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EMBRYONIC STEM CELLS: CONCISE REVIEW

Epigenetic Modification Is Central to Genome Reprogramming in Somatic Cell Nuclear Transfer


LYLE ARMSTRONG,a,b MAJLINDA LAKO,a,b WENDY DEAN,c MIODRAG STOJKOVICa,b Centre for Stem Cell Biology and Developmental Genetics and bInstitute of Human Genetics, University of Newcastle, Central Parkway, Newcastle upon Tyne, United Kingdom; cLaboratory of Developmental Genetics and Imprinting, The Babraham Institute, Cambridge, United Kingdom
Key Words. Embryonic stem cells Genome reprogramming Epigenetic modification Somatic cell nuclear transfer
a

ABSTRACT
The recent high-profile reports of the derivation of human embryonic stem cells (ESCs) from human blastocysts produced by somatic cell nuclear transfer (SCNT) have highlighted the possibility of making autologous cell lines specific to individual patients. Cell replacement therapies have much potential for the treatment of diverse conditions, and differentiation of ESCs is highly desirable as a means of producing the ranges of cell types required. However, given the range of immunophenotypes of ESC lines currently available, rejection of the differentiated cells by the host is a potentially serious problem. SCNT offers a means of circumventing this by producing ESCs of the same genotype as the donor. However, this technique is not without problems because it requires resetting of the gene expression program of a somatic cell to a state consistent with embryonic development. Some remodeling of parental DNA does occur within the fertilized oocyte, but the somatic genome presented in a radically different format to those of the gametes. Hence, it is perhaps unsurprising that many genes are expressed aberrantly within cloned embryos and the ESCs derived from them. Epigenetic modification of the genome through DNA methylation and covalent modification of the histones that form the nucleosome is the key to the maintenance of the differentiated state of the cell, and it is this that must be reset during SCNT. This review focuses on the mechanisms by which this is achieved and how this may account for its partial failure in the cloning process. We also highlight the potential dangers this may introduce into ESCs produced by this technology. STEM CELLS 2006;24: 805 814

AN OVERVIEW

OF THE

CLONING PROCESS

It has long been known that certain invertebrate species can be duplicated or cloned simply by dividing them into two pieces and allowing the separated halves to grow into a complete organism. However, this cannot be applied to vertebrates. It is more than 50 years since the groundbreaking studies of Briggs and King [1 4] demonstrated that somatic cell nuclear transfer (SCNT) could be used to clone frogs. Using oocytes and donor nuclei from Rana pipiens, they found that the reconstructed embryos were capable of development to at least the early cleavage stages and in some cases as far along as the tadpole stage. The use of blastomere nuclei was possibly instrumental in this process because such cells are relatively unspecialized [5, 6]. In retrospect, it was not surprising that early attempts to use SCNT to clone frogs from adult somatic cells met with failure. Later work by Gurdon [7] using intestinal cells from tadpoles demonstrated that differentiated somatic cells were capable of producing viable embryos. These observations

suggested that, in principle, the genome could be reset to a totipotent state. In contrast, SCNT in vertebrate cells was rather more difficult and for many years it was believed that the cells of adult vertebrates were simply too specialized to revert to a totipotent state. This opinion was decisively contradicted with the cloning of Dolly in 1996 [8] by fusion of a mammary gland epithelial cell from a Finn Dorset ewe with the enucleated oocyte from a separate donor (Fig. 1). Many studies have confirmed the feasibility of SCNT-based cloning of bovine, mouse, and pig [9 11] and that the cytoplasm of oocytes from sheep, cow, and rabbit is capable of reprogramming somatic cells from other species and supporting the growth of such interspecies-cloned embryos to blastocysts [12]. Moreover, the recent success of producing a cloned human blastocyst derived from donated oocytes fused with their associated cumulus cells provides further evidence of the broad applicability of the SCNT process [13]. These results potentially have paved the way for human therapeutic cloning by deriving patient-specific em-

Correspondence: Lyle Armstrong, Ph.D., Centre for Stem Cell Biology and Developmental Genetics, University of Newcastle, International Centre for Life, Newcastle NE1 3BZ, U.K. Telephone: 44 0191 241 8695; Fax: 44 0191 241 8666; e-mail: lyle.armstrong@ncl.ac.uk Received July 29, 2005; accepted for publication November 2, 2005; first published online in STEM CELLS EXPRESS November 10, 2005. AlphaMed Press 1066-5099/2006/$20.00/0 doi: 10.1634/stemcells.2005-0350

