epigenetic modification is central to genome reprogramming.pdf

Epigenetic Modification Is Central to Genome Reprogrammingin Somatic Cell Nuclear Transfer

LYLE ARMSTRONG,a,b MAJLINDA LAKO,a,b WENDY DEAN,c MIODRAG STOJKOVICa,b

aCentre 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

ABSTRACT

The recent high-profile reports of the derivation of humanembryonic stem cells (ESCs) from human blastocysts pro-duced by somatic cell nuclear transfer (SCNT) have high-lighted the possibility of making autologous cell lines specificto individual patients. Cell replacement therapies have muchpotential for the treatment of diverse conditions, and differ-entiation of ESCs is highly desirable as a means of produc-ing the ranges of cell types required. However, given therange of immunophenotypes of ESC lines currently avail-able, rejection of the differentiated cells by the host is apotentially serious problem. SCNT offers a means of circum-venting this by producing ESCs of the same genotype as thedonor. However, this technique is not without problemsbecause it requires resetting of the gene expression programof a somatic cell to a state consistent with embryonic devel-

opment. Some remodeling of parental DNA does occurwithin the fertilized oocyte, but the somatic genome pre-sented in a radically different format to those of the gametes.Hence, it is perhaps unsurprising that many genes are ex-pressed aberrantly within “cloned” embryos and the ESCsderived from them. Epigenetic modification of the genomethrough DNA methylation and covalent modification of thehistones that form the nucleosome is the key to the mainte-nance of the differentiated state of the cell, and it is this thatmust be reset during SCNT. This review focuses on themechanisms by which this is achieved and how this mayaccount for its partial failure in the “cloning” process. Wealso highlight the potential dangers this may introduce intoESCs produced by this technology. STEM CELLS 2006;24:805–814

AN OVERVIEW OF THE CLONING PROCESSIt has long been known that certain invertebrate species can be“duplicated” or “cloned” simply by dividing them into twopieces and allowing the separated halves to grow into a com-plete organism. However, this cannot be applied to vertebrates.

It is more than 50 years since the groundbreaking studies ofBriggs and King [1–4] demonstrated that somatic cell nucleartransfer (SCNT) could be used to clone frogs. Using oocytes anddonor nuclei from Rana pipiens, they found that the “recon-structed” embryos were capable of development to at least theearly cleavage stages and in some cases as far along as thetadpole stage. The use of blastomere nuclei was possibly instru-mental in this process because such cells are relatively unspe-cialized [5, 6]. In retrospect, it was not surprising that earlyattempts to use SCNT to clone frogs from adult somatic cellsmet with failure. Later work by Gurdon [7] using intestinal cellsfrom tadpoles demonstrated that differentiated somatic cellswere capable of producing viable embryos. These observations

suggested that, in principle, the genome could be “reset” to atotipotent state. In contrast, SCNT in vertebrate cells was rathermore difficult and for many years it was believed that the cellsof adult vertebrates were simply too specialized to revert to atotipotent state. This opinion was decisively contradicted withthe cloning of “Dolly” in 1996 [8] by fusion of a mammarygland epithelial cell from a Finn Dorset ewe with the enucleatedoocyte from a separate donor (Fig. 1). Many studies haveconfirmed the feasibility of SCNT-based cloning of bovine,mouse, and pig [9–11] and that the cytoplasm of oocytes fromsheep, cow, and rabbit is capable of reprogramming somaticcells from other species and supporting the growth of suchinterspecies-cloned embryos to blastocysts [12]. Moreover, therecent success of producing a cloned human blastocyst derivedfrom donated oocytes fused with their associated cumulus cellsprovides further evidence of the broad applicability of the SCNTprocess [13]. These results potentially have paved the way forhuman 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:[email protected] Received July 29, 2005; accepted for publication November 2, 2005; first published online in STEMCELLS EXPRESS November 10, 2005. ©AlphaMed Press 1066-5099/2006/$20.00/0 doi: 10.1634/stemcells.2005-0350

EMBRYONIC STEM CELLS: CONCISE REVIEW

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

bryonic stem cells (ESCs) with the ability to produce essen-tially unlimited supplies of tissues for cell replacement ther-apy [14 –16].

