REPRODUCTION

REVIEW

Epigenetic reprogramming during early development in

mammals
Fátima Santos and Wendy Dean
Laboratory of Developmental Genetics and Imprinting, Developmental Genetics Programme,
The Babraham Institute, Cambridge CB2 4AT, UK
Correspondence should be addressed to Wendy Dean; Email: [email protected]

Abstract
Epigenetic modifications serve as an extension of the information content by which the underlying genetic code may be inter-
preted. These modifications mark genomic regions and act as heritable and stable instructions for the specification of chroma-
tin organisation and structure that dictate transcriptional states. In mammals, DNA methylation and the modification of
histones account for the major epigenetic alterations. Two cycles of DNA methylation reprogramming have been character-
ised. During germ cell development, epigenetic reprogramming of DNA methylation resets parent-of-origin based genomic
imprints and restores totipotency to gametes. On fertilisation, the second cycle is triggered resulting in an asymmetric differ-
ence between parental genomes. Further epigenetic asymmetry is evident in the establishment of the first two lineages at the
blastocyst stage. This differentiative event sets the epigenetic characteristics of the lineages as derivatives of the inner cell
mass (somatic) and trophectoderm (extra-embryonic). It is the erasure and subsequent re-tracing of the epigenetic checkpoints
that pose the most serious obstacles to somatic nuclear transfer. Elaboration of the mechanisms of these interactions will be
invaluable in our fundamental understanding of biological processes and in achieving substantial therapeutic advances.
Reproduction (2004) 127 643–651

Introduction information is faithfully and heritably reiterated according

to their early lineage origin.
This year marks the twentieth anniversary of the early work
The focus of this review is to recount the current state
from the laboratories of Surani and Solter (Surani et al.
of understanding of epigenetic modifications and some of
1984, McGrath & Solter 1984) that led to the biological
the activities thought to be involved in the establishment
principle of pronuclear non equivalency at fertilisation.
of epigenetic marks during preimplantation development
These studies set the scene for a mechanistic explanation
in mammals. The consequence of reprogramming of
of the differential and complimentary potentials of
somatic nuclei through investigations of epigenetic mark-
gametes. In brief, monoparental mouse embryos generated
ing in cloned embryos during this early phase of develop-
by micromanipulation manifest strict opposing pheno-
ment is highlighted.
types. Gynogenetic embryos (diploid maternal) character-
istically are growth restricted and fail to derive a functional
placenta. In contrast, androgenetic embryos (diploid
DNA methylation
paternal) while profoundly growth retarded, hyperprolife-
rate extra-embryonic tissues. The essence of this difference Epigenetic reprogramming can be defined as any meiotic
was the understanding that during maturation of gametes or mitotic alteration that does not result in a change in
there is marking of specific regions of the genome for later DNA sequence but will have a significant impact on the
differential expression (Surani et al. 1990). This parent-of- development of the organism (Russo et al. 1996). The
origin mark, or genomic imprint, has led to the realisation most prominent form of epigenetic alteration in mammals
that epigenetic differences play an absolutely critical role is the symmetric methylation of cytosine in the 50 position
in the re-establishment of totipotency of the zygote in CpG dinucleotides. DNA methylation can be thought
(McGrath & Solter 1984). Furthermore, these early differ- to have a twofold biological significance, gene regulation
ences are reflected later during the establishment of the and structural fidelity (Bestor 2000, Robertson & Wolffe
first lineages at the blastocyst stage. Thereafter, epigenetic 2000). DNA methylation has been firmly established as

