The effects of culture on genomic imprinting profiles in human embryonic and fetal mesenchymal stem cells

Human embryonic stem (hES) cells and fetal mesenchymal stem cells (fMSC) offer great potential for regenerative therapy strategies. It is therefore important to characterise the properties of these cells in vitro. One major way the environment impacts on cellular physiology is through changes to epigenetic mechanisms. Genes subject to epigenetic regulation via genomic imprinting have been characterised extensively. The integrity of imprinted gene expression therefore provides a measurable index for epigenetic stability. Allelic expression of 26 imprinted genes and DNA methylation at associated differentially methylated regions (DMRs) was measured in fMSC and hES cell lines. Both cell types exhibited monoallelic expression of 13 imprinted genes, biallelic expression of six imprinted genes, and there were seven genes that differed in allelic expression between cell lines. fMSCs exhibited the differential DNA methylation patterns associated with imprinted expression. This was unexpected given that gene expression of several imprinted genes was biallelic. However, in hES cells, differential methylation was perturbed. These atypical methylation patterns did not correlate with allelic expression. Our results suggest that regardless of stem cell origin, in vitro culture affects the integrity of imprinted gene expression in human cells. We identify biallelic and variably expressed genes that may inform on overall epigenetic stability. As differential methylation did not correlate with imprinted expression changes we propose that other epigenetic effectors are adversely influenced by the in vitro environment. Since DMR integrity was maintained in fMSC but not hES cells, we postulate that specific hES cell derivation and culturing practices result in changes in methylation at DMRs.     

The effects of culture on genomic imprinting profiles in human embryonic and fetal mesenchymal stem cells

Human embryonic stem (hES) cells and fetal mesenchymal stem cells (fMSC) offer great potential for regenerative therapy strategies. It is therefore important to characterize the properties of these cells in vitro. One major way the environment impacts on cellular physiology is through changes to epigenetic mechanisms. Genes subject to epigenetic regulation via genomic imprinting have been characterized extensively. The integrity of imprinted gene expression therefore provides a measurable index for epigenetic stability. Allelic expression of 26 imprinted genes and DNA methylation at associated differentially methylated regions (DMRs) was measured in fMSC and hES cell lines. Both cell types exhibited monoallelic expression of 13 imprinted genes, biallelic expression of six imprinted genes, and there were seven genes that differed in allelic expression between cell lines. fMSC s exhibited the differential DNA methylation patterns associated with imprinted expression. This was unexpected given that gene expression of several imprinted genes was biallelic. However, in hES cells, differential methylation was perturbed. These atypical methylation patterns did not correlate with allelic expression. Our results suggest that regardless of stem cell origin, in vitro culture affects the integrity of imprinted gene expression in human cells. We identify biallelic and variably expressed genes that may inform on overall epigenetic stability. As differential methylation did not correlate with imprinted expression changes we propose that other epigenetic effectors are adversely influenced by the in vitro environment. Since DMR integrity was maintained in fMSC but not hES cells, we postulate that specific hES cell derivation and culturing practices result in changes in methylation at DMRs.Key words: genomic imprinting, embryonic stem cells, mesenchymal stem cells, differentiation, methylation, epigenetic stability     

Differentially methylated regions of imprinted genes in prenatal, perinatal and postnatal human tissues

