[Cell Cycle 4:6, 760-763; June 2005]; ©2005 Landes Bioscience

Maintenance of Epigenetic Memory in Cloned Embryos

Perspectives

Ray K. Ng* ABSTRACT

J. B. Gurdon Different cell types have characteristic patterns of gene expression. Once a cell has
differentiated, its daughter cells nearly always differentiate in the same way. The mainte-
Wellcome Trust/Cancer Research UK Gurdon Institute; Department of Zoology;

.
University of Cambridge; Cambridge UK
nance of cell lineage involves either instructions from a cell’s surroundings or the inheritance

E
of memory from a parent cell. In normal development, the differentiation state of a cell is

*Correspondence to: Ray K. Ng; Wellcome Trust/Cancer Research UK Gurdon
Institute; Tennis Court Road; Cambridge CB2 1QN, UK; Tel.: +44.1223.334.090;
Fax:+44.1223.334.185; Email: j.gurdon@gurdon.cam.ac.uk
UT
remarkably stable and irreversible. However the transplantation of a somatic cell nucleus
to an enucleated egg often leads to a complete reprogramming of gene expression. We

RIB
summarize here the results of some Amphibian nuclear transfer experiments that reveal a
Received 04/13/05; Accepted 04/14/05 memory of gene expression. This and some other experiments exemplify epigenetic
Previously published online as a Cell Cycle E-publication: memory that persists through many cell divisions. In the case of nuclear transfer experiments,

IST
http://www.landesbioscience.com/journals/cc/abstract.php?id=1743 the actively transcribed state of a gene can be propagated through many cell divisions
in the absence of the stimulus that first induced the activity of this gene. We discuss the
KEY WORDS

D
possible basis of these two examples of persistent epigenetic memory, namely changes
at DNA methylation and histone modifications.

OT
nuclear transplantation, cloning, epigenetic
memory, differentiation, gene expression, DNA

INTRODUCTION
methylation, histone.

ABBREVIATIONS
ON
The process of cell differentiation is very stable. In most cases, progenitor or stem cells
.D
PcG polycomb group produce daughter cells of the same kind, and only rarely if ever does a differentiated cell
trxG trithorax group change to another type, or give daughter cells of an unrelated kind. There are two likely
HMT histone methyltransferase
CE

explanations for this stability; one is that, when a cell divides, its daughter cells receive
PGC primordial germ cells
edd endodermin
instructions from the surroundings, e.g., signaling molecules, so that they are directed to
IEN

differentiate in the same way as the parent cell. The other is that daughter cells have a
ACKNOWLEDGEMENTS
memory of the parent cell, so that they inherit a differentiated state.
We know, from Amphibian nuclear transfer experiments, that the memory of a differ-
SC

We acknowledge support from the Wellcome entiated state can be completely reversed, when the nucleus of a differentiating or differ-
Trust and the Cambridge Overseas Trust. entiated cell is transplanted to egg cytoplasm. In 1952, Briggs and King achieved the first
BIO

success of nuclear transfer by transplanting a blastula nucleus into an enucleated unfertilized

egg of Rana pipens and about 40% of the nucleus-transplanted eggs developed normally
into tadpoles.1 Following that, Gurdon et al were able to produce normal fertile Xenopus
ES

frogs by using nuclei from differentiated donor cells of intestinal epithelial origin.2 This
demonstrated that nuclei from fully differentiated cells are capable of promoting the
ND

development of a whole organism. This therefore established the general principle of the
conservation of genome. It also showed that any memory processed by a nucleus can be
reversed, although this happens progressively less efficiently when cells become increasingly
LA

specialized.3

EPIGENETIC MEMORY OF DIFFERENTIATION STATUS

05
20

Nuclear transplantation is accompanied by a dramatic alteration of gene expression in

a transplanted somatic cell nucleus. For successful reprogramming, genes normally
©

expressed in embryos need to be switched on, while genes not normally expressed in
embryos need to be switched off in transplanted nuclei. Failure in the reprogramming of
gene expression can be a major reason for the low viability of cloned animals. For example,
it has been shown that the gene oct4, which is required for embryonic development and
for the maintenance of stem cell pluripotent status, is inefficiently activated in some
cloned mice.4,5 Another example of “off-state” genes is the imprinted genes in mammalian
genomes, e.g., H19 and Igf2. In normal development, the erasure of existing imprints
occurs in primordial germ cells (PGCs) during gametogenesis and is followed by the
initiation of a new set of imprints in the male and female germ lines. However, in some