q 2004 Society for Reproduction and Fertility DOI: 10.1530/rep.1.00221

ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org
644 F Santos and W Dean

playing a critical part in transcriptional repression. This somatic tissues that derive from the ICM are the highly
has been described in the context of development and methylated PGCs which arise , day 7 in the extra-
establishes the allele-specific expression status in many embryonic mesoderm of the developing embryo (Ginsburg
imprinted loci through differential DNA methylation of et al. 1990). Their migration via the allantois to the devel-
parental alleles (i.e. differential methylated regions, oping germinal ridges, where they will eventually differen-
DMRs) (Li et al. 1993). DNA methylation has been impli- tiate into mature gametes, completes the cycle of
cated in ‘genome defence’ associated with the silencing epigenetic reprogramming.
of parasitic retrotransposons (Yoder et al., 1997) and to a Despite the genome-wide decline in DNA methylation,
function in the maintenance of the structural integrity of certain sequences remain refractory to the general
chromosomes and prevention of chromosomal rearrange- demethylation during preimplantation development. Of
ments (Chen et al. 1998). special interest is the observation that DMRs of imprinted
genes and certain classes of repeat sequences are exempt
from these demethylation events (Reik & Walter 2001a).
DNA methylation reprogramming
Methylation analysis of bisulphite-treated DNA isolated
Mammalian development is characterised by bimodal from oocytes and sperm suggest that intracisternal A par-
DNA methylation reprogramming that occurs initially ticles maintain their methylation (Lane et al. 2003). This
during germ cell development and then during preimplan- interesting observation begs the question of the role of
tation (Fig. 1) (Reik & Walter 2001a). Primordial germ both active and passive demethylation during preimplan-
cells (PGCs) enter the developing germinal ridge and tation development. The significance of active demethyla-
begin differentiation and expansion. At this time, the tion has been at the centre of considerable debate in the
highly methylated PGCs undergo rapid genome-wide field of epigenetics. Two hypotheses have been tabled to
demethylation such that by day 12.5 most of the methyl- explain the need for active demethylation. Paternal-
ation is lost (Reik & Walter 2001b). This reprogramming specific loss of methylation may simply be required to
phase coincides with the erasure and resetting of parent- allow for generalised de-repression of paternal alleles to
of-origin specific marks that include DNA methylation of accommodate the minor transcriptional burst at the end of
imprinted DMRs associated with allele-specific gene the first cell cycle (Aoki et al. 1997). Perhaps more provo-
expression (Tucker et al. 1996). The exact timing of de cative is the implication of active demethylation in the
novo methylation has not been firmly established but is resolution of parent-offspring conflict that is exerted via
initiated in males at , 14.5 dpc (days post-coitum) and imprinted gene expression (Moore & Haig 1991). This pro-
thereafter in females such that the mature gametes of both posal embodies an evolutionary principle purporting that
sexes will eventually become highly methylated. each parent battles to maximise their own genetic fitness
The second phase of methylation reprogramming occurs by ensuring the transmission of their genetic legacy. Thus,
between fertilisation and formation of the blastocyst. On paternal interests seek to exert control over genes that
fertilisation a rapid paternal-specific asymmetric loss of maximise growth and survival of individual offspring
methylation is observed (Mayer et al. 2000, Dean et al. while maternal interests must try to moderate the growth
2003). This process takes place in the absence of tran- of single individuals for the benefit of the entire litter. This
scription or DNA replication and is termed active mediation might be possible through selective methylation
demethylation. Thereafter, there is a step-wise decline in and demethylation of growth regulatory genes. In this
methylation until the morula stage (Dean et al. 2001, San- regard many imprinted genes identified to date have
tos et al. 2002). This decline occurs as a result of the growth regulatory functions or influence postnatal suck-
absence of the primary DNA methyl transferase, Dnmt1, ling behaviour (Beechey et al. 2003).
during DNA replication (Bestor 2000). Thus, the newly A number of predictions arise from this hypothesis. In
replicated strand fails to become methylated and the level support of the notion that active demethylation arose in
of methyl cytosine per nucleus declines. This replication- the context of imprinting in mammals is the observation
dependent loss of DNA methylation is referred to as pas- that neither Xenopus (Stancheva et al. 2002) nor zebra
sive demethylation (Rougier et al. 1998). The initiation of fish (Macleod et al. 1999) show active demethylation. Fur-
the de novo methylation occurs after the fifth cell cycle thermore, it would be expected that active demethylation
and coincides with the time of the first differentiative should be conserved in mammals and should take place
event. The establishment of the first two cell lineages in the first cell cycle rather than immediately prior to
results in yet another significant asymmetry. The inner cell zygotic genome activation. Active paternal-specific
mass (ICM), which gives rise to all the tissues of the adult, demethylation has been confirmed in several other mam-
becomes hypermethylated, while the trophectoderm (TE), mals including the rat, pig and bovine, and, partially, in
that forms most of the structure of the placenta, is under- sheep (Dean et al. 2003, F Santos, unpublished data).
methylated (Dean et al. 2001, Santos et al. 2002). This However, what remains illusive is the identity of this
differential is maintained and reflected in highly methyl- active demethylase. In order to learn more about this pro-
ated somatic tissues and the distinctively hypomethylated cess we have undertaken an in-depth study of the first cell
extra-embryonic tissues of the placenta. Among the cycle of the mouse with respect to paternal-specific