STEM CELLS 2006;24:805 814 www.StemCells.com

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Figure 1. The method used to create Dolly the sheep. The nuclear material was removed from an oocyte taken from an adult female and replaced by that of a somatic cell from another animal. Fusion and activation of this reconstructed zygote gave rise to an embryo that was surgically transferred to a surrogate mother wherein development to term was completed.

bryonic stem cells (ESCs) with the ability to produce essentially unlimited supplies of tissues for cell replacement therapy [14 16]. Clearly, there are still many problems associated with SCNT. The majority of cloned embryos (irrespective of species) do not survive to birth, whereas those that do often demonstrate a variety of defects that greatly reduce the probability of their survival to adulthood [1719]. The occurrence of many of these defects has been attributed to incomplete reprogramming of the somatic donor nucleus and a failure of appropriately orchestrated gene expression required for embryonic development [20, 21]. In this review, we will concentrate on the mechanisms that establish a normal epigenotype and consider the critical areas in which these differ between SCNT-derived and naturally fertilized embryos.

HOW IS SCNT ACHIEVED?


There are two basic strategies for the cloning of mammals by SCNT that are able to produce embryos capable of development to term. Both of these techniques require the removal of the nuclear material from the oocyte and differ only in the way in which the nuclear material of the donor cell is introduced and the subsequent activation of the reconstructed embryo. Enucleation of the MII oocyte may be achieved by a number of techniques; the most popular is capillary incision of the zona pellucida, using a micromanipulator followed by removal of the polar body and adjacent metaphase chromosomes by suction into a glass pipette [22]. Additional methods include enucleation by centrifugation [23] and bisection of the oocyte followed by removal of fragment containing the

nuclear material [24 26] (the so-called handmade cloning method). Although this technique has the advantage of simplicity, it does remove more oocyte cytoplasm and therefore it may reduce the amounts of proteins needed for reprogramming and early embryonic development. Introduction of the donor nucleus can be performed using a variety of methods that aim to optimize the successful production of offspring by altering several factors. These techniques rely on either microinjection of the donor cell or its isolated nucleus into the oocyte cytoplasm, or fusion of the donor cell with the enucleated oocyte is achieved through appropriately timed electrical pulses. Of key importance is cell cycle synchrony of the donor cells. Full-term cloned animals have been obtained most consistently from donor cells in a quiescent state (G0 or G1), which may be induced in cultured cells by serum starvation [8, 27] or by using cells, such as granulosa cells, that are naturally quiescent in the donor organism. The usefulness of quiescent cells has been attributed to their reduced transcriptional activity and chromatin modifications that are associated with cells in G0, which may enhance their epigenetic plasticity. Normally quiescent cells (e.g., resting lymphocytes) have lower levels of histone methylation than their cycling counterparts, a circumstance that led some workers to suggest that such cells may be more easily reprogrammable. The modulation of repressive histone modifications such as methylation of lysine 9 on histone H3 is a facet of genome reprogramming in normal embryos [28] that is not reflected in SCNT. Thus, the marked reduction of this modification in noncycling cells may account, in part, for their greater usefulness in animal cloning. Recent evidence in support of this arises from the observation that when