Epigenetic plasticity in relation to in utero exposures may mechanistically explain observed differences in the likelihood of developing common complex diseases including hypertension, diabetes and cardiovascular disease through the cumulative effects of subtle alterations in gene expression. Imprinted genes are essential mediators of growth and development and are characterized by differentially methylated regulatory regions (DMRs) that carry parental allele-specific methylation profiles. This theoretical 50% level of methylation provides a baseline from which endogenously- or exogenously-induced deviations in methylation can be detected. We quantified DNA methylation at imprinted gene DMRs in a large panel of human conceptal tissues, in matched buccal cell specimens collected at birth and at one year of age, and in the major cell fractions of umbilical cord blood to assess the stability of methylation at these regions. DNA methylation was measured using validated pyrosequencing assays at seven DMRs regulating the IGF2/H19, DLK1/MEG3, MEST, NNAT and SGCE/PEG10 imprinted domains. DMR methylation did not significantly differ for the H19, MEST and SGCE/PEG10 DMRs across all conceptal tissues analyzed (ANOVA p>0.10). Methylation differences at several DMRs were observed in tissues from brain (IGF2 and MEG3-IG DMRs), liver (IGF2 and MEG3 DMRs) and placenta (both DLK1/MEG3 DMRs and NNAT DMR). In most infants, methylation profiles in buccal cells at birth and at one year of age were comparable, as was methylation in the major cell fractions of umbilical cord blood. Several infants showed temporal deviations in methylation at multiple DMRs. Similarity of inter-individual and intra-individual methylation at some, but not all of the DMRs analyzed supports the possibility that methylation of these regions can serve as useful biosensors of exposure.     

Establishment of paternal allele-specific DNA methylation at the imprinted mouse Gtl2 locus

The monoallelic expression of imprinted genes is controlled by epigenetic factors including DNA methylation and histone modifications. In mouse, the imprinted gene Gtl2 is associated with two differentially methylated regions: the IG-DMR, which serves as a gametic imprinting mark at which paternal allele-specific DNA methylation is inherited from sperm, and the Gtl2-DMR, which acquires DNA methylation on the paternal allele after fertilization. The timeframe during which DNA methylation is acquired at secondary DMRs during post-fertilization development and the relationship between secondary DMRs and imprinted expression have not been well established. In order to better understand the role of secondary DMRs in imprinting, we examined the methylation status of the Gtl2-DMR in pre- and post-implantation embryos. Paternal allele-specific DNA methylation of this region correlates with imprinted expression of Gtl2 during post-implantation development but is not required to implement imprinted expression during pre-implantation development, suggesting that this secondary DMR may play a role in maintaining imprinted expression. Furthermore, our developmental profile of DNA methylation patterns at the Cdkn1c- and Gtl2-DMRs illustrates that the temporal acquisition of DNA methylation at imprinted genes during post-fertilization development is not universally controlled.     

DHPLC-based method for DNA methylation analysis of differential methylated regions from imprinted genes

The bisulfite genomic sequencing method is one of the most widely used techniques for methylation analysis in heterogeneous unbiased PCR, amplifying for both methylated and unmethylated alleles simultaneously. However, it requires labor-intensive and time-consuming cloning and sequencing steps. In the current study, we used a denaturing high-performance liquid chromatography (DHPLC) procedure in a complementary way with the bisulfite genomic sequencing to analyze the methylation of differentially methylated regions (DMRs) of imprinted genes. We showed reliable and reproducible results in distinguishing overall methylation profiles of DMRs regions of human SNRPN, H19, MEST/PEG1, LIT1, IGF2, TSSC5, WT1 antisense, and mouse H19, Mest/Peg1, Igf2R imprinted genes. These DHPLC profiles were in accordance with bisulfite genomic sequencing data and may serve as a type of "fingerprint," revealing the overall methylation status of DMRs associated with sample heterogeneity. We conclude that DHPLC analysis could be used to increase the throughput efficiency of methylation pattern analysis of imprinted genes after the bisulfite conversion of genomic DNA and unbiased PCR amplification.     