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 Epigenetic reprogramming in early development 645

Figure 1 Methylation reprogramming during mouse development. The diagram depicts methylation levels of various classes of genes during
germ cell and embryonic development (methylated imprinted genes (black line) and non-imprinted genetic sequences (red, maternal; blue,
paternal)). (A) Highly methylated primordial germ cells enter the germinal ridge and undergo loss and reacquisition of methylation during their
expansion phase. Examples of these cells (day 11.5, 13.5 and 14.5) stained for alkaline phosphatase, a PGC marker, are pictured above.
The horizontal time axis and the vertical axis indicating the relative methylation levels are not to scale (modified from Reik & Walter 2001a).
Genome-wide DNA methylation reprogramming is represented by indirect immunofluorescence of fertilised oocytes (B) and embryos (C) using
an antibody to 5-methyl cytidine (5MeC). (B) Active demethylation: the first cell cycle. In the fertilised mouse oocyte a rapid and asymmetric
loss of DNA methylation (red signal) can be observed in the male (green in lower merge panel) but not in the female pronucleus. Genome-wide
loss of DNA methylation starts at sperm decondensation (left) and continues until it is undetectable in the paternal compartment (right), the pro-
cess taking about 6 h. Lower panels show a merge (yellow) between the DNA methylation (red) and DNA stain (green). hpf, hours post fertilisa-
tion. Scale bar 25 mm (Santos et al. 2002). (C) Passive demethylation phase. From the 2-cell stage (left) to the morula (right) the DNA
methylation (red) is passively lost due to the exclusion of DNA methyltransferase 1 (Dnmt1) from the nucleus. By the blastocyst stage lineage-
specific de novo methylation is apparent with the inner cell mass (ICM) being highly methylated (red) while the trophectoderm remains hypo-
methylated. Mouse embryos are depicted. Lower panels show a merge (yellow) between the DNA methylation (red) and DNA stain (green).
Scale bar 25 mm (Santos et al. 2002).

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646 F Santos and W Dean

demethylation using an exquisitely sensitive and specific meiotic maturation (Fig. 2A). The female is arrested in MII
antibody to 5-methyl cytidine (5MeC) (Reynaud et al. metaphase awaiting the signal for the completion of meio-
1992). To achieve this high-resolution profile, materials sis. The male, while haploid, is complexed in a nearly
were generated by in vitro fertilisation (IVF). Thus, control inert toroidal configuration unique to the presence of pro-
over very early post-fertilisation events was possible and tamines (Braun 2001). Thus, in order to restore diploid
both an estimate of the rate and a more exact time point complement to the zygote extensive remodelling must
for demethylation could be determined. At fertilisation, occur. This involves decondensation and nucleoprotamine
the male and female gametes are at different stages of exchange for nucleohistone. Nucleohistone exchange has

