DNA methylation patterns in tissues from mid-gestation bovine foetuses produced by somatic cell nuclear transfer show subtle abnormalities in nuclear reprogramming
© Couldrey and Lee; licensee BioMed Central Ltd. 2010
Received: 19 August 2009
Accepted: 7 March 2010
Published: 7 March 2010
Cloning of cattle by somatic cell nuclear transfer (SCNT) is associated with a high incidence of pregnancy failure characterized by abnormal placental and foetal development. These abnormalities are thought to be due, in part, to incomplete re-setting of the epigenetic state of DNA in the donor somatic cell nucleus to a state that is capable of driving embryonic and foetal development to completion. Here, we tested the hypothesis that DNA methylation patterns were not appropriately established during nuclear reprogramming following SCNT. A panel of imprinted, non-imprinted genes and satellite repeat sequences was examined in tissues collected from viable and failing mid-gestation SCNT foetuses and compared with similar tissues from gestation-matched normal foetuses generated by artificial insemination (AI).
Most of the genomic regions examined in tissues from viable and failing SCNT foetuses had DNA methylation patterns similar to those in comparable tissues from AI controls. However, statistically significant differences were found between SCNT and AI at specific CpG sites in some regions of the genome, particularly those associated with SNRPN and KCNQ1OT1, which tended to be hypomethylated in SCNT tissues. There was a high degree of variation between individuals in methylation levels at almost every CpG site in these two regions, even in AI controls. In other genomic regions, methylation levels at specific CpG sites were tightly controlled with little variation between individuals. Only one site (HAND1) showed a tissue-specific pattern of DNA methylation. Overall, DNA methylation patterns in tissues of failing foetuses were similar to apparently viable SCNT foetuses, although there were individuals showing extreme deviant patterns.
These results show that SCNT foetuses that had developed to mid-gestation had largely undergone nuclear reprogramming and that the epigenetic signature at this stage was not a good predictor of whether the foetus would develop to term or not.
Somatic cell nuclear transfer (SCNT) has been used to successfully produce cloned animals from several mammalian species since a sheep was cloned using a differentiated somatic donor cell . However, to date widespread application of SCNT in agricultural breeding programs has not yet been captured because the technology remains inefficient despite more than 10 years of research. Irrespective of the species being cloned, there is still a high rate of pregnancy failure throughout gestation [2–6]. The most common SCNT foetal phenotypes across species are foetal overgrowth and loss of allometric growth regulation (collectively known as "large offspring syndrome"), musculoskeletal defects, and acute, excessive accumulation of allantoic fluid (hydrallantois or hydrops) accompanied by perturbations in the composition of this fluid . In cattle, the large offspring syndrome appears to be independent of the donor cell genetics.
Many of the developmental defects observed in cloned bovine foetuses suggest the involvement of growth regulating genes, particularly those known to be imprinted. Some of these genes play key roles in regulating cellular proliferation, growth and development of the foetus and the placenta (reviewed ). The phenotypes commonly observed in SCNT foetuses bear many similarities to some of those seen in experimentally-created imprinting disruptions in mice (silencing of both alleles or biallelic expression of imprinted genes), or to naturally-occurring human syndromes, such as Beckwith-Wiedemann syndrome (BWS) [9–13]. These similarities suggest that the expression of some of these imprinted genes is abnormal and/or that these genes are not appropriately reprogrammed following SCNT. Furthermore, SCNT calves dying shortly after birth were shown to have abnormal expression of imprinted genes in a variety of organs when compared to controls generated by AI ; this was not the case in surviving adult clones [15, 16]. During the development of multicellular organisms, different cells and tissues acquire different programs of gene expression. It is thought that a substantial part of this gene regulation is mediated through epigenetic modifications such as DNA methylation, histone tail modifications and the binding of non-histone proteins to chromatin [17–19] so that each somatic cell in the organism has its own epigenetic signature (epigenome) which reflects its genotype, developmental history and environmental influences, which ultimately determines the phenotype of the cell and the organism. This is clearly illustrated in the events following fertilization, where the majority of the genome undergoes active paternal demethylation, then passive maternal demethylation. Re-methylation of the genome then occurs during repeated mitosis as cells progress towards lineage commitment and the development of the embryo proper and the placenta [18, 20–22]. How the developmental programs are coordinated and orchestrated from the genomic blueprint is still poorly understood, even in normal development.