Establishment of paternal allele-specific DNA methylation at the imprinted mouse Gtl2 locus

The monoallelic expression of imprinted genes is controlled by epigenetic factors including DNA methylation and histone modifications. In mouse, the imprinted gene Gtl2 is associated with two differentially methylated regions: the IG-DMR, which serves as a gametic imprinting mark at which paternal allele-specific DNA methylation is inherited from sperm, and the Gtl2-DMR, which acquires DNA methylation on the paternal allele after fertilization. The timeframe during which DNA methylation is acquired at secondary DMRs during post-fertilization development and the relationship between secondary DMRs and imprinted expression have not been well established. In order to better understand the role of secondary DMRs in imprinting, we examined the methylation status of the Gtl2-DMR in pre- and post-implantation embryos. Paternal allele-specific DNA methylation of this region correlates with imprinted expression of Gtl2 during post-implantation development but is not required to implement imprinted expression during pre-implantation development, suggesting that this secondary DMR may play a role in maintaining imprinted expression. Furthermore, our developmental profile of DNA methylation patterns at the Cdkn1c- and Gtl2-DMRs illustrates that the temporal acquisition of DNA methylation at imprinted genes during post-fertilization development is not universally controlled.Key words: genomic imprinting, DNA methylation, Gtl2, secondary DMR, epigenetics     

Germline-derived DNA methylation and early embryo epigenetic reprogramming: The selected survival of imprints

DNA methylation is an essential epigenetic mechanism involved in many essential cellular processes. During development epigenetic reprograming takes place during gametogenesis and then again in the pre-implantation embryo. These two reprograming windows ensure genome-wide removal of methylation in the primordial germ cells so that sex-specific signatures can be acquired in the sperm and oocyte. Following fertilization the majority of this epigenetic information is erased to give the developing embryo an epigenetic profile coherent with pluripotency. It is estimated that ∼65% of the genome is differentially methylated between the gametes, however following embryonic reprogramming only parent-of-origin methylation at known imprinted loci remains. This suggests that trans-acting factors such as Zfp57 can discriminate imprinted differentially methylated regions (DMRs) from the thousands of CpG rich regions that are differentially marked in the gametes. Recently transient imprinted DMRs have been identified suggesting that these loci are also protected from pre-implantation reprograming but succumb to de novo remethylation at the implantation stage. This highlights that “ubiquitous” imprinted loci are also resilient to gaining methylation by protecting their unmethylated alleles. In this review I examine the processes involved in epigenetic reprograming and the mechanisms that ensure allelic methylation at imprinted loci is retained throughout the life of the organism, discussing the critical differences between mouse and humans.This article is part of a Directed Issue entitled: Epigenetics Dynamics in development and disease.     

Bovine DNA methylation imprints are established in an oocyte size-specific manner, which are coordinated with the expression of the DNMT3 family proteins

A subset of genes, known as imprinted genes, is present in the mammalian genome. Genomic imprinting governs the monoallelic expression of these genes, depending on whether the gene was inherited from the sperm or the egg. This parent-of-origin specific gene expression is generally dependent on the epigenetic modification, DNA methylation, and the DNA methylation status of CpG dinucleotides residing in loci known as differentially methylated regions (DMRs). The enzymatic machinery responsible for the addition of methyl (-CH(3)) groups to the cytosine residue in the CpG dinucleotides are known as DNA methyltransferases (DNMTs). Correct establishment and maintenance of methylation patterns at imprinted genes has been associated with placental function and regulation of embryonic/fetal development. Much work has been carried out on imprinted genes in mouse and human; however, little is known about the methylation dynamics in the bovine oocyte. The primary objective of the present study was to characterize the establishment of methylation at maternally imprinted genes in bovine growing oocytes and to determine if the expression of the bovine DNMTs-DNMT3A, DNMT3B, and DNMT3L-was coordinated with DNA methylation during oocyte development. To this end, a panel of maternally imprinted genes was selected (SNRPN, MEST, IGF2R, PEG10, and PLAGL1) and putative DMRs for MEST, IGF2R, PEG10, and PLAGL1 were identified within the 5' regions for each gene; the SNRPN DMR has been reported previously. Conventional bisulfite sequencing revealed that methylation marks were acquired at all five DMRs investigated in an oocyte size-dependent fashion. This was confirmed for a selection of genes using pyrosequencing analysis. Furthermore, mRNA expression and protein analysis revealed that DNMT3A, DNMT3B, and DNMT3L are also present in the bovine oocyte during its growth phase. This study demonstrates for the first time that an increase in bovine imprinted gene DMR methylation occurs during oocyte growth, as is observed in mouse.     