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 Epigenetic reprogramming in early development 647

already taken place by the earliest time that antibody Several candidates have been suggested for the active
accessibility can be demonstrated in the decondensing demethylase. Szyf and colleagues (Bhattacharya et al.
mouse sperm (Perreault 1992, McLay & Clarke 2003). 1999) reported an activity for methyl binding domain pro-
Paternal-specific demethylation is observed as early as 3 h tein 2 (MBD2) which fulfilled the criterion for the active
and completed within 6 h of IVF, several hours in advance demethylase (Cedar & Verdine 1999). Amidst much scep-
of the initiation of DNA replication (Santos et al. 2002). tical acceptance, MBD2 was thought to be a possible can-
didate in early embryos. Homozygous oocytes stained
with antibody for 5MeC and were found to undergo active
Active demethylation: mechanisms and candidate demethylation (Fig. 3B). Methyl binding domain protein 4
activities (MBD4), a uridine deglycosylase, has been proposed as a
potential demethylase activity owing to its role in DNA
In contrast to other vertebrate model organisms such as
repair (Hendrich & Tweedie 2003). Fertilised oocytes
Xenopus, the small size and restriction on the number of
homozygous for the deletion stained in a pattern indistin-
female gametes poses considerable experimental chal-
guishable from the control (Fig. 3B). This clear result
lenges to some biochemical questions in the mouse. While
suggested that MBD4 was not the activity responsible for
isolation of this demethylase activity directly from mouse
active demethylation in the early mouse embryo (F Santos,
oocytes was not feasible, some experimental manipulation
unpublished data). Currently, other candidates are being
was accessible. To this end we attempted to titrate the
pursued and we look forward with interest to the outcome
demethylase activity in mouse oocytes using conditions
of these studies.
permissive for polyspermy (Santos et al. 2002). Under
Rehydration of sperm DNA during pronuclear matu-
appropriate conditions, up to five sperm can be matured in
ration presents an intriguing proposition for paternal-
MII oocytes (Perreault 1992). Despite clear evidence of
specific active demethylation. The packaging of a haploid
titration of other components required for pronuclear for-
complement of DNA into the mature sperm is itself a feat
mation, demethylation of supernumerary male pronuclei
of engineering triumph (Ward & Coffey 1991). The
took place without impediment (Fig. 3A). This unexpected
extreme reduction in size due to the exclusion of water
result suggests that the oocytes were extremely well sup-
molecules provides an interesting source of ‘free energy’
plied with demethylase and that the specificity was tightly
on sperm decondensation. This process is energy produ-
regulated as the female pronucleus remained unaffected.
cing and in the context of coupled reactions may be
Three basic mechanisms have been proposed for active
capable of supplying the energy needed for demethyla-
demethylation. The first is the direct removal of the methyl
tion. ATP-coupled chromatin remodelling activities such
group from the major groove. This ‘snipping’ away of the
as those provided by lymphoid-specific helicase (LSH), a
side group would require the breaking of carbon bonds
member of the SNF/helicase family, are essential for nor-
and the production of methanol (Bhattacharya et al. 1999,
mal murine development and are associated with estab-
Bird 2002). The second group of reactions would either
lishing methylation patterns (Dennis et al. 2001). It is
specifically remove the methyl cytosine replacing it with a
tempting to speculate that within this remodelling com-
cytosine (Klimasauskas et al. 1994) or remove the CpG
plex is an activity that functions at this early stage in a
dinucleotide by nucleotide excision, a replication-depen-
unique capacity as a DNA demethylase.
dent DNA repair process. A third possibility is hydrolytic
deamination which results in the conversion of 5-methyl- Epigenetic reprogramming beyond the first cell
cytosine to thymine that subsequently becomes repaired in
cycle
the next replication cycle. Energetically this last option is
highly unfavourable; however; this does open the field for The decline in DNA methylation, largely from maternally
the consideration of reactions that achieve deamination derived sequences, continues in a step-wise fashion until
via enzymatic pathways. the morula stage when very little signal can be detected