Clearly, there are still many problems associated withSCNT. The majority of cloned embryos (irrespective of species)do not survive to birth, whereas those that do often demonstratea variety of defects that greatly reduce the probability of theirsurvival to adulthood [17–19]. The occurrence of many of thesedefects has been attributed to incomplete reprogramming of thesomatic donor nucleus and a failure of appropriately orches-trated gene expression required for embryonic development [20,21]. In this review, we will concentrate on the mechanisms thatestablish a normal epigenotype and consider the critical areas inwhich these differ between SCNT-derived and naturally fertil-ized embryos.

HOW IS SCNT ACHIEVED?There are two basic strategies for the cloning of mammals bySCNT that are able to produce embryos capable of develop-ment to term. Both of these techniques require the removal ofthe nuclear material from the oocyte and differ only in theway in which the nuclear material of the donor cell isintroduced and the subsequent activation of the reconstructedembryo. Enucleation of the MII oocyte may be achieved by anumber of techniques; the most popular is capillary incisionof the zona pellucida, using a micromanipulator followed byremoval of the polar body and adjacent metaphase chromo-somes by suction into a glass pipette [22]. Additional meth-ods include enucleation by centrifugation [23] and bisectionof the oocyte followed by removal of fragment containing the

nuclear material [24 –26] (the so-called “handmade” cloningmethod). Although this technique has the advantage of sim-plicity, it does remove more oocyte cytoplasm and thereforeit may reduce the amounts of proteins needed for reprogram-ming and early embryonic development.

Introduction of the donor nucleus can be performed using avariety of methods that aim to optimize the successful produc-tion of offspring by altering several factors. These techniquesrely on either microinjection of the donor cell or its isolatednucleus into the oocyte cytoplasm, or fusion of the donor cellwith the enucleated oocyte is achieved through appropriatelytimed electrical pulses. Of key importance is cell cycle syn-chrony of the donor cells. Full-term cloned animals have beenobtained most consistently from donor cells in a quiescent state(G0 or G1), which may be induced in cultured cells by serumstarvation [8, 27] or by using cells, such as granulosa cells, thatare naturally quiescent in the donor organism. The usefulness ofquiescent cells has been attributed to their reduced transcrip-tional activity and chromatin modifications that are associatedwith cells in G0, which may enhance their epigenetic plasticity.Normally quiescent cells (e.g., resting lymphocytes) have lowerlevels of histone methylation than their cycling counterparts, acircumstance that led some workers to suggest that such cellsmay be more easily reprogrammable. The modulation of repres-sive histone modifications such as methylation of lysine 9 onhistone H3 is a facet of genome reprogramming in normalembryos [28] that is not reflected in SCNT. Thus, the markedreduction of this modification in noncycling cells may account,in part, for their greater usefulness in animal cloning. Recentevidence in support of this arises from the observation that when

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

806 Epigenetic Modification in Nuclear Transfer

the nuclei of quiescent T cells from mice carrying the enhancedgreen fluorescent protein (EGFP) transgene (which is normallyinactive in these cells) are transferred into one-cell stage em-bryos, approximately three times as many embryos re-expressedEGFP than when activated T cells were used [29]. An advantageof using G0 synchronized donor cells is that they are diploid, arequisite for normal development of the embryo. Tetraploiddonor cells at G2/M may also be used in a specialized strategyinvolving activation of the reconstructed embryo in the absenceof cytochalasin B which permits expulsion of the excess nuclearmaterial into a pseudo-polar body, thereby returning the ploidyof the embryo to 2C [30].

Not all cells that would normally be nondividing are usefuldonors for SCNT. Neurons and other types of terminally differ-entiated cells are generally rather poor candidates for cloningstudies; this may reflect the lack of developmental plasticity oftheir genomes. Such cells are believed to repress many moregenes than cycling or less differentiated cells and as such arethought to be extremely difficult to reprogram [31].

However, other types of cells that are not terminally differ-entiated may make better donors. There is considerable evidenceto suggest that the use of ESCs as nuclear donors gives rise toviable offspring with greater efficiency than many somatic celltypes [32, 33]. This depends on the particular ESC lines used (inmouse at least) and the number of passages over which theyhave been cultured. ESCs are subject to epigenetic changes withprolonged culture, affecting their ability to contribute to embry-onic development perhaps by modification of genes essential forembryonic and fetal development. Among these classes ofgenes, those associated with pluripotency as well as the parent-of-origin marked imprinted genes have been best studied. Theimportance of such genes to development is highlighted by theunsuitability of primordial germ cells as nuclear donors beyondembryonic day 12; this unsuitability has been attributed to theerasure of parental imprints during gametogenesis [34]. Somaticstem cells are also possible donors as demonstrated by the use ofmesenchymal stem cells from adult mouse bone marrow [35].Interestingly, these cells seem to offer little advantage (in termsof development to term) over more readily accessible cell typessuch as skin fibroblasts.