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the nuclei of quiescent T cells from mice carrying the enhanced green fluorescent protein (EGFP) transgene (which is normally inactive in these cells) are transferred into one-cell stage embryos, approximately three times as many embryos re-expressed EGFP than when activated T cells were used [29]. An advantage of using G0 synchronized donor cells is that they are diploid, a requisite for normal development of the embryo. Tetraploid donor cells at G2/M may also be used in a specialized strategy involving activation of the reconstructed embryo in the absence of cytochalasin B which permits expulsion of the excess nuclear material into a pseudo-polar body, thereby returning the ploidy of the embryo to 2C [30]. Not all cells that would normally be nondividing are useful donors for SCNT. Neurons and other types of terminally differentiated cells are generally rather poor candidates for cloning studies; this may reflect the lack of developmental plasticity of their genomes. Such cells are believed to repress many more genes than cycling or less differentiated cells and as such are thought to be extremely difficult to reprogram [31]. However, other types of cells that are not terminally differentiated may make better donors. There is considerable evidence to suggest that the use of ESCs as nuclear donors gives rise to viable offspring with greater efficiency than many somatic cell types [32, 33]. This depends on the particular ESC lines used (in mouse at least) and the number of passages over which they have been cultured. ESCs are subject to epigenetic changes with prolonged culture, affecting their ability to contribute to embryonic development perhaps by modification of genes essential for embryonic and fetal development. Among these classes of genes, those associated with pluripotency as well as the parentof-origin marked imprinted genes have been best studied. The importance of such genes to development is highlighted by the unsuitability of primordial germ cells as nuclear donors beyond embryonic day 12; this unsuitability has been attributed to the erasure of parental imprints during gametogenesis [34]. Somatic stem cells are also possible donors as demonstrated by the use of mesenchymal stem cells from adult mouse bone marrow [35]. Interestingly, these cells seem to offer little advantage (in terms of development to term) over more readily accessible cell types such as skin fibroblasts. Whichever method is used to transfer the donor nucleus, it undergoes disassembly in response to the high levels of maturation promoting factor (MPF) found in the MII-stage oocyte cytoplasm. Reassembly of the nucleus occurs after artificial activation of the reconstructed oocyte. An extension of the time between introduction of the donor nucleus and activation may be beneficial to the development of live clones [36, 37], although this is not absolutely essential. However, there is no definite consensus on the effect of MPF levels on somatic nuclear reprogramming. The high MPF activity of MII oocytes induces nuclear envelope breakdown and premature chromosome condensation, which have beneficial or harmful effects upon the reconstructed embryo depending on the cell cycle stage of the somatic donor [38, 39]. Donor cells in S phase also undergo premature chromosome condensation, but this results in a pulverized chromatin appearance [40] that may cause damage to the DNA duplexes of the donor nuclei. Cytoplasts derived from pre-activated oocytes do not induce nuclear envelope breakdown [41] and premature chromosome condensation, and thus donor cells can be used from any stage of the cell cycle; www.StemCells.com

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however, the rate of progression of these embryos to blastocysts using this method is low for differentiated somatic cells. Preactivated cytoplasts work better with blastomeres, which (because these are less developmentally committed) would tend to suggest that pre-activation removes some of the oocyte-derived factors that are capable of reprogramming the somatic genome. The inference from this is that the chromatin modifications induced in the somatic genome by premature chromosome condensation in the MII cytoplast may facilitate its reprogramming to a totipotent state. It is not clear how long the somatic nucleus should be exposed to high MPF levels to complete reprogramming because various groups report a broad range of findings (as little as 15 minutes [36] to 6 hours [40]) between introduction of the donor cell and activation, but it is possible that the differentiation state of the donor cell has a significant impact on this timing. The nature of the oocyte-derived factors responsible for reprogramming is largely unknown, although it is clear from activation studies that their existence is transitory [42, 43]. From the point of view of the normally fertilized oocyte, their limited persistence is undoubtedly sufficient for the task of rapidly demethylating the incoming paternal DNA, but the highly differentiated state of a transplanted somatic donor karyoplast may be more problematic. Donor cells from early preimplantationstage embryos may be more easily reprogrammed [44] because they are pluripotent and have a lower level of genomic DNA methylation per blastomere [45]. These nuclei may require less reprogramming of the genes required for early embryo development than the types of donor cells which are most accessible for the purposes of creating nuclear transfer (NT) ESC lines (e.g., fibroblasts will not share this advantage [46]). Four-cell embryo nuclei, arrested at metaphase, were successfully used to generate cloned mice by a serial NT technique [47], which resulted in higher rates of progression to blastocysts (83%) and 57% development of offspring to term. It has been suggested that such serial transfer allowed more time for reprogramming of the metaphase nucleus to take place (i.e., the initial reprogramming undertaken in the enucleated oocyte and up to the four-cell stage is augmented by another passage of the donor nucleus through another round of early embryonic development). It would be tempting to speculate that a serial transfer technique would be capable of solving many of the problems associated with cloning in mammals by more effectively removing the somatic memory of the donor nuclei. However, it has yet to be established that this technique offers any advantages for the isolation of ESC lines for therapeutic applications. Of course, the use of twice the number of oocytes would be a major obstacle for use in human studies in which oocyte availability is limited. Even if this obstacle were removed, it is essential to ensure that any human ESC lines produced by SCNT are capable of use in cell replacement therapy with minimal potential risk to the patient. For this reason, it is essential to increase our understanding of the nature of genome reprogramming in both normal and SCNT embryos.