Figure 2 Asymmetric chromatin remodelling is a requisite for fertilisation. (A) At fertilisation the oocyte chromatin is organised in a somatic con-
figuration with methylated (red) DNA complexed with histones (abundant methylation of NH2 termini). This status remains unchanged through-
out female pronuclear maturation. In contrast, sperm chromatin is fully gametic, with protamines instead of histones, and highly dehydrated.
Exchange of nucleoprotamine for nucleohistone has to occur during the first hour after fertilisation. Histones are acquired with acetylated lysines
in the amino terminal tails. Meanwhile, DNA becomes demethylated (black) through an active process. The mature male pronucleus has only
residual DNA methylation (centromeric region) and acetylated nucleosomes. This asymmetric distribution of chromatin organisation in the ferti-
lised oocyte raises a number of interesting points. In order to achieve a somatic organisation of the male chromatin the transition will first
require the removal of the acetylation group catalysed by histone deacetylases (HDACs), followed by the recruitment of specific histone methyl-
transferases (HMTs). Mechanistically, the inability to make this transition from acetylated to methylated histone may be the cause of the loss of
DNA methylation specifically from the male pronucleus (chromatin in the zygote adapted from Braun, 2001). hpf, hours post fertilisation. (B) At
syngamy, the maternal and paternal chromosomes align themselves in a unique configuration maintaining their parental associations. Maternal
chromosomes show DNA methylation along the entire chromosome while paternal chromosomes (encircled) retain only centromeric methyl-
ation. This highlights the epigenetic differences between maternal and paternal chromosomes and points to interesting chromatin differences
that may well remain throughout the preimplantation period of development.

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648 F Santos and W Dean

by 5MeC antibody staining (Rougier et al. 1998). This pas- Ferguson-Smith 2001, Howell et al. 2001). What is special
sive loss of methylation occurs as the oocyte form of the about the eight-cell stage? Perhaps the answer lies in the
primary methyl transferase (Dnmt1o) is actively excluded changes in totipotency that occur at this time when
from the nucleus despite the extraordinary abundance of the blastomeres of the embryo exhibit different cellular
the protein inherited in the cytoplasm (Howell et al. features and are known to acquire an ‘inside-outside
2001). In a most remarkable and specific form of temporal identity’, arguably the very first cell determination event
regulation, the Dnmt1o is excluded except for a single cell (Johnson 1986). These changes lead to the formation of
cycle at the eight-cell stage. Interestingly, deletion of the presumptive embryonic (inside; ICM) and presumptive
Dnmt1o 50 exon results in the abolition of imprinted meth- extra-embryonic (outside; TE) lineages (Johnson 1986). By
ylation marks that cannot thereafter be restored (Dean & the 5th cell cycle the final phase of methylation repro-
gramming is initiated. De novo methylation is evident in
both cell lineages of the blastocyst. However, it is the ICM
that becomes hypermethylated in comparison with the TE.
This establishes the asymmetric level of methylation seen
in somatic cells as ICM derivatives and in the placenta as
a TE derivative (Reik et al. 2003).

Epigenetic remodelling in the early embryo:

implication for somatic nuclear transfer
Since the establishment of paternal-specific demethylation
in the fertilised oocyte, the question of the associated
chromatin structure and organisation permissive for this
event has become the focus of our attention. In organisms
which possess DNA methylation, an inter-reliance exists
between these epigenetic marking systems to specify tran-
scriptional states and structural features of chromatin
(Strahl & Allis 2000, Jenuwein & Allis 2001).
The 50 amino-terminal tails of the core histones of the
nucleosome lie in the major groove of the double helix
and can be covalently modified by post-translational
addition including methylation, acetylation, phosphoryl-
ation, ubiquitination and ADP-ribosylation to lysine, ser-
ine and arginine residues. This led to the postulate that the
interpretation of the underlying genetic code was, in part,
achieved through the combinatorial modifications of key
amino acid residues of core histones or a ‘histone code’
(Strahl & Allis 2000, Turner 2000, Jenuwein & Allis 2001).
Switching between these modifications was associated
with specific chromatin states and the transitions from

Figure 3 Active demethylation: mechanisms and candidate activities.

