resistant to Hpall digestion. When the site is unmethylated, amplification is not possible after digestion with
Hpall. Thus, amplification can not occur after Hpall
digestion of male DNA (single active X chromosome)
or female DNA at a time when both X chromosomes
are active. However, when one X becomes methylated
at this site in the female, amplification will be 50 per
cent that observed in control undigested female DNA
(as half the X chromosomes will be methylated and
resistant to cutting by Hpall). The amplification
procedures must be quantitative and this is made
possible by incorporating an internal standard in the
amplification reaction (Singer-Sam et al. 1989;
1990a,6).
Eggs, sperm, preimplantation embryonic stages,
germ cells, and dissected tissues of ealy post-implantation stages are examined in this way. A small sample
of DNA from single embryo samples allows determination of embryo sex by PCR using Y-specific primers
(Nagamine etal. 1989; Mardon and Page, 1989).
Samples are then cut with either Hpall or Mspl and the
PGK region containing the diagnostic Hpall site is
amplified. The results (see Table 2 and Fig. 4), allow
the following conclusions: (1) since the CpG site is not
methylated in sperm and eggs, at least this site is not
serving as the primary imprint to distinguish the
maternal and paternal X chromosomes when inactivation of expression occurs in the extra-embryonic
tissues; (2) the CpG site on the inactive X is already
methylated by 6.5 days' gestation whether X-inactivation is random, (as it is in embryonic tissue) or
preferential paternal X-inactivation (in extra-embryonic tissues). We still do not know precisely if
methylation precedes or follows transcriptional silenc-
60
M. Monk and M. Grant
PCR PGK before ( - ) and after (+)
Hpa II digestion
Fig. 4. Ethidium bromide
stained gels of an amplified
$
emb
epc
($
emb
e.e.e.
epc
e.e.e.
fragment spanning the
£
^ w ..
Hpall site 7 in the 5' region
mm^m
mmm
mm
»
H«i»
*~ —
^
^
of the X-linked PGK gene
g
""
(E). A standard cloned
_ +
_ _^
_ j _
_
^
_
DNA is included in each
+
PCR reaction to allow
quantitation of experimental (E) compared to standard (S) amplification. The lanes show amplification of the sequence in
DNA from dissected regions of 6.5 day embryos, extra-embryonic ectoplacental cone (epc), extra-embryonic ectoderm
(eee) and embryonic (emb) tissues. DNA was extracted, digested with Hpall ( + ) or mock digested ( —). the standard
DNA added and the PCR reactions carried out as described in Singer-Sam et al. (1989; \990a,b).
ing, but we do know that the events are close in time.
Methylation of this informative site is certainly occurring about three days earlier than the timing proposed
by Lock et al. (1987) for HPRT.
differences. In interspecific crosses between fish, Whitt
et al. (1977) have observed that the more distantly
related the parents in the cross the greater the
imprinting effects (non-reciprocal lethality, developmental abnormalities, preferential allele expression).
Methylation of CpG sites in an imprinted transgene
We conclude with a working hypothesis incorporatCAT17 transgenic mice are now being studied by Wolf
ing all these seemingly diverse phenomena - the crossReik, Sarah Howlett and ourselves, to determine
talk hypothesis (see Monk, 1990) which proposes the
whether a methylation difference exists in eggs and following: (1) communication between homologous
sperm and, if not, to determine the timing of the regions of each chromosome pair allows a comparison
establishment of the female transmitted methylation
of degree of similarity or difference; (2) response to
imprint in development. Also, using the PCR ap- differences detected by modification of one or both (or
proach, we can ask whether the methylation imprint is
neither) homologous regions, resulting in 'repair' by
present in the germ line of the progeny, and, if so, when
recombination or gene conversion, duplication, or
it is erased. Preliminary results suggest that, like the X- differential expression (which may be partial or
inactivation picture, methylation is not the primary
absolute).
imprint in the sperm and egg, and in addition, the
Irregularities between homologous chromosome
methylation imprint does not appear to be present in
regions may be modifications themselves, e.g. due to
the primordial germ cells.
insertion (transgenes or repeat sequences), rearrangement or deletion, perhaps resulting in cis-positioneffect variegation; they could be accumulated epigenConcluding remarks
etic modifications transmitted through the germ line
(ancestral differences), or DNA sequence differences
We have considered the choice of which X chromosome
(e.g. in interspecific crosses). In the case of gameteis to be active and the role of methylation in the specific imprinting, the differences may be memories of
imprinting mechanism determining paternal X-inactidifferential packaging and programming of gene exvation in extra-embryonic tissues of the mouse. It is pression in the gametes. Just how homologous chromoclear that the primary imprint in the gametes is not
somes (X chromosomes or autosomes) communicate
methylation of a key site relating to PGK inactivation in
somatic tissues. The methylation difference for the and perceive these differences is open to speculation at
this stage.
PGK gene on the active and inactive X chromosomes is
due to de novo methylation in early development and it
We thank Judy Singer-Sam, Jeanne Le Bon and Art Riggs
is in place in both embryonic (random inactivation) and
for the invaluable contribution of the quantitative single site
extra-embryonic (paternal X-inactivation) lineages by methylation technique and their collaboration in the study of
6.5 days' gestation, close to the time of X-inactivation
CpG site methylation in the early embryo.
itself. Preliminary experiments looking at methylation
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