In reproductive cloning by SCNT, the epigenetic signature of a differentiated somatic cell must be reset to a state resembling totipotency, capable of driving full development after fusion of the cell with an enucleated oocyte cytoplast. Incomplete nuclear reprogramming is widely postulated to be a major contributor to the low developmental success rate following SCNT. Evidence to support this include observed hypo-methylation [23–26], hyper-methylation [27, 28], or mosaic methylation states  in tissue samples collected from abnormal foetuses or cloned calves that died shortly after birth. Normal methylation following SCNT has also been reported [28, 30, 31], suggesting a degree of stochasticity in nuclear reprogramming. The variable findings from different studies are due to different genes or genomic regions examined, different tissues used and controls that were not gestation- or age-matched. This has made comparisons between previous studies difficult.
Cloned animals that reach maturity are able to reproduce normally and give rise to normal offspring without the high rate of pregnancy failure or large offspring syndrome associated with SCNT [32, 33], suggesting that underlying cause/s of the abnormalities associated with SCNT is/are epigenetic.
In this study, we examined the DNA methylation patterns in a panel of candidate genes using tissues from three foetal organs (liver, kidney and adrenal glands) of similar gestations generated by either SCNT or AI. These organs were selected because of the pivotal roles they play in foetal metabolism, nutrient sensing and hematopoiesis (liver), regulation of blood pressure and foetal fluid homeostasis (kidney) and foetal endocrinology (adrenal glands). As most cases of hydrallantois occur from or just after mid-gestation, the selection of this stage of gestation allowed us to compare samples from foetuses that showed clear physiological and anatomical abnormalities with those that had not yet exhibited these symptoms and thus, had the potential to develop to further.
The genes selected consisted of those known to be imprinted in other species, non-imprinted genes and repeat sequences (satellites 1, 2 and alpha). The imprinted genes include those associated with BWS, such as IGF2, KCNQ1, CDKN1C, KCNQ1OT1; others such as ASCL2, HAND1, DIO3; and SNRPN, a gene in the Prader-Willi and Angelman syndrome locus. The non-imprinted genes include colony stimulating factor (CSF-1), STAT5a, DKK-1, and GR, which codes for the glucocorticoid receptor, that mediates the action of glucocorticoids and mineralcorticoids, both important in regulation of fluid composition. The CpG islands examined included those located upstream of transcriptional start sites or within the gene itself or in equivalent regions shown in other species to be differentially methylated regions (DMRs) and normally associated with imprinted genes. The SNRPN site is equivalent to the imprinting centre (IC) of the human gene  which has been shown to be aberrantly methylated in the Prader-Willi (PWS) and Angelman syndromes . The KCNQ1OT1 region is equivalent to the human KCNQ1OT1 DMR which was found to be hypomethylated at increased frequency in human IVF offspring [12, 36, 37]. Together, this selection of genomic sequences allowed us to assess how well these different regions were re-programmed after nuclear transfer in embryos that were capable of developing at least to mid-gestation.
Pregnancy rates in SCNT and AI
From the first ultrasound scan at Day 35, 29 of the 42 (69%) SCNT recipient dams that received a Day 7 blastocyst were found to be pregnant. By Day 130, just 10 days prior to the development of the first SCNT hydrops case, only 14 were still pregnant (33%). Four subsequently developed clinical hydrops and were slaughtered. For the AI group, 13 of the 18 (72%) recipients inseminated were pregnant at Day 35, 11 of these still pregnant (61%) at Day 130; none showed signs of abnormal fluid accumulation.
DNA methylation analysis
DNA methylation analysis at each of the listed genomic regions was performed from the same genomic DNA for each sample. Because the cleavage of the transcribed RNA is sequence-specific, some fragments may contain only one CpG site whilst others may contain up to 6 CpG sites where the CpG dinucleotides were arranged consecutively in the sequence or close together. Where multiple CpG sites occurred within a fragment, the methylation level reported by the EpiTYPER software was that of the most highly methylated site. Where is was not possible to resolve two fragments with the same mass but with different sequences, the methylation level is recorded as an average of the two fragments. The number of CpG sites or group of sites that could be analyzed for each region is given in each figure legend.