(A) Polyspermic fertilisation of zona-free mouse oocytes. To evaluate
whether the demethylation of the paternal allele was titratable,
oocytes were exposed to capacitated sperm after removal of the
zonae and incubated for 9.5 h prior to fixation and staining with anti-
body to 5MeC. This procedure produced fertilised oocytes containing
multiple extra male pronuclei. The results show some of these poly-
spermic fertilised embryos with one male pronucleus, and three and
five male pronuclei. Titration of components necessary for pronuclear
formation is evident as the size of the pronucleus is inversely pro-
portional to the number of supernumerary male pronuclei (Santos
et al. 2002). (B) Distribution of CpG methylation during pronuclear
stages in the MBD2 and MBD4 null mouse fertilised oocytes using
indirect immunofluorescence with 5MeC antibody. Demethylation of
the male pronucleus was indistinguishable from that of appropriate
controls. Scale bar 20 mm.

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 Epigenetic reprogramming in early development 649

active to inactive configurations. In general, methylation Given the asymmetric loss of DNA methylation from the
of residues is associated with transcriptional repression paternal compartment, investigation of chromatin modifi-
while acetylation is known to accompany actively tran- cations in the one-cell fertilised oocytes is of particular
scribing regions. With this possibility in mind investi- interest (Arney et al. 2002, Cowell et al. 2002). Using a
gations have focussed intensively around residues of core heterochromatin-specific antibody to dimethylated H3-K9
histones that may adopt either modification. Thus a vast (anti-a-4x-methH3-K9), it was demonstrated that the
and expanding literature is evolving around the signifi- decondensing sperm and maturing male pronuclei are
cance of chromatin modification of lysine 9 of histone H3 negative for this modification (Reik et al. 2003). In con-
(H3-K9) (reviewed in Lachner et al. 2003). trast, the female pronucleus stains intensely for the same
configuration, which remains throughout the first cell
cycle. Despite this histone methylation asymmetry, acetyl-
ated H3-K9 was found to be abundant in both pronuclei,
in agreement with earlier reports (Adenot et al. 1997).
Remodelling of the sperm nucleus is a requisite for ferti-
lisation (Fig. 2A). Exchange of nucleoprotamine for
nucleohistone occurs during the first hour after fertilisation
(Perreault 1992). Acetylation of histones takes place in the
cytoplasm mediated by histone acetyl transferases (HATs)
and thus histones are acquired with acetylated lysines in
the amino terminal tails (Turner 2000). Methylation takes
place in the nucleus mediated by histone methyl trans-
ferases (HMTs). The transition first involves the removal of
the acetylation group catalysed by histone deacetylases
(HDACs). Thus the smooth transition from an active
(acetylated) to an inactive and potentially silent (methyl-
ated) chromatin configuration requires a series of specific
enzymatic activities. At present, no lysine-specific histone
demethylase activities have been identified and thus the
only mechanism of reactivation of silent loci may be by
replacement of histones (Lachner et al. 2003).
The asymmetric distribution of chromatin organised in
the fertilised oocyte raises a number of interesting points.
The heterochromatin-specific antibody is known to associ-
ate with peri- and centromeric satellite sequences and
other substantial stretches of heterochromatin throughout

Figure 4 Genome-wide DNA methylation in IVF produced and

cloned bovine embryos. (A) In bovine the paternal genome (blue)
undergoes active demethylation while the maternal genome (red) is
passively demethylated until the 8-cell stage when de novo methyl-
ation (black line) is observed (upper panel). At the blastocyst stage
there is an asymmetric distribution of DNA methylation between the
ICM (embryonic) and TE (extra-embryonic) lineages. This pattern is
altered on somatic nuclear transfer. Cloned embryos (lower panel)
show active but not passive demethylation activity. However, preco-
cious de novo methylation is observed by the 4-cell stage suggesting
that it may occur as early as the second cell cycle. (B) Organisation
of DNA methylation in normal and cloned bovine embryos. Between
the 8- and 16-cell stage a heterogeneous population of nuclei is
observed in control embryos (left panel). Cloned embryos (right
panel) are highly homogeneous with a methylation pattern strongly
reminiscent of the donor nuclei (lower panel) (Dean et al. 2001). (C)
Cloned embryos fail to achieve the asymmetry of both DNA and H3-
K9 methylation characteristic of un-manipulated control blastocysts.
Aberrant hypermethylation of the trophectoderm in cloned embryos
may be an early marker of the placental abnormalities frequently
reported. The figure depicts the merged channels of DNA and H3-K9
methylation (modified from Santos et al. 2003).