EPIGENETIC MODIFICATION IS TO REPROGRAMMING

CENTRAL

Upon transfer of a somatic nucleus to an oocyte during the cloning process, several essential changes must ensue. First, the somatic nucleus must cease to express its unique repertoire of gene products. Second, that nucleus must become subject to the

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cyst from which ESCs are derived. However, it would be surprising if imprinted genes were the only loci affected by incomplete epigenetic reprogramming given its genome-wide role in controlling gene expression. Indeed, there is evidence to support this latter view; microarray analysis has demonstrated that approximately 4% of a panel of 10,000 murine genes showed abnormal expression levels in the placenta of NT mice. Perhaps surprising was that although the livers of cloned animals also showed gene dysregulation, this was less extensive than in the placenta, affecting a different set of genes [61]. In a companion study, gene expression patterns of NT clones derived from ESCs were compared with clones derived from cumulus cells as the somatic donors. This study found that a smaller subset of genes were affected in clones derived from ESCs compared with clones derived from cumulus cells, in keeping with the earlier suggestion that embryonic cells may require less reprogramming to reestablish totipotency. The fact that such errors occur at all in the ICM should make us exercise an element of caution when considering the use of NT-derived ESCs in regenerative medicine.

instructions provided by the oocyte cytoplasm to unfold a new pattern of development-specific gene transcripts, and third, the heritable memory endowed by the chromatin that ensured the characteristics of the donor tissue must be erased. All these changes involve a remodeling, not of the underlying genetic sequences that comprise the genome, but of the epigenetic features that overwrite the gene sequences and find interpretation in new gene expression [48]. Epigenetic reprogramming is an essential feature of normal development and is associated with the erasure of some of the epigenetic modifications inherited from the gametes [49]. A major epigenetic modification in mammals is the addition of a methyl group to the 5 position in the symmetrical CpG dinucleotide. Alteration of the 5-methylcytosine content of specific regions of the genome is thought to be important in controlling gene expression that must undergo radical changes in both normally fertilized embryos and those reconstructed by NT. DNA methylation is thought to be important for the regulation of a number of different groups of genes and genomic sequences [50, 51]. Among the most important are the long terminal repeat of endogenous retroviruses, in which DNA methylation plays an essential role in the silencing of these retrotransposons, thereby maintaining genome-defense, the differentially methylated regions of imprinted genes, and the inactive X chromosome [52, 53]. The pattern of DNA methylation is an indicator of the differentiation state of the cell although there is a paucity of information concerning the rules governing this pattern. Imprinted genes are a unique group of genes that are important for fetal growth and development, especially in the placenta, as well as for postnatal behavior and cognition. The expression of imprinted genes does not follow a mendelian pattern of inheritance but instead depends on the parent-oforigin to dictate its expression [54 57]. Regulatory regions of such genes are typically methylated in the silent allele and are exempt from the large-scale genome-wide demethylation that occurs during pre-implantation development. Imprinted genes are particularly sensitive to environmental changes. T9ese can vary in severity from simply altering culture conditions to SCNT [58, 59]. Thus, it is not surprising that embryos produced using assisted reproduction technologies in humans or NT of several species show widespread methylation defects in imprinted genes [60, 61]. Furthermore, genome-wide patterns have been reported to be aberrant on SCNT, with nearly 50% of cloned bovine and sheep blastocysts showing gross genomewide errors in both DNA methylation and histone acetylation and a high degree of methylation errors at specific loci [28, 62, 63]. This high error rate may represent a potentially fundamental obstacle and preclude the use of NT to derive patient-specific ESCs even though such cells may appear to be pluripotent under our current definitions of their ability to differentiate into multiple cell lineages. Cells may be derived from these lines which have the morphological and immunological characteristics that define them as therapeutically useful types, but these may not be able to function in the same way as similar indigenous cells when introduced into a patient if they are unable to express certain genes at their correct levels. A contrary viewpoint could be that because many of the epigenetic errors that result from NT affect imprinted genes typically involved in extra embryonic development, they would be less likely to affect the inner cell mass (ICM) of the blasto-