Placental 5-methylcytosine and 5-hydroxymethylcytosine patterns associate with size at birth

Altered placental function as a consequence of aberrant imprinted gene expression may be one mechanism mediating the association between low birth weight and increased cardiometabolic disease risk. Imprinted gene expression is regulated by epigenetic mechanisms, particularly DNA methylation (5mC) at differentially methylated regions (DMRs). While 5-hydroxymethylcytosine (5hmC) is also present at DMRs, many techniques do not distinguish between 5mC and 5hmC. Using human placental samples, we show that the expression of the imprinted gene CDKN1C associates with birth weight. Using specific techniques to map 5mC and 5hmC at DMRs controlling the expression of CDKN1C and the imprinted gene IGF2, we show that 5mC enrichment at KvDMR and DMR0, and 5hmC enrichment within the H19 gene body, associate positively with birth weight. Importantly, the presence of 5hmC at imprinted DMRs may complicate the interpretation of DNA methylation studies in placenta; future studies should consider using techniques that distinguish between, and permit quantification of, both modifications.     

Methylation variation at IGF2 differentially methylated regions and maternal folic acid use before and during pregnancy

Folic acid (FA) supplementation before and during pregnancy has been associated with decreased risk of neural tube defects although recent reports suggest it may also increase the risk of other chronic diseases. We evaluated exposure to maternal FA supplementation before and during pregnancy in relation to aberrant DNA methylation at two differentially methylated regions (DMRs) regulating Insulin-like Growth Factor 2 (IGF2) expression in infants. Aberrant methylation at these regions has been associated with IGF2 deregulation and increased susceptibility to several chronic diseases. Using a self-administered questionnaire, we assessed FA intake before and during pregnancy in 438 pregnant women. Pyrosequencing was used to measure methylation at two IGF2 DMRs in umbilical cord blood leukocytes. Mixed models were used to determine relationships between maternal FA supplementation before or during pregnancy and DNA methylation levels at birth. Average methylation at the H19 DMR was 61.2%. Compared to infants born to women reporting no FA intake before or during pregnancy, methylation levels at the H19 DMR decreased with increasing FA intake (2.8%, p=0.03, and 4.9%, p=0.04, for intake before and during pregnancy, respectively). This methylation decrease was most pronounced in male infants (p=0.01). Methylation alterations at the H19 DMR are likely an important mechanism by which FA risks and/or benefits are conferred in utero. Because stable methylation marks at DMRs regulating imprinted genes are acquired before gastrulation, they may serve as archives of early exposures with the potential to improve our understanding of developmental origins of adult disease.     

Dynamic stage-specific changes in imprinted differentially methylated regions during early mammalian development and prevalence of non-CpG methylation in oocytes

Mammalian imprinted genes are associated with differentially methylated regions (DMRs) that are CpG methylated on one of the two parental chromosomes. In mice, at least 21 DMRs acquire differential methylation in the germline and many of them act as imprint centres. We previously reported the physical extents of differential methylation at 15 DMRs in mouse embryos at 12.5 days postcoitum. To reveal the ontogeny of differential methylation, we determined and compared methylation patterns of the corresponding regions in sperm and oocytes. We found that the extent of the gametic DMRs differs significantly from that of the embryonic DMRs, especially in the case of paternal gametic DMRs. These results suggest that the gametic DMR sequences should be used to extract the features specifying methylation imprint establishment in the germline: from this analysis, we noted that the maternal gametic DMRs appear as unmethylated islands in male germ cells, which suggests a novel component in the mechanism of gamete-specific marking. Analysis of selected DMRs in blastocysts revealed dynamic changes in allelic methylation in early development, indicating that DMRs are not fully protected from the major epigenetic reprogramming events occurring during preimplantation development. Furthermore, we observed non-CpG methylation in oocytes, but not in sperm, which disappeared by the blastocyst stage. Non-CpG methylation was frequently found at maternally methylated DMRs as well as non-DMR regions, suggesting its prevalence in the oocyte genome. These results provide evidence for a unique methylation profile in oocytes and reveal the surprisingly dynamic nature of DMRs in the early embryo.     