760 Cell Cycle 2005; Vol. 4 Issue 6

 Maintenance of Epigenetic Memory in Cloned Embryos

A
cases of nuclear transfer, the
imprints in the donor somatic
nuclei are erased while in others
some are maintained
unchanged.6,7 Another observa-
tion relates to the inactive X
chromosome. In cloned mouse
embryos, a random X chromo-
some inactivation takes place in
the inner cell mass, but in the
trophoectoderm, the repressed
X chromosome is always the
same that of the donor nucleus.8
This is therefore another example
of the persistence of the “off-
state” of gene expression after
nuclear transfer. The persistence
of “off-state” genes in cloned
embryos can possibly be
explained by the failure of
demethylation of regulatory
gene regions, e.g., promoter.
Recently, a study demonstrates
that the reactivation of oct4 gene
expression in nuclear transplant
oocytes is regulated by demethy-
lation of the oct4 promoter.9

B
Therefore, it suggests that the
inefficient activation of oct4 in
some of the cloned embryos
may due to failure of demethy-
lation of the oct4 promoter and
as a result, lead to the binding of
methyl-CpG binding proteins
such as MeCP2, MBD1 and
MBD2 to the methylated oct4
promoter and silence its tran-
scriptional activity.

Figure 1. Hypothetical models for

epigenetic memory. (A) The promoter
region of repressor gene is methy-
lated in donor cells and therefore
has no inhibitory effect on donor
gene expression. Incomplete repro-
gramming after nuclear transfer results
in failure of demethylation of the
promoter region of a repressor gene,
leading a continuous expression of
the donor-specific gene in nuclear
transplant cells. (B) The active gene
state of a donor-specific gene in
donor cells is due to the association
of the modified histones, e.g., acety-
lated H3K9, with the promoter
region. Incomplete reprogramming
after nuclear transfer results in
continuous association of these
modified histones, thereby leading
to the epigenetic memory of donor
gene expression in nuclear transplant
cells. “Donor gene” refers to a gene
which has specific expression in the
donor cell type.