IGF2 exon 10
A cleavage fragment containing CpG sites 7, 8, and 9 was consistently methylated to ≤10% in every sample analysed. To eliminate the possibility that this was due to unknown SNPs resulting in a C to T conversion and thus loss of CpG sites, a subset of 10 DNA samples were amplified across this region and the amplicons sequenced. All samples contained the expected CGCGCG sequence.
Mean methylation levels were similar to those reported for Day 26 trophoblast tissue , with all CpG sites methylated to ~10%, the exception being CpG31, where mean methylation levels were 40-50%. There were no significant differences among the three groups.
DNA Satellites I, II and alpha
In this study, we addressed the question of whether nuclear reprogramming has occurred appropriately after SCNT by examining DNA methylation patterns in tissue samples collected from three organs from mid-gestation foetuses. Being able to sub-divide the SCNT group into those that appeared "viable" at slaughter and those that were failing as a result of clinical hydrops (SCNT-hydrops) allowed us to determine if the failing foetuses were methylated differently compared with those that had the potential to develop further. As there are no means to determine, with certainty, the outcome from a foetus that looked "viable" at mid-gestation (SCNT or AI), we can only make the assumption that these SCNT foetuses have the potential, like the AI foetuses, to develop further. Previous studies have indicated that 50-75% of foetuses surviving to mid-gestation will result in a live calf [40–43]. In the majority of regions examined, the DNA from the SCNT or SCNT-hydrops samples were appropriately methylated when compared with control AI samples. Generally, there were no differences in the mean methylation patterns and levels between the SCNT and SCNT-hydrops groups despite evident phenotypic differences. However, close inspection of individual profiles revealed that within the SCNT-hydrops group, there were individuals who showed aberrant hypo- or hypermethylation, particularly in the IGF2 exon 10 DMR, KCNQ1OT1, SNRPN and HAND1 regions. Aberrant methylation in any of those genes was not always accompanied by aberrant methylation in other regions. Furthermore, two individual that are genetically identical could be aberrantly methylated in different ways, demonstrating the stochasticity of the reprogramming defects. At specific CpG sites, significant differences between SCNT and AI samples were detected in some genes. It is not possible to say if these sites represent "hot-spots" for methylation variation or whether these methylation aberrations are the cause of some of the abnormal phenotype seen in SCNT foetuses.
Genes in the BWS locus were of particular interest because of the variable overgrowth phenotypes seen in SCNT foetuses and how some of the phenotypes resemble BWS in humans. The methylation profiles of CpG islands associated with the KCNQ1 and CDKN1C genes were practically identical between the AI control and both groups of SCNT, in liver, kidney and adrenal samples. This suggests that the methylation at these sites is tightly regulated and they were appropriately methylated in these SCNT organs, even in those from failing hydrops pregnancies. Similarly, no differences were observed for the region associated with ASCL2. This tight regulation did not extend to the KCNQ1OT1 region located close to the CDKN1C gene in this imprinted cluster. CpG sites in the KCNQ1OT1 region showed substantial variation between individuals and a tendency for SCNT samples to be less methylated at almost every analyzable CpG site. Taken together, these results suggest that even within the same locus, some CpG islands are methylated appropriately in SCNT tissues while others are less so. As it was not possible to distinguish between the maternal and paternal allele in cattle, we were unable to determine if the aberrant methylation was restricted to one allele or if both were aberrantly methylated.
Similarly, at the region corresponding to the SNRPN imprint control region in humans, the methylation levels were also highly variable between individuals in all groups. Mean methylation at the majority of CpG sites in both SCNT groups tended to be lower when compared with the AI group, although not as dramatically hypomethylated as in early extraembryonic tissues, (Couldrey and Lee, unpublished data and that of others ). Although there was a tendency for this region to be incorrectly methylated following SCNT, not all CpG sites in this region were affected in the same way. An example of this is the cleavage fragment containing CpG7-9 which was consistently unmethylated for every sample in each of the three tissues in all treatment groups. It is possible that these CpG sites are invariably protected from epigenetic modification.