EPIGENETIC REPROGRAMMING AND NT EMBRYOS

IN

NORMAL

An understanding of the mechanisms that govern epigenetic reprogramming during normal development and how they might differ in the context of SCNT is central to our ambition to derive epigenetically normal ESCs. Upon fertilization, there is a series of events that involve the incoming sperm as it encounters the egg cytoplasm. The initial event after fertilization is the decondensation of the sperm nucleus, resulting in the unwinding of the tightly packaged sperm DNA held in a unique, almost toroidal, conformation by the sperm-specific protamines (Fig. 2) [64]. So highly ordered is this chromatin organization in sperm that it is effectively dehydrated, and hence rehydration is an essential, very early event. Upon decondensation, protamines are replaced rapidly by nucleohistones derived from the oocyte cytoplasm, usually in the first hour after fertilization [65], and the DNA is wound onto the histone octamers in an ATP-dependent process [66]. It is during this same time period that rapid and paternal-specific demethylation of the genome takes place in the absence of transcription or DNA synthesis (active demethylation). The octamer of each nucleosome comprises histones H2A, H2B, H3, and H4, and their interaction may be stabilized by the presence of the linker histone H1oo (oocyte-specific). This distinct H1 protein remains associated with the DNA at least until the two-cell stage of the embryo and is normally replaced with the somatic H1 by the four-cell stage [67]. This remodeling of the sperm nucleus into an accessible, transcriptionally competent chromatin configuration is coincident with the formation of the pronuclear membrane and demethylation of the paternal genome. As the end of telophase approaches, centromeric proteins A and B, which function as part of the kinetochore complexes, are assembled onto the DNA. Upon completion of active demethylation, and with the initiation of S phase, transcription factors (e.g., TATA box protein and Sp1) bind to prime the genome for transcription late in the first cell cycle [68] (Fig. 3). The exact nature of the active demethylation is not well understood. Active demethylation is operatively defined as loss

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Figure 2. Remodeling of paternal chromatin after fertilization until the first cell division. Sperm DNA is highly compacted due to association with protamine. Removal of protamine is followed by binding of the DNA by acetylated histones that help to maintain the newly formed chromatin in an open conformation. Reprogramming of the genome by progressive demethylation of DNA is accompanied by histone modifications, loss of oocyte-specific histone H1oo, and recruitment of nonhistone proteins to prepare DNA for transcription.

Figure 3. Methylation levels throughout pre-implantation development of normal and nuclear transfer-derived embryos. The paternal genome (purple) of normally derived embryos undergoes rapid active demethylation, whereas the maternal genome (yellow) undergoes passive demethylation until the morula stage of pre-implantation development, when de novo methylation commences. Cloned embryos (turquoise) undergo a reduced passive demethylation.

of methylation in the absence of DNA replication. The speed with which this process occurs strongly suggested that it is mediated enzymatically. Identification of a putative demethylase enzyme(s), especially the active demethylase in the oocyte, has met with some controversy. As yet, the origin of this activity has not been unequivocally assigned to either the oocyte cytowww.StemCells.com

plasm or the sperm itself; however, indirect evidence of partial demethylation on SCNT points to the activity residing in the oocyte cytoplasm. Demethylation of up to five supernumerary male pronuclei obtained by polyspermic fertilization of zonafree mouse oocytes suggests a high abundance of this activity [69]. Several candidate proteins have been proposed. Three basic mechanisms have been proposed. The first and most simplistic would involve direct removal of methyl groups from the major groove of DNA. Although the mechanism by which this is achieved is uncertain, methyl binding domain protein (MBD2) has been shown to possess demethylase activity [70], with methanol as the stable leaving group. Independent attempts at verification of this result, by two different groups, failed to find demethylase activity for MBD2. Moreover, immunofluorescent analysis of 5methyl cytosine in MBD2 null crosses indicated no difference in the paternal-specific loss of methylation at the one-cell stage [71]. A second possible mechanism envisages the replacement of 5-methylcytosine by cytosine or removal of the CpG dinucleotide by either base or nucleotide excision repair [72]. For this reason, the uridine deglycosylase enzyme methyl binding domain protein binding 4 (MBD4) was proposed as a potential demethylase because of its role in DNA repair [73] although paternal-specific demethylation appeared to occur normally in MDB4 null fertilized oocytes [69]. A third possibility proposes hydrolytic deamination of 5-methylcytosine resulting in the conversion of 5MeC to thymidine; however, this process would require considerable energy input and as such is the least likely mechanism. A recent report of enzymatic deami-