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 Maintenance of Epigenetic Memory in Cloned Embryos
The other aspect of reprogramming differentiated donor nuclei is
to switch off the expressing tissue-specific genes. The first indication
of a continuous “on-state” of gene expression in nuclear transplanted
embryos was reported by Briggs and King in 1957. They observed
that, in Rana pipiens, endoderm-derived nuclear transplant embryos
showed a preferential deterioration of their ectoderm, compared to
their endoderm.10 They therefore suggested that donor endoderm
nuclei may “remember” their endoderm origin and behave like
endoderm in nonendoderm tissues in nuclear transplant embryos,
thereby resulting in the early deterioration of ectoderm tissues.
However, the ectoderm differentiates before the endoderm, and it is
known that the ectoderm also deteriorates first in nuclear transplant
embryos derived from donor nuclei of ectoderm or other cell-types.11
Recently, we found that genes specific to the donor cell type of a
transplanted nucleus are continuously expressed in the donor irrelevant
tissue (wrong cell lineage) in some of the cloned embryos after
Xenopus somatic cell nuclear transfer.12 The mis-expression of the
donor “on-state” genes in the wrong germ layer, i.e., endodermin
(edd) in the animal region of endoderm-derived cloned embryos and
Sox2 in the vegetal region of neuroectoderm-derived cloned
embryos, demonstrated a persistent of epigenetic memory of the
active gene state in transplanted nuclei. The extent of epigenetic
memory was found to be highly variable among cloned embryos.
Remarkably, in some cases, there is almost complete memory of the
donor “on-state” of gene expression in the wrong cell lineage in
cloned embryos, after more than 12 mitotic cell divisions. In normal
Amphibian development, there is no transcription between fertilization
and the blastula stage. Also transplanted somatic cell nuclei discontinue
all transcription from the time of transfer into recipient egg and the
blastula stage. In spite of this cessation of transcription by transplanted
nuclei for about 12 cell cycles, in many cases such nuclei strongly
express a gene characteristic of their germ layer origin when they
resume transcription at the blastula stage. This result implies that
this memory is very stable throughout development and is possibly
linked to the maintenance of the donor differentiation status.
Moreover, this memory does not affect the expression of signal factor
responsive genes, which are in “dormant” state in cloned embryos.
MOLECULAR MECHANISMS OF EPIGENETIC MEMORY
The memory of an “on-state” of gene expression in our experiments
cannot be explained by external signals (see Introduction above),
because appropriate external signals do not exist in the wrong cell
lineage. Therefore epigenetic memory must depend, in this case, on
heritable epigenetic marks. In order to transmit a specific gene
expression pattern to the daughter cell lineage, these marks must be
stable through many rounds of DNA replication and cell division.
We know of two classes of epigenetic modification that can
regulate gene transcription: (1) methylation of DNA and (2) modifi-
cation of chromatin binding proteins. To explain epigenetic memory
in nuclear transplant embryos, two hypothetical models are purposed.
One model (Fig. 1A) depends on the methylated state of the pro-
moter region of a negative regulatory gene. Methylation of the
cytosine of a CpG dinucleotide is known to be involved in transcrip-
tional silencing. Failure of demethylation of the promoter region of
such gene due to incomplete reprogramming would result in no
expression of that negative regulatory protein. Therefore there is no
inhibitory mechanism to block the expression of donor differentiation
genes. The CpG methylation state of parent cells would be able to
pass on to the daughter cells by a semi-conservative mechanism during
762 Cell Cycle
cell replication.13 Nevertheless, this model makes the assumption that
the expression of every early differentiation genes takes place by the
negative effect of repressor proteins, but there is no evidence for this
so far.
The other model (Fig. 1B) depends on modified histones binding
to the promoter of donor expressing genes. It has been found that
some histone proteins carrying specific modifications on their N-ter-
minals are closely related to the transcriptionally active/inactive state
of the genome. A well known example is histone 3 lysine 9 (H3K9)
acetylation. Incomplete reprogramming might involve continuous
association of modified histones with those donor specific gene
promoters and could consequently lead to epigenetic memory of
donor gene expression. It has been suggested that histone modifications
can be transmitted through semi-conservative replication of nucleo-
somes during cell division and therefore might act as heritable
epigenetic marks for cell lineage memory.14 However, there is another
study suggesting that the replacement of histone variants during
nucleosome replication may mark different transcriptional states.15
As the Drosophila genome has no recognized covalent modification
like DNA methylation, the heritable epigenetic marks for maintaining
the cell differentiation state in this case are believed to be achieved
by an interaction between modified histones and protein complexes
such as those of the Polycomb group (PcG) and trithorax group
(trxG).16 PcG proteins are required for transcriptional repression
while trxG proteins are associated with the active state of gene
transcription. It is reported that both groups of proteins can take
part in histone modifications and chromatin remodeling. For example,
trxG proteins TRX and ASH1 are histone methyltransferases (HMTs)
and able to methylate lysine 4 of histone 3 (H3K4); the PRC2 PcG
complex contains E(z) subunits which can methylate H3K9 and
H3K27.17 These two models of epigenetic memory are not mutually
exclusive and probably both are required for the inheritance of cell
lineage gene expression patterns.
CONCLUSION AND PERSPECTIVE
Differentiated cells have lost their pluripotency and developmental
plasticity. By means of nuclear transfer experiments, these non-
pluripotent cells can regain their pluripotency through epigenetic
reprogramming. Alterations to epigenetic modifications allow a
switch in patterns of gene expression, that is required for changes in
cell differentiation. However, a certain degree of epigenetic memory
of development history is retained and is not erased completely
through nuclear transfer. It is therefore essential to identify the precise
nature of this epigenetic memory of the differentiated state in order
to understand the maintenance of cell lineage during normal devel-
opment. At the same time, it is important to elucidate the mechanisms
by which epigenetic memory is established, and consequently to
achieve a higher cloning efficiency by more effective reprogramming.
This will also reveal the potential use of cells generated by nuclear
transplantation for cell replacement therapy. By understanding the
normality of cells derived by nuclear transfer and their cellular
responses to different developmental signals, it may be possible to
generate a population of a particular cell type. The potential value of
cloning will depend largely on understanding various mechanisms
that regulate the epigenetic expression of appropriate genes.
2005; Vol. 4 Issue 6
 Maintenance of Epigenetic Memory in Cloned Embryos

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