Differentially methylated regions in maternal and paternal uniparental disomy for chromosome 7

DNA methylation is a hallmark of genomic imprinting and differentially methylated regions (DMRs) are found near and in imprinted genes. Imprinted genes are expressed only from the maternal or paternal allele and their normal balance can be disrupted by uniparental disomy (UPD), the inheritance of both chromosomes of a chromosome pair exclusively from only either the mother or the father. Maternal UPD for chromosome 7 (matUPD7) results in Silver-Russell syndrome (SRS) with typical features and growth retardation, but no gene has been conclusively implicated in SRS. In order to identify novel DMRs and putative imprinted genes on chromosome 7, we analyzed eight matUPD7 patients, a segmental matUPD7q31-qter, a rare patUPD7 case and ten controls on the Infinium HumanMethylation450K BeadChip with 30?017 CpG methylation probes for chromosome 7. Genome-scale analysis showed highly significant clustering of DMRs only on chromosome 7, including the known imprinted loci GRB10, SGCE/PEG10, and PEG/MEST. We found ten novel DMRs on chromosome 7, two DMRs for the predicted imprinted genes HOXA4 and GLI3 and one for the disputed imprinted gene PON1. Quantitative RT-PCR on blood RNA samples comparing matUPD7, patUPD7, and controls showed differential expression for three genes with novel DMRs, HOXA4, GLI3, and SVOPL. Allele specific expression analysis confirmed maternal only expression of SVOPL and imprinting of HOXA4 was supported by monoallelic expression. These results present the first comprehensive map of parent-of-origin specific DMRs on human chromosome 7, suggesting many new imprinted sites.     

Differentially methylated regions in maternal and paternal uniparental disomy for chromosome 7

DNA methylation is a hallmark of genomic imprinting and differentially methylated regions (DMRs) are found near and in imprinted genes. Imprinted genes are expressed only from the maternal or paternal allele and their normal balance can be disrupted by uniparental disomy (UPD), the inheritance of both chromosomes of a chromosome pair exclusively from only either the mother or the father. Maternal UPD for chromosome 7 (matUPD7) results in Silver-Russell syndrome (SRS) with typical features and growth retardation, but no gene has been conclusively implicated in SRS. In order to identify novel DMRs and putative imprinted genes on chromosome 7, we analyzed eight matUPD7 patients, a segmental matUPD7q31-qter, a rare patUPD7 case and ten controls on the Infinium HumanMethylation450K BeadChip with 30 017 CpG methylation probes for chromosome 7. Genome-scale analysis showed highly significant clustering of DMRs only on chromosome 7, including the known imprinted loci GRB10, SGCE/PEG10, and PEG/MEST. We found ten novel DMRs on chromosome 7, two DMRs for the predicted imprinted genes HOXA4 and GLI3 and one for the disputed imprinted gene PON1. Quantitative RT-PCR on blood RNA samples comparing matUPD7, patUPD7, and controls showed differential expression for three genes with novel DMRs, HOXA4, GLI3, and SVOPL. Allele specific expression analysis confirmed maternal only expression of SVOPL and imprinting of HOXA4 was supported by monoallelic expression. These results present the first comprehensive map of parent-of-origin specific DMRs on human chromosome 7, suggesting many new imprinted sites.     