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histone modifications on the chromatin associated with the methylated allele provide a specific mark that ensures maintenance of methylation. The imprint control regions of the inactive (methylated alleles) are known to have methylation of lysine 9 on histone H3 (methyl H3K9) and hypoacetylation of H3 and H4, whereas the active (unmethylated allele) is characterized by H3/H4 hyperacetylation and H3 K4 methylation [86]. As yet, no underlying biochemical mechanism has been described to link these histone modifications to any specific means for maintenance of chromatin states in imprinted and nonimprinted regions alike.

nation by Aid during T-cell development has introduced the possibly that this activity is also important during the first cell cycle [74]. Mechanistically, the loss of a methyl group poses a considerable enzymatic challenge, and these activities and their epigenetic regulation are a research hotspot at present. Why the embryo needs to actively demethylate the paternal genome so rapidly after fertilization remains a mystery. It has been suggested that de-repression of a number of paternal alleles is required to accommodate the burst of transcriptional activity that occurs at the end of the first cell cycle [75]. An alternative hypothesis has been suggested largely focusing on imprinted alleles. It suggests that active demethylation arose as a protective response of the maternal genome to reduce the influence of the paternal genome, which may have alleles optimized to the production of larger, more competitive offspring, thus serving the maternal interest in ensuring survival of larger numbers of offspring overall [76].

EPIGENETIC INFORMATION FIDELITY FAILS DURING CLONING


Imprinted DNA methylation of loci is very often disrupted in NT embryos, affecting the extra-embryonic tissues more frequently than those of the embryo. However, although this may have certain consequences for embryonic survival and/or growth, imprinted loci do not represent the bulk of the mammalian genome, with more than 70 imprinted loci known to date [http://www.mgu.har.mrc.ac.uk/research/imprinting]. The studies of several groups have shown that the somatic genome used in NT does not respond so readily to the demethylation activity of the oocyte [63, 87, 88], and in most cases the level of methylated DNA remains much higher than in normal embryos (Fig. 3), a state more reminiscent of somatic cells. In addition to reduced passive loss of DNA methylation, the onset of de novo methylation frequently begins much earlier (four-cell stage) than in normal embryos, suggesting that incomplete remodeling of the donor nucleus impairs the normal temporal progression of epigenetic reprogramming leading to transcriptional misregulation. One might expect that given the apparent abundance of demethylating activity in the oocyte, the somatic genome would be rapidly demethylated; clearly, this is not the case. A number of explanations may account for inadequate epigenetic remodeling of the donor nucleus. First, the enucleation process may remove significant essential components intimately associated with the MII chromosomes which are required for demethylation. Precedence for an essential component of the mitotic apparatus has been reported in nonhuman primates [89]. Removal of a critical cytoplasm factor may also account for impaired demethylation, but because NT has a low efficiency irrespective of the cloning protocol, this seems unlikely. Continued expression of demethylating proteins can be discounted because very little transcription occurs at this time [90]. This leaves the possibilities that the endogenous Dnmts continue to methylate target sites, coupled with the likely explanation that the chromatin of a differentiated cell differs from that of the diploid zygote and hence is resistant to the demethylating activity of the oocyte. If this were true, one would expect that NT embryos would retain some of the characteristics of the somatic cell donors. Indeed, this is observed in preimplantation NT embryos derived from myoblast donor cells [91]. Curiously, progression of these embryos to the blastocyst stage was favored in media normally used for the culture of myoblasts, indicating a preference for the nutritional requirements of the somatic donor cell. This was supported by the expression of GLUT4 (a myoblast expressed protein) and precocious localization of GLUT1 to the plasma membrane, resulting in enhanced glucose uptake in the cloned embryos, indicat-