Whichever method is used to transfer the donor nucleus, itundergoes disassembly in response to the high levels of matu-ration promoting factor (MPF) found in the MII-stage oocytecytoplasm. Reassembly of the nucleus occurs after artificialactivation of the reconstructed oocyte. An extension of the timebetween introduction of the donor nucleus and activation maybe beneficial to the development of live clones [36, 37], al-though this is not absolutely essential. However, there is nodefinite consensus on the effect of MPF levels on somaticnuclear reprogramming. The high MPF activity of MII oocytesinduces nuclear envelope breakdown and premature chromo-some condensation, which have beneficial or harmful effectsupon the reconstructed embryo depending on the cell cycle stageof the somatic donor [38, 39]. Donor cells in S phase alsoundergo premature chromosome condensation, but this results ina “pulverized” chromatin appearance [40] that may cause dam-age to the DNA duplexes of the donor nuclei. Cytoplasts derivedfrom pre-activated oocytes do not induce nuclear envelopebreakdown [41] and premature chromosome condensation, andthus donor cells can be used from any stage of the cell cycle;

however, the rate of progression of these embryos to blastocystsusing this method is low for differentiated somatic cells. Pre-activated cytoplasts work better with blastomeres, which (be-cause these are less developmentally committed) would tend tosuggest that pre-activation removes some of the oocyte-derivedfactors that are capable of reprogramming the somatic genome.The inference from this is that the chromatin modificationsinduced in the somatic genome by premature chromosome con-densation in the MII cytoplast may facilitate its reprogrammingto a totipotent state. It is not clear how long the somatic nucleusshould be exposed to high MPF levels to complete reprogram-ming because various groups report a broad range of findings(as little as 15 minutes [36] to 6 hours [40]) between introduc-tion of the donor cell and activation, but it is possible that thedifferentiation state of the donor cell has a significant impact onthis timing. The nature of the oocyte-derived factors responsiblefor reprogramming is largely unknown, although it is clear fromactivation studies that their existence is transitory [42, 43]. Fromthe point of view of the normally fertilized oocyte, their limitedpersistence is undoubtedly sufficient for the task of rapidlydemethylating the incoming paternal DNA, but the highly dif-ferentiated state of a transplanted somatic donor karyoplast maybe more problematic. Donor cells from early preimplantation-stage embryos may be more easily reprogrammed [44] becausethey are pluripotent and have a lower level of genomic DNAmethylation per blastomere [45]. These nuclei may require lessreprogramming of the genes required for early embryo devel-opment than the types of donor cells which are most accessiblefor the purposes of creating nuclear transfer (NT) ESC lines(e.g., fibroblasts will not share this advantage [46]). Four-cellembryo nuclei, arrested at metaphase, were successfully used togenerate cloned mice by a serial NT technique [47], whichresulted in higher rates of progression to blastocysts (83%) and57% development of offspring to term. It has been suggestedthat such serial transfer allowed more time for reprogrammingof the metaphase nucleus to take place (i.e., the initial repro-gramming undertaken in the enucleated oocyte and up to thefour-cell stage is augmented by another passage of the donornucleus through another round of early embryonic develop-ment). It would be tempting to speculate that a serial transfertechnique would be capable of solving many of the problemsassociated with cloning in mammals by more effectively remov-ing the somatic “memory” of the donor nuclei. However, it hasyet to be established that this technique offers any advantagesfor the isolation of ESC lines for therapeutic applications. Ofcourse, the use of twice the number of oocytes would be a majorobstacle for use in human studies in which oocyte availability islimited. Even if this obstacle were removed, it is essential toensure that any human ESC lines produced by SCNT are capa-ble of use in cell replacement therapy with minimal potentialrisk to the patient. For this reason, it is essential to increase ourunderstanding of the nature of genome reprogramming in bothnormal and SCNT embryos.