DNA methylation variability at growth-related imprints does not contribute to overweight in monozygotic twins discordant for BMI

Defective genomic imprinting is often associated with syndromes that include abnormal growth as a clinical phenotype. However, whether differential methylation at imprinted loci also contributes to nonsyndromic abnormal body weight regulation is yet unknown. In this study, we investigated a potential contribution of aberrant DNA methylation at nine differentially methylated regions (DMRs) to the development of nonsyndromic overweight. Sixteen monozygotic (MZ) twins discordant for BMI (BMI difference ranging from 2.9-9.5 kg/m(2)) were recruited from the East Flanders Prospective Twin Survey. DNA extracted from saliva samples was bisulfite-treated followed by PCR amplification of target regions in DMRs most representative for abnormal growth syndromes: KvDMR1, H19 CTCF4, H19 CTCF6, IGF2 DMR0, IGF2 DMR2, GRB10, MEST, SNRPN, GNAS XL-α-s and GNAS Exon1A. At the DMRs analyzed, methylation-dependent primer extension experiments revealed only small intrapair differences in methylation indexes (MI) between the heavy and lean co-twins. In addition, no significant correlations between intrapair BMI differences and intrapair differences in MI were observed. In conclusion, DNA methylation variability at the nine DMRs analyzed does not seem to contribute to the discordancy in BMI observed in these MZ twins.     

Bisulfite sequencing and dinucleotide content analysis of 15 imprinted mouse differentially methylated regions (DMRs): paternally methylated DMRs contain less CpGs than maternally methylated DMRs

Imprinted genes in mammals show monoallelic expression dependent on parental origin and are often associated with differentially methylated regions (DMRs). There are two classes of DMR: primary DMRs acquire gamete-specific methylation in either spermatogenesis or oogenesis and maintain the allelic methylation differences throughout development; secondary DMRs establish differential methylation patterns after fertilization. Targeted disruption of some primary DMRs showed that they dictate the allelic expression of nearby imprinted genes and the establishment of the allelic methylation of secondary DMRs. However, how primary DMRs are recognized by the imprinting machinery is unknown. As a step toward elucidating the sequence features of the primary DMRs, we have determined the extents and boundaries of 15 primary mouse DMRs (including 12 maternally methylated and three paternally methylated DMRs) in 12.5-dpc embryos by bisulfite sequencing. We found that the average size of the DMRs was 3.2 kb and that their average G+C content was 54%. Dinucleotide content analysis of the DMR sequences revealed that, although they are generally CpG rich, the paternally methylated DMRs contain less CpGs than the maternally methylated DMRs. Our findings provide a basis for the further characterization of DMRs.     

Influence of the Prader-Willi syndrome imprinting center on the DNA methylation landscape in the mouse brain

Reduced representation bisulfite sequencing (RRBS) was used to analyze DNA methylation patterns across the mouse brain genome in mice carrying a deletion of the Prader-Willi syndrome imprinting center (PWS-IC) on either the maternally- or paternally-inherited chromosome. Within the ∼3.7 Mb imprinted Angelman/Prader-Willi syndrome (AS/PWS) domain, 254 CpG sites were interrogated for changes in methylation due to PWS-IC deletion. Paternally-inherited deletion of the PWS-IC increased methylation levels ∼2-fold at each CpG site (compared to wild-type controls) at differentially methylated regions (DMRs) associated with 5′ CpG island promoters of paternally-expressed genes; these methylation changes extended, to a variable degree, into the adjacent CpG island shores. Maternal PWS-IC deletion yielded little or no changes in methylation at these DMRs, and methylation of CpG sites outside of promoter DMRs also was unchanged upon maternal or paternal PWS-IC deletion. Using stringent ascertainment criteria, ∼750,000 additional CpG sites were also interrogated across the entire mouse genome. This analysis identified 26 loci outside of the imprinted AS/PWS domain showing altered DNA methylation levels of ≥25% upon PWS-IC deletion. Curiously, altered methylation at 9 of these loci was a consequence of maternal PWS-IC deletion (maternal PWS-IC deletion by itself is not known to be associated with a phenotype in either humans or mice), and 10 of these loci exhibited the same changes in methylation irrespective of the parental origin of the PWS-IC deletion. These results suggest that the PWS-IC may affect DNA methylation at these loci by directly interacting with them, or may affect methylation at these loci through indirect downstream effects due to PWS-IC deletion. They further suggest the PWS-IC may have a previously uncharacterized function outside of the imprinted AS/PWS domain.     