PASSIVE DEMETHYLATION
The rapid genome-wide loss of 5-methylcytosine from the paternal genome, with the exception of some elite sequences (e.g., imprinted genes centromeric satellites and some endogenous retroviruses [77]), is followed by a slower passive demethylation of the genome. Passive demethylation is the replicationdependent result of the exclusion of DNA methyltransferase 1 (Dnmt1) during preimplantation development in mammals [78]. The exception to this pattern is the eight-cell stage when Dnmt1o, the oocyte form of the methyltransferase, enters the nucleus. Thereafter, the enzyme remains relegated to the cytoplasm until sometime on day 7 when the somatic form is first detectable [79]. Failed maintenance of methylation on newly synthesized DNA strands accounts for the stepwise decline in DNA methylation, reaching its low point at the morula stage [80]. De novo methylation begins coincident with the first differentiation event within the embryo which establishes the cell lineages that will give rise to the ICM and trophectoderm of the blastocyst. Imprinted genes are exempt from this, and in mouse and human embryos, the oocyte-specific form of Dnmt1 (Dnmt1o) is thought to be involved in the maintenance of the imprint [81]. This is probably of much greater importance to the maternal genome because it has by far the greater number of methylated imprinted genes, but the mechanism by which this methylation is maintained remains unknown. It has been suggested that binding of nonhistone proteins to individual imprinting control regions could prevent their methylation in either the paternal or maternal germ line cells [82]. This hypothesis is supported by the binding of the CCCTC binding factor protein to the unmethylated maternal allele of the H19/Igf2 locus in mouse, which seems to maintain its unmethylated status. Introduction of mutations into the binding site for this protein prevented binding of CCCTC binding factor and led to methylation on the maternal allele [83, 84] although it is unclear whether there are factors in the male germline cells which could direct DNA methylation to this imprinting control region [85] or whether the methylated state is simply established by default. Similarly, it is possible that nonhistone proteins could be responsible for the maintenance of imprinting during embryonic development or to preserve the differentiated state (the cellular memory) of somatic cells. An additional possibility is that

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ing the continuation of somatic gene expression, a hallmark of incomplete memory erasure. There is a growing body of evidence to support such retention of some epigenetic memory of the somatic donor cell [92, 93]. Prime examples are the inefficient reactivation of the pluripotency gene Oct4 in cloned preimplantation embryos as discussed earlier in this text [42, 94] which has been linked to demethylation of the Oct4 promoter [95] and random erasure of methylation from imprinted genes in embryos derived from NTs [61]. The suggestion of a histone code controlling the expression or repression of genes by altering the conformation of the chromatin has gained widespread attention [96, 97]. This code represents an additional layer of information controlling the activity of the genome and involves combinatorial patterns of covalent modifications to the N-terminal tails of histone molecules which potentially provides a heterogeneous identity for each nucleosome within the genome [98]. The protrusion of the histone N-terminal tail sequences into the DNA major groove [99] makes them excellent targets for modifying the binding of control proteins at these sites. Epigenetic marking systems have been shown to mutually reinforce transcription states such that specific histone modifications and DNA methylation based information are likely to co-localize [100, 101]. Is it possible that methylation of somatic-specific genes could be marked by histone N-terminal tail modifications in a manner that also makes them more resistant to reprogramming by the oocyte? Acetylation of H3 and H4 is most commonly linked with active gene expression [102], whereas deacetylation correlates with gene repression and is linked with DNA methylation via methyl CpG binding proteins that attract the transcriptional repressor modeling complexes [103]. However, the critical group of histonemodifying activities are the histone methyl transferases (HMTs). These act during the transition between gene activation and repression at critical lysine residues. Genes actively transcribing are typically acetylated on lysine residues by histone acetyl transferases (HATs). To turn off gene expression, histones are deacetylated by histone deacetylases. These deacetylated residues are substrates for HMT, leaving methyl groups on key lysine residues. This configuration is associated with gene inactivation; however, for heritable gene silencing, a hallmark of differentiated cells, DNA methylation is then targeted and thereafter faithfully maintained with each replication cycle. Mammalian oocytes typically express very low levels of histone deacetylases [104] throughout the preimplantation period and hence favor the sustained transcription SCNT donor genes because gametes are transcriptionally silent during most of the first cell cycle. This may be at odds with the silencing of cell type-specific gene products necessary if reprogramming of the genome and the reinitiation of an embryonic pattern of gene expression occur. Conversely, oocytes do express higher levels of the histone acetyltransferases HAT1 and GCN5, so one might imagine that remodeling of chromatin at inactive genes could be possible although this would potentially require the presence of histone demethylases. Early pluripotency genes like Oct4 would necessarily have to become reactivated involving DNA demethylation as well as chromatin remodeling. There are other histone modifications that are associated with transcription or repression. Methylation of H3 lysine 9 (H3K9) is associated with repression, whereas H3 K4 methylation corresponds to activation. To date, at least five methylatable lysine positions exist in www.StemCells.com