EPIGENETIC MODIFICATION IS CENTRALTO REPROGRAMMINGUpon transfer of a somatic nucleus to an oocyte during thecloning process, several essential changes must ensue. First, thesomatic nucleus must cease to express its unique repertoire ofgene products. Second, that nucleus must become subject to the

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instructions provided by the oocyte cytoplasm to unfold a newpattern of development-specific gene transcripts, and third, theheritable memory endowed by the chromatin that ensured thecharacteristics of the donor tissue must be erased. All thesechanges involve a remodeling, not of the underlying geneticsequences that comprise the genome, but of the epigeneticfeatures that overwrite the gene sequences and find interpreta-tion in new gene expression [48].

Epigenetic reprogramming is an essential feature of normaldevelopment and is associated with the erasure of some of theepigenetic modifications inherited from the gametes [49]. Amajor epigenetic modification in mammals is the addition of amethyl group to the 5� position in the symmetrical CpG dinu-cleotide. Alteration of the 5-methylcytosine content of specificregions of the genome is thought to be important in controllinggene expression that must undergo radical changes in bothnormally fertilized embryos and those reconstructed by NT.DNA methylation is thought to be important for the regulationof a number of different groups of genes and genomic sequences[50, 51]. Among the most important are the long terminal repeatof endogenous retroviruses, in which DNA methylation plays anessential role in the silencing of these retrotransposons, therebymaintaining “genome-defense,” the differentially methylated re-gions of imprinted genes, and the inactive X chromosome [52,53]. The pattern of DNA methylation is an indicator of thedifferentiation state of the cell although there is a paucity ofinformation concerning the rules governing this pattern.

Imprinted genes are a unique group of genes that are im-portant for fetal growth and development, especially in theplacenta, as well as for postnatal behavior and cognition. Theexpression of imprinted genes does not follow a mendelianpattern of inheritance but instead depends on the parent-of-origin to dictate its expression [54–57]. Regulatory regions ofsuch genes are typically methylated in the silent allele and areexempt from the large-scale genome-wide demethylation thatoccurs during pre-implantation development. Imprinted genesare particularly sensitive to environmental changes. T9ese canvary in severity from simply altering culture conditions toSCNT [58, 59]. Thus, it is not surprising that embryos producedusing assisted reproduction technologies in humans or NT ofseveral species show widespread methylation defects in im-printed genes [60, 61]. Furthermore, genome-wide patterns havebeen reported to be aberrant on SCNT, with nearly 50% ofcloned bovine and sheep blastocysts showing gross genome-wide errors in both DNA methylation and histone acetylationand a high degree of methylation errors at specific loci [28, 62,63]. This high error rate may represent a potentially fundamentalobstacle and preclude the use of NT to derive patient-specificESCs even though such cells may appear to be pluripotent underour current definitions of their ability to differentiate into mul-tiple cell lineages. Cells may be derived from these lines whichhave the morphological and immunological characteristics thatdefine them as therapeutically useful types, but these may not beable to function in the same way as similar indigenous cellswhen introduced into a patient if they are unable to expresscertain genes at their correct levels.

A contrary viewpoint could be that because many of theepigenetic errors that result from NT affect imprinted genestypically involved in extra embryonic development, they wouldbe less likely to affect the inner cell mass (ICM) of the blasto-

cyst from which ESCs are derived. However, it would besurprising if imprinted genes were the only loci affected byincomplete epigenetic reprogramming given its genome-widerole in controlling gene expression. Indeed, there is evidence tosupport this latter view; microarray analysis has demonstratedthat approximately 4% of a panel of 10,000 murine genesshowed abnormal expression levels in the placenta of NT mice.Perhaps surprising was that although the livers of cloned ani-mals also showed gene dysregulation, this was less extensivethan in the placenta, affecting a different set of genes [61]. In acompanion study, gene expression patterns of NT clones derivedfrom ESCs were compared with clones derived from cumuluscells as the somatic donors. This study found that a smallersubset of genes were affected in clones derived from ESCscompared with clones derived from cumulus cells, in keepingwith the earlier suggestion that embryonic cells may require lessreprogramming to reestablish totipotency. The fact that sucherrors occur at all in the ICM should make us exercise anelement of caution when considering the use of NT-derivedESCs in regenerative medicine.

EPIGENETIC REPROGRAMMING IN NORMALAND NT EMBRYOSAn understanding of the mechanisms that govern epigeneticreprogramming during normal development and how they mightdiffer in the context of SCNT is central to our ambition to deriveepigenetically normal ESCs.