Small but statistically significant differences in methylation levels between SCNT and the control group were detected at specific CpG sites in HAND1, ASCL2 and the KCNQ1 promoter regions in various tissues. These differences were very small and the overall methylation status of the entire region examined was not altered so their biological significance is uncertain. The occurrence of these very small differences is no higher than would be expected purely by chance at the 5% level. Only the HAND1 region showed tissue specific methylation differences as the adrenal tissues have a noticeably different pattern compared with the other two tissues. In these regions, the similarity in methylation pattern between SCNT and normal tissues suggests that this region has undergone appropriate reprogramming and that tissue-specific methylation patterns were successfully established in this organ. Where there were no tissue-specific methylation differences, the absence of difference in methylation between AI and SCNT samples is either an indication of successful reprogramming or that these regions normally escape global demethylation [23, 29, 44] in the pre-implantation embryo and therefore did not require extensive reprogramming after SCNT.
For imprinted genes, the imprints that mark the parental origin of each allele are normally established during gametogenesis, leading to differential methylation of the male and female gametic DNA in DMRs. The methylation levels in DMRs have previously been reported to be ~50%. This level of methylation was believed to be due to the almost complete methylation of one parental allele versus the non-methylation of the other allele. We found that for the IGF2 exon 10 DMR, the majority of the CpG sites were methylated to between 70 to nearly 100% in the three foetal tissues examined, even in the controls. Assuming that the paternal allele was completely methylated, this suggests that the maternal allele was progressively methylated during development. One possible explanation for this is that after additional epigenetic marks that dictate allele-specific expression are established during early embryogenesis, there is no longer the requirement to maintain the differential marking at the DMRs so most of the CpG sites in somatic cells then become progressively methylated. Alternatively, it may be that only a few CpG sites in the region are required to be differentially methylated to distinguish the parental alleles so the non-essential sites become methylated through methylation spreading.
Comparison of the methylation profiles in non-imprinted genes and the repetitive DNA satellite regions showed that in general, these regions appeared to be appropriately methylated in the SCNT tissue samples examined. Although satellite sequences are non-coding and are thought to be kept highly methylated in the genome, not every CpG site was methylated to the same extent across the region. Despite significant differences in methylation levels at specific CpG sites between treatment groups, these differences are very small and the significance in unclear.
An intriguing observation from this study is that in some genes, there is a surprisingly large variation in the methylation levels between individuals at practically every CpG site in the region examined, even between individuals in the normal control group. This suggests that there is tolerance for a range of DNA methylation levels in some genomic regions; whether this translates to phenotypic variability is unknown. This variability could be in part, explained by the inherent lower fidelity (compared with DNA replication) of the DNA maintenance methylation mechanism, which is estimated to be about 95% for methylation of the newly unmethylated strand . However, this does not explain why the methylation of certain regions is so tightly regulated. This variability is not due to heterogeneous tissue sampling as the analyses for multiple genes were carried out on the same bisulfite-treated DNA sample for each individual. In these same samples, other genomic regions showed very tight invariant methylation at almost all CpG sites in every individual. In general, consistent with previous findings  CpG islands upstream of transcriptional start sites or near promoters were less methylated (5-20%) than the two putative DMRs and satellite sequences, which tended to be methylated to 40-100%.
Previous studies assessing the reprogramming of the donor nucleus after SCNT have used antibodies raised against methylcytosine residues to compare global methylation in the nuclei of SCNT and normal embryos . This technique only allows visualization of highly methylated regions which are likely to be repeat sequences that do not code for functional genes. Subtle differences in regions present at two copies per genome will be masked by the overall methylation of highly repetitive elements. Other techniques which average DNA methylation levels over the genomic region analyzed have led to the misconception that DNA methylation levels are similar at each CpG site across the entire CpG island. In contrast, the MassARRAY technology is able, in many instances, to calculate DNA methylation levels at individual CpG sites reproducibly down to 5% for each informative CpG unit . However, because the MassARRAY method depends on sequence-specific cleavage of derived RNA products, this technology is unable to analyze the methylation at every CpG site when suitable cleavage sites are unavailable such as in high density CpG regions. Bisulfite sequencing will be a useful adjunct when these regions warrant further examination. The success of both techniques however, is dependent on being able to design primers that flank ~500 bp of CpG-rich sequences but the primers themselves must bind to regions which do not contain CpG sites. For some CpG islands that are large (up to ~8 kb), this is not always possible and other methods must be devised to study such regions.