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H3 (K4, K9, K27, K36, and K79) and one on H4 (K20) [105]. Methylation of H3K27, in particular, is an epigenetic mark resulting in the recruitment of polycomb group proteins implicated in gene silencing [106]. An additional layer of complexity is provided by the existence of three distinct methylation states in which the appropriate lysine may be mono-, di-, or trimethylated. These states may be involved in controlling the transcriptional competence of particular loci; at H3K27, for example, both di- and trimethylation are observed but it is only the trimethylated state that recruits polycomb complexes and induced stable gene silencing [107]. To date, only one histone demethylase activity has been described associated to histone lysine 4 methylation and gene activation [108]. Transcriptionally active promoter sequences are associated with H3K4 trimethylation, whereas dimethylation at this position appears to represent a transcription competent state from which transcription does not necessarily take place [109], so it seems that lysine trimethylation represents stability of either expression or silencing. It is uncertain whether the oocyte has the capability to reprogram these types of chromatin modifications. The pattern of asymmetric DNA methylation in the newly fertilized mouse oocyte is also observed for some methylated states of K9 and K27 residues [110]. Although this correlates with the general remodeling of the paternal genome, the activity responsible for inducing this modification in the maternal genome is present at the germinal vesicle stage and disappears soon after fertilization. The specific temporal regulation of this activity was demonstrated experimentally by explanting a male pronucleus that underwent rapid H3K9 methylation when introduced into an enucleated germinal vesicle stage oocyte [111]. Recent reports implicate a number of HMT activities in the oocyte and early embryo with specific sequence targets and nuclear compartments [112]. Perhaps successful reprogramming during NT relies upon inducing histone modifications targeted to critical genomic regions more readily associated with germline resetting and not ordinarily expressed in the oocyte. At present, it is not yet clear whether the oocyte is uniquely competent to remodel and reprogram the wide variety of chromatin modifications, both nucleosomal and otherwise, in a more efficient manner. Perhaps the focus of attention should continue with presenting inherently more compatible donors during SCNT. Irrespective of the limitations to reprogramming, a low number of NT embryos do survive, suggesting that in rare cases it is capable of at least partial resetting of the genome. It may be the case that the reprogramming activity is simply overwhelmed by the enormous task of having to modify or replace somatic histones, remove polycomb complex proteins, and demethylate areas of the genome that may be a lot less accessible than the corresponding areas in gamete-derived genomes. Alternatively, it may be that such reprogramming is actually forbidden for the genomes of somatic cells and it is only when the mechanism controlling this malfunctions that successful clones arise.

EPIGENETIC ALTERATIONS REMODEL SOMATIC NUCLEAR DONORS


Studies in Xenopus have indicated that such repressive complexes do not disassemble easily [113], so it may simply be that although the oocyte attempts to reprogram the somatic genome, there is not enough time during embryonic development for this to be completed. Some insight into this may be derived from

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must apply this basic information to understand the extraordinary situation when the oocyte, challenged with a somatic nucleus, attempts to erase somatic epigenotypes to initiate development. It may even be possible to design strategies for epigenetic intervention which give the reprogramming process a helping hand by partially resetting the epigenotype of the somatic donor cell to a more embryonic state. It is to hoped that ultimately the investigation of epigenetic reprogramming in NT will give us sufficient understanding to manipulate this process in somatic cells by an epigenetic engineering approach so that we can produce therapeutically useful pluripotent cells directly.

studies of cell fusion between differentiated somatic cells and pluripotent ESCs. Monitoring the reactivation of an Oct4-green fluorescent protein transgene in hybrids derived from ESC-thymocytes indicates a loss of the dimethyl H3K9 associated with gene silencing in association with allele-specific reactivation of Oct4 mRNA. Acquisition of the active chromatin mark dimethyl H3K4 accompanied this transition. They conclude that a small number of critical genes are fully competent to establish and maintain a pluripotent epigenotype. Therefore, should Oct4 undergo correct reprogramming, they predict that other key activities will have similar appropriate new chromatin profiles [114].

FUTURE PROSPECTS
The use of SCNT to produce patient-specific ESC lines holds great promise for the development of individually tailored cell replacement therapy and regenerative medicine. However, we must proceed carefully, establishing the balance in which the potential benefits will consistently far outweigh prospective risks. It is clear that significant improvements must be made in understanding the ordinary process whereby an oocyte remodels a sperm nucleus, restoring totipotency to the diploid zygote. We

ACKNOWLEDGMENTS
This work was supported by Medical Research Council UK, One North East, Biotechnology and Biological Sciences Research Council, the Leukemia Research Foundation, and the UK Department of Health (Life Knowledge Park).

DISCLOSURES
The authors indicate no potential conflicts of interest.

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