Upon fertilization, there is a series of events that involve theincoming sperm as it encounters the egg cytoplasm. The initialevent after fertilization is the decondensation of the spermnucleus, resulting in the unwinding of the tightly packagedsperm DNA held in a unique, almost toroidal, conformation bythe sperm-specific protamines (Fig. 2) [64]. So highly ordered isthis chromatin organization in sperm that it is effectively dehy-drated, and hence rehydration is an essential, very early event.Upon decondensation, protamines are replaced rapidly by nu-cleohistones derived from the oocyte cytoplasm, usually in thefirst hour after fertilization [65], and the DNA is wound onto thehistone octamers in an ATP-dependent process [66]. It is duringthis same time period that rapid and paternal-specific demeth-ylation of the genome takes place in the absence of transcriptionor DNA synthesis (active demethylation). The octamer of eachnucleosome comprises histones H2A, H2B, H3, and H4, andtheir interaction may be stabilized by the presence of the linkerhistone H1oo (oocyte-specific). This distinct H1 protein remainsassociated with the DNA at least until the two-cell stage of theembryo and is normally replaced with the somatic H1 by thefour-cell stage [67].

This remodeling of the sperm nucleus into an accessible,transcriptionally competent chromatin configuration is coinci-dent with the formation of the pronuclear membrane and de-methylation of the paternal genome. As the end of telophaseapproaches, centromeric proteins A and B, which function aspart of the kinetochore complexes, are assembled onto the DNA.Upon completion of active demethylation, and with the initia-tion of S phase, transcription factors (e.g., TATA box proteinand Sp1) bind to prime the genome for transcription late in thefirst cell cycle [68] (Fig. 3).

The exact nature of the active demethylation is not wellunderstood. Active demethylation is operatively defined as loss


of methylation in the absence of DNA replication. The speedwith which this process occurs strongly suggested that it ismediated enzymatically. Identification of a putative demethyl-ase enzyme(s), especially the active demethylase in the oocyte,has met with some controversy. As yet, the origin of this activityhas not been unequivocally assigned to either the oocyte cyto-

plasm or the sperm itself; however, indirect evidence of partialdemethylation on SCNT points to the activity residing in theoocyte cytoplasm. Demethylation of up to five supernumerarymale pronuclei obtained by polyspermic fertilization of zona-free mouse oocytes suggests a high abundance of this activity[69]. Several candidate proteins have been proposed. Threebasic mechanisms have been proposed. The first and mostsimplistic would involve direct removal of methyl groups fromthe major groove of DNA. Although the mechanism by whichthis 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 attemptsat verification of this result, by two different groups, failed tofind demethylase activity for MBD2. Moreover, immunofluo-rescent analysis of 5methyl cytosine in MBD2 null crossesindicated no difference in the paternal-specific loss of methyl-ation at the one-cell stage [71]. A second possible mechanismenvisages the replacement of 5-methylcytosine by cytosine orremoval of the CpG dinucleotide by either base or nucleotideexcision repair [72]. For this reason, the uridine deglycosylaseenzyme methyl binding domain protein binding 4 (MBD4) wasproposed as a potential demethylase because of its role in DNArepair [73] although paternal-specific demethylation appeared tooccur normally in MDB4 null fertilized oocytes [69]. A thirdpossibility proposes hydrolytic deamination of 5-methylcytosineresulting in the conversion of 5MeC to thymidine; however, thisprocess would require considerable energy input and as such isthe least likely mechanism. A recent report of enzymatic deami-

Figure 2. Remodeling of paternal chromatin after fertilization until the first cell division. Sperm DNA is highly compacted due to association withprotamine. 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 ofoocyte-specific histone H1oo, and recruitment of nonhistone proteins to prepare DNA for transcription.

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


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nation by Aid during T-cell development has introduced thepossibly that this activity is also important during the first cellcycle [74].

Mechanistically, the loss of a methyl group poses a consid-erable enzymatic challenge, and these activities and their epi-genetic regulation are a research “hotspot” at present. Why theembryo needs to actively demethylate the paternal genome sorapidly after fertilization remains a mystery. It has been sug-gested that de-repression of a number of paternal alleles isrequired to accommodate the burst of transcriptional activitythat occurs at the end of the first cell cycle [75]. An alternativehypothesis has been suggested largely focusing on imprintedalleles. It suggests that active demethylation arose as a protec-tive response of the maternal genome to reduce the influence ofthe paternal genome, which may have alleles optimized to theproduction of larger, more competitive offspring, thus servingthe maternal interest in ensuring survival of larger numbers ofoffspring overall [76].