Methylation variation at IGF2 differentially methylated regions and maternal folic acid use before and during pregnancy

Folic acid (FA) supplementation before and during pregnancy has been associated with decreased risk of neural tube defects although recent reports suggest it may also increase the risk of other chronic diseases. We evaluated exposure to maternal FA supplementation before and during pregnancy in relation to aberrant DNA methylation at two differentially methylated regions (DMRs) regulating insulin-like growth factor 2 (IGF2) expression in infants. Aberrant methylation at these regions has been associated with IGF2 deregulation and increased susceptibility to several chronic diseases. Using a self-administered questionnaire, we assessed FA intake before and during pregnancy in 438 pregnant women. Pyrosequencing was used to measure methylation at two IGF2 DMRs in umbilical cord blood leukocytes. Mixed models were used to determine relationships between maternal FA supplementation before or during pregnancy and DNA methylation levels at birth. Average methylation at the H19 DMR was 61.2%. Compared to infants born to women reporting no FA intake before or during pregnancy, methylation levels at the H19 DMR decreased with increasing FA intake (2.8%, p = 0.03 and 4.9%, p = 0.04, for intake before and during pregnancy, respectively). This methylation decrease was most pronounced in male infants (p = 0.01). Methylation alterations at the H19 DMR are likely an important mechanism by which FA risks and/or benefits are conferred in utero. Because stable methylation marks at DMRs regulating imprinted genes are acquired before gastrulation, they may serve as archives of early exposures with the potential to improve our understanding of developmental origins of adult disease.Key words: folic acid, epigenetics, IGF2, periconception, prenatal, exposure     

Erasing genomic imprinting memory in mouse clone embryos produced from day 11.5 primordial germ cells

Genomic imprinting is an epigenetic mechanism that causes functional differences between paternal and maternal genomes, and plays an essential role in mammalian development. Stage-specific changes in the DNA methylation patterns of imprinted genes suggest that their imprints are erased some time during the primordial germ cell (PGC) stage, before their gametic patterns are re-established during gametogenesis according to the sex of individuals. To define the exact timing and pattern of the erasure process, we have analyzed parental-origin-specific expression of imprinted genes and DNA methylation patterns of differentially methylated regions (DMRs) in embryos, each derived from a single day 11.5 to day 13.5 PGC by nuclear transfer. Cloned embryos produced from day 12.5 to day 13.5 PGCs showed growth retardation and early embryonic lethality around day 9.5. Imprinted genes lost their parental-origin-specific expression patterns completely and became biallelic or silenced. We confirmed that clones derived from both male and female PGCs gave the same result, demonstrating the existence of a common default state of genomic imprinting to male and female germlines. When we produced clone embryos from day 11.5 PGCs, their development was significantly improved, allowing them to survive until at least the day 11.5 embryonic stage. Interestingly, several intermediate states of genomic imprinting between somatic cell states and the default states were seen in these embryos. Loss of the monoallelic expression of imprinted genes proceeded in a step-wise manner coordinated specifically for each imprinted gene. DNA demethylation of the DMRs of the imprinted genes in exact accordance with the loss of their imprinted monoallelic expression was also observed. Analysis of DNA methylation in day 10.5 to day 12.5 PGCs demonstrated that PGC clones represented the DNA methylation status of donor PGCs well. These findings provide strong evidence that the erasure process of genomic imprinting memory proceeds in the day 10.5 to day 11.5 PGCs, with the timing precisely controlled for each imprinted gene. The nuclear transfer technique enabled us to analyze the imprinting status of each PGC and clearly demonstrated a close relationship between expression and DNA methylation patterns and the ability of imprinted genes to support development.