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650 F Santos and W Dean

the genome (Peters et al. 2002). Ordinarily, the presence Epigenetic profiles of cloned embryos produced from
of methH3-K9 is associated with transcriptional repres- two different donor sources indicated that quantitative and
sion. The conflict arises from the observation that although qualitative deregulation of DNA and histone methylation
both the male and female pronuclei are transcriptionally occurred in reconstructed embryos. Perhaps the most strik-
silent, only the female has chromatin in a form associated ing aspect of these embryos was the resemblance of the
with gene repression. A minor burst of transcriptional pattern of DNA methylation and heterochromatic organis-
reactivation occurs at the end of the first cell cycle with ation to that of the donor cells (Fig. 4B) and the loss of
paternally derived alleles expressed in advance (Aoki et al. lineage based asymmetric patterns of epigenetic marks
1997). These discrepancies suggest that the codified (Fig. 4C). Furthermore, there was a correlation between
interpretation of histone modifications may be specific normal epigenotype and rate of development to the blas-
and distinctive in gametes and embryos, as compared tocyst stage indicating it was predictive for normality (San-
with somatic cells where totipotency has been progress- tos et al. 2003).
ively restricted. These results reinforce the fact that one of the key fea-
It is intriguing that the female pronucleus remains DNA tures of somatic nuclear transfer will be the establishment
methylated and possesses methyl histone modifications of conditions that allow for erasure of heritable memory
suggestive of a mechanistic link between these two systems which are a hallmark feature of differentiated cell
epigenetic systems. This suggests at least two possible types (Wade & Kikyo 2002). In addition, selection of
interpretations. Either the chromatin organisation of the donor populations that are intrinsically more reprogram-
female pronucleus is resistant to an activity found in both mable by the oocyte cytoplasm will be essential (Solter
compartments or the male is specifically susceptible and 2000). These criteria are at present described only by epi-
targeted for demethylation. genetic profiling using antibodies; however, they are
powerful tools that permit rapid screening with a mini-
mum of tissue, a persistent challenge to the mammalian
Epigenetic reprogramming on somatic nuclear developmental epigeneticist.
Further investigations using genetically defined
transfer
mutations will be essential to tease apart the complex fea-
The strict progression of events that ordinarily take place tures of the dynamically evolving organisation of chroma-
and the precise and asymmetric outcomes of reprogram- tin in germ cells, embryos and their differentiated
ming highlight some epigenetic resetting features that pose antecedents. We anticipate a deluge of information in the
a potential obstacle to somatic nuclear transfer (Wade & next few years that will begin to explain the interrelated
Kikyo 2002). Must the highly methylated (DNA) nucleus functions of epigenetic marks and the instructions they
of a differentiated somatic cell undergo reprogramming on provide for the seamless unfolding of cellular function and
cloning in order to develop to term? If so, how is the cellu- developmental processes.
lar memory system that ensures fidelity of the differen-
tiated cell type circumvented? Does the oocyte, primed to Acknowledgements
remodel nucleoprotamine, recognise the chromatin con-
The work carried out by the authors takes place in the Lab-
figuration of the somatic cell and establish an epigenetic oratory of Developmental Genetics and Imprinting, Babra-
state equivalent to that of the fertilised oocyte? ham Institute. We would like to thank Wolf Reik for allowing
We have begun to investigate some of these questions us to embark on this exciting research area. We apologise in
associated with somatic nuclear transfer in mammals by advance that many original references have been omitted
focusing attention on cloned preimplantation stage owing to space restrictions. This work is funded by the
embryos. Owing to the persistent difficulties encountered BBSRC and MRC.
when attempting cloning in the mouse, we have exploited
the fact that somatic nuclear transfer in cattle is well References
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