PASSIVE DEMETHYLATIONThe rapid genome-wide loss of 5-methylcytosine from the pa-ternal genome, with the exception of some elite sequences (e.g.,imprinted genes centromeric satellites and some endogenousretroviruses [77]), is followed by a slower passive demethyl-ation of the genome. Passive demethylation is the replication-dependent 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 whenDnmt1o, the oocyte form of the methyltransferase, enters thenucleus. Thereafter, the enzyme remains relegated to the cyto-plasm until sometime on day 7 when the somatic form is firstdetectable [79]. Failed maintenance of methylation on newlysynthesized DNA strands accounts for the stepwise decline inDNA methylation, reaching its low point at the morula stage[80]. De novo methylation begins coincident with the firstdifferentiation event within the embryo which establishes thecell lineages that will give rise to the ICM and trophectoderm ofthe blastocyst. Imprinted genes are exempt from this, and inmouse and human embryos, the oocyte-specific form of Dnmt1(Dnmt1o) is thought to be involved in the maintenance of theimprint [81]. This is probably of much greater importance to thematernal genome because it has by far the greater number ofmethylated imprinted genes, but the mechanism by which thismethylation is maintained remains unknown. It has been sug-gested that binding of nonhistone proteins to individual imprint-ing control regions could prevent their methylation in either thepaternal or maternal germ line cells [82]. This hypothesis issupported by the binding of the CCCTC binding factor proteinto the unmethylated maternal allele of the H19/Igf2 locus inmouse, which seems to maintain its unmethylated status. Intro-duction of mutations into the binding site for this protein pre-vented binding of CCCTC binding factor and led to methylationon the maternal allele [83, 84] although it is unclear whetherthere are factors in the male germline cells which could directDNA methylation to this imprinting control region [85] orwhether the methylated state is simply established by default.Similarly, it is possible that nonhistone proteins could be re-sponsible for the maintenance of imprinting during embryonicdevelopment or to preserve the differentiated state (the cellularmemory) of somatic cells. An additional possibility is that

histone modifications on the chromatin associated with themethylated allele provide a specific mark that ensures mainte-nance of methylation. The imprint control regions of the inactive(methylated alleles) are known to have methylation of lysine 9on histone H3 (methyl H3K9) and hypoacetylation of H3 andH4, whereas the active (unmethylated allele) is characterized byH3/H4 hyperacetylation and H3 K4 methylation [86]. As yet, nounderlying biochemical mechanism has been described to linkthese histone modifications to any specific means for mainte-nance of chromatin states in imprinted and nonimprinted re-gions alike.

EPIGENETIC INFORMATION FIDELITY FAILSDURING CLONINGImprinted DNA methylation of loci is very often disrupted inNT embryos, affecting the extra-embryonic tissues more fre-quently than those of the embryo. However, although this mayhave certain consequences for embryonic survival and/orgrowth, imprinted loci do not represent the bulk of the mam-malian genome, with more than 70 imprinted loci known to date[http://www.mgu.har.mrc.ac.uk/research/imprinting]. The stud-ies of several groups have shown that the somatic genome usedin NT does not respond so readily to the demethylation activityof the oocyte [63, 87, 88], and in most cases the level ofmethylated 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, theonset of de novo methylation frequently begins much earlier(four-cell stage) than in normal embryos, suggesting that incom-plete remodeling of the donor nucleus impairs the normal tem-poral progression of epigenetic reprogramming leading to tran-scriptional misregulation. One might expect that given theapparent abundance of demethylating activity in the oocyte, thesomatic genome would be rapidly demethylated; clearly, this isnot the case. A number of explanations may account for inad-equate epigenetic remodeling of the donor nucleus. First, theenucleation process may remove significant essential compo-nents intimately associated with the MII chromosomes whichare required for demethylation. Precedence for an essentialcomponent of the mitotic apparatus has been reported in non-human primates [89]. Removal of a critical cytoplasm factormay also account for impaired demethylation, but because NThas a low efficiency irrespective of the cloning protocol, thisseems unlikely. Continued expression of demethylating proteinscan be discounted because very little transcription occurs at thistime [90]. This leaves the possibilities that the endogenousDnmts continue to methylate target sites, coupled with the likelyexplanation that the chromatin of a differentiated cell differsfrom that of the diploid zygote and hence is resistant to thedemethylating activity of the oocyte. If this were true, onewould expect that NT embryos would retain some of the char-acteristics of the somatic cell donors. Indeed, this is observed inpreimplantation NT embryos derived from myoblast donor cells[91]. Curiously, progression of these embryos to the blastocyststage was favored in media normally used for the culture ofmyoblasts, indicating a preference for the nutritional require-ments of the somatic donor cell. This was supported by theexpression of GLUT4 (a myoblast expressed protein) and pre-cocious localization of GLUT1 to the plasma membrane, result-ing in enhanced glucose uptake in the cloned embryos, indicat-


ing the continuation of somatic gene expression, a hallmark ofincomplete memory erasure. There is a growing body of evi-dence to support such retention of some epigenetic memory ofthe somatic donor cell [92, 93]. Prime examples are the ineffi-cient reactivation of the pluripotency gene Oct4 in cloned pre-implantation 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 inembryos derived from NTs [61].

The suggestion of a “histone code” controlling the expres-sion or repression of genes by altering the conformation of thechromatin has gained widespread attention [96, 97]. This coderepresents an additional layer of information controlling theactivity of the genome and involves combinatorial patterns ofcovalent modifications to the N-terminal tails of histone mole-cules which potentially provides a heterogeneous identity foreach nucleosome within the genome [98]. The protrusion of thehistone N-terminal tail sequences into the DNA major groove[99] makes them excellent targets for modifying the binding ofcontrol proteins at these sites. Epigenetic marking systems havebeen shown to mutually reinforce transcription states such thatspecific histone modifications and DNA methylation based in-formation are likely to co-localize [100, 101]. Is it possible thatmethylation of somatic-specific genes could be marked by his-tone N-terminal tail modifications in a manner that also makesthem more resistant to reprogramming by the oocyte? Acetyla-tion of H3 and H4 is most commonly linked with active geneexpression [102], whereas deacetylation correlates with generepression and is linked with DNA methylation via methyl CpGbinding proteins that attract the transcriptional repressor mod-eling complexes [103]. However, the critical group of histone-modifying activities are the histone methyl transferases (HMTs).These act during the transition between gene activation andrepression at critical lysine residues. Genes actively transcribingare typically acetylated on lysine residues by histone acetyltransferases (HATs). To turn off gene expression, histones aredeacetylated by histone deacetylases. These deacetylated resi-dues are substrates for HMT, leaving methyl groups on keylysine residues. This configuration is associated with gene in-activation; however, for heritable gene silencing, a hallmark ofdifferentiated cells, DNA methylation is then targeted and there-after faithfully maintained with each replication cycle. Mamma-lian oocytes typically express very low levels of histonedeacetylases [104] throughout the preimplantation period andhence favor the sustained transcription SCNT donor genes be-cause gametes are transcriptionally silent during most of the firstcell cycle. This may be at odds with the silencing of celltype-specific gene products necessary if reprogramming of thegenome and the reinitiation of an embryonic pattern of geneexpression occur. Conversely, oocytes do express higher levelsof the histone acetyltransferases HAT1 and GCN5, so one mightimagine that remodeling of chromatin at inactive genes could bepossible although this would potentially require the presence ofhistone demethylases. Early pluripotency genes like Oct4 wouldnecessarily have to become reactivated involving DNA demeth-ylation as well as chromatin remodeling. There are other histonemodifications that are associated with transcription or repres-sion. Methylation of H3 lysine 9 (H3K9) is associated withrepression, whereas H3 K4 methylation corresponds to activa-tion. To date, at least five methylatable lysine positions exist in

H3 (K4, K9, K27, K36, and K79) and one on H4 (K20) [105].Methylation of H3K27, in particular, is an epigenetic markresulting in the recruitment of polycomb group proteins impli-cated in gene silencing [106]. An additional layer of complexityis provided by the existence of three distinct methylation statesin which the appropriate lysine may be mono-, di-, or trimethy-lated. These states may be involved in controlling the transcrip-tional “competence” of particular loci; at H3K27, for example,both di- and trimethylation are observed but it is only thetrimethylated state that recruits polycomb complexes and in-duced stable gene silencing [107]. To date, only one histonedemethylase activity has been described associated to histonelysine 4 methylation and gene activation [108]. Transcription-ally active promoter sequences are associated with H3K4 trim-ethylation, whereas dimethylation at this position appears torepresent a transcription competent state from which transcrip-tion does not necessarily take place [109], so it seems that lysinetrimethylation represents stability of either expression or silenc-ing. It is uncertain whether the oocyte has the capability toreprogram these types of chromatin modifications.

The pattern of asymmetric DNA methylation in the newlyfertilized mouse oocyte is also observed for some methylatedstates of K9 and K27 residues [110]. Although this correlateswith the general remodeling of the paternal genome, the activityresponsible for inducing this modification in the maternal ge-nome is present at the germinal vesicle stage and disappearssoon after fertilization. The specific temporal regulation of thisactivity was demonstrated experimentally by explanting a malepronucleus that underwent rapid H3K9 methylation when intro-duced into an enucleated germinal vesicle stage oocyte [111].Recent reports implicate a number of HMT activities in theoocyte and early embryo with specific sequence targets andnuclear compartments [112]. Perhaps successful reprogrammingduring NT relies upon inducing histone modifications targetedto critical genomic regions more readily associated with germ-line resetting and not ordinarily expressed in the oocyte.

At present, it is not yet clear whether the oocyte is uniquelycompetent to remodel and reprogram the wide variety of chro-matin modifications, both nucleosomal and otherwise, in a moreefficient manner. Perhaps the focus of attention should continuewith presenting inherently more compatible donors duringSCNT. Irrespective of the limitations to reprogramming, a lownumber of NT embryos do survive, suggesting that in rare casesit is capable of at least partial resetting of the genome. It may bethe case that the reprogramming activity is simply overwhelmedby the enormous task of having to modify or replace somatichistones, remove polycomb complex proteins, and demethylateareas of the genome that may be a lot less accessible than thecorresponding areas in gamete-derived genomes. Alternatively,it may be that such reprogramming is actually “forbidden” forthe genomes of somatic cells and it is only when the mechanismcontrolling this malfunctions that successful clones arise.

EPIGENETIC ALTERATIONS REMODEL SOMATICNUCLEAR DONORSStudies in Xenopus have indicated that such repressive com-plexes do not disassemble easily [113], so it may simply be thatalthough the oocyte attempts to reprogram the somatic genome,there is not enough time during embryonic development for thisto be completed. Some insight into this may be derived from


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studies of cell fusion between differentiated somatic cells andpluripotent ESCs. Monitoring the reactivation of an Oct4-greenfluorescent protein transgene in hybrids derived from ESC-thymo-cytes indicates a loss of the dimethyl H3K9 associated with genesilencing in association with allele-specific reactivation of Oct4mRNA. Acquisition of the active chromatin mark dimethyl H3K4accompanied this transition. They conclude that a small number ofcritical genes are fully competent to establish and maintain apluripotent epigenotype. Therefore, should Oct4 undergo correctreprogramming, they predict that other key activities will havesimilar appropriate new chromatin profiles [114].

FUTURE PROSPECTSThe use of SCNT to produce patient-specific ESC lines holds greatpromise for the development of individually “tailored” cell replace-ment therapy and regenerative medicine. However, we must pro-ceed carefully, establishing the balance in which the potentialbenefits will consistently far outweigh prospective risks.

It is clear that significant improvements must be made inunderstanding the ordinary process whereby an oocyte remodelsa sperm nucleus, restoring totipotency to the diploid zygote. We

must apply this basic information to understand the extraordi-nary situation when the oocyte, challenged with a somaticnucleus, attempts to erase somatic epigenotypes to initiate de-velopment. It may even be possible to design strategies forepigenetic intervention which give the reprogramming process a“helping hand” by partially resetting the epigenotype of thesomatic donor cell to a more embryonic state. It is to hoped thatultimately the investigation of epigenetic reprogramming in NTwill give us sufficient understanding to manipulate this processin somatic cells by an “epigenetic engineering” approach so thatwe can produce therapeutically useful pluripotent cells directly.

ACKNOWLEDGMENTSThis work was supported by Medical Research Council UK,One North East, Biotechnology and Biological Sciences Re-search Council, the Leukemia Research Foundation, and the UKDepartment of Health (Life Knowledge Park).

DISCLOSURESThe authors indicate no potential conflicts of interest.

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