The ability to analyze large numbers of samples and genes and quantify the level of methylation at specific CpG sites allows a more accurate assessment of methylation profiles in the populations of interest. The technology has enabled us to appreciate detail previously unrealized: a) some CpG sites are always protected from methylation whilst others tend to be highly methylated; b) methylation at some CpG site(s) within a region show high variability between individuals whilst others are invariably methylated in every individual; c) some regions, such as the KCNQ1OT1 site in adrenal tissues and DIO3 in all tissues show apparent periodicity in the methylation profile, with highly-methylated sites interspersed with lowly-methylated sites; d) tissue-specific methylation patterns were uncommon. It remains for the biological significance of these observations to be determined.
The detail revealed by this method of DNA methylation analysis calls into question whether the practice of reporting the average methylation level across all CpG sites within a region and comparing this value between experimental samples is meaningful. This could potentially mask methylation differences between experimental groups at CpG sites that may be important for regulating chromatin structure and hence, gene expression. CpG sites that are invariably protected from methylation, or those which are always methylated are not evident when averaging methylation levels across a region. Subtle tissue-specific differences may also be masked. Furthermore, the biological significance of averaged methylation levels in a region is unclear.
We have used the MassARRAY technology to look at multiple regions in the genome and found that for SCNT foetuses that survived to mid-gestation, albeit with phenotypic abnormalities in some cases, the methylation patterns were very similar to those of naturally conceived foetuses, at least for the three organs examined. This suggests that in those foetuses, the majority of these sites in the genome have been appropriately "reprogrammed". However, there were two regions located in imprinted gene clusters (BWS and PWS loci) where SCNT samples tended to be hypomethylated. This implies the importance of these two region in regulating normal foetal development and growth. Similar observations of aberrant methylation in the BWS locus in children arising from human IVF is further evidence of the susceptibility of this region to external influence. We cannot yet with confidence predict the developmental outcome of a SCNT foetus from its epigenetic state at any stage; it is just a snap-shot of the dynamic nature of the epigenetic status of the genome. We are far from understanding how DNA methylation patterns relate to phenotypic outcomes in entire organisms.
Production and collection of foetal tissues
All manipulations of animals involved in the present study were conducted in accordance with the regulations of the New Zealand Animal Welfare Act of 1999. SCNT embryos were produced essentially as previously described . An adult skin fibroblast cell line (AESF-1) from a high genetic merit Friesian bull was used as nuclear donor. After in vitro culture for 7 days, the embryos were transferred to synchronized recipients and pregnancy establishment determined at Day 35 of gestation by trans-rectal ultrasound scanning. Pregnancies were monitored monthly by ultrasound scanning until Day 120. From then, the animals were monitored closely by rectal palpation for the development of hydrallantois. SCNT pregnancies that were diagnosed with hydrops were terminated by slaughter at the abattoir. Viable SCNT pregnancies from the same cohort but without hydrops were also terminated at around the same gestation as those with hydrops. Additional samples from SCNT-hydrops pregnancies at similar stages of gestation generated from several other donor cell lines were included in the study. Thus, the group SCNT-hydrops consisted of SCNT foetuses derived from the AESF-1 line as well as from five other cell lines of both sexes and of different breeds. This allowed us to investigate whether the DNA methylation status is similar or different in SCNT-hydrops foetuses with different genetic backgrounds. Control pregnancies were generated by artificial insemination (AI) with frozen semen from the bull which provided the AESF-1 donor cells and foetal tissues were collected at the equivalent stage of gestation as the SCNT foetuses. The uteri and its contents were recovered after slaughter of the recipient dams. Gross foetal and placental morphology was recorded and foetal and placental tissue samples collected and snap-frozen in liquid nitrogen. For this study, three organs, the kidney, liver and adrenal glands, which commonly show growth disregulation in SCNT foetuses, were used (AI, n = 5; SCNT, n = 6 and SCNT-hydrops, n = 4 from the AESF-1 line and n = 6 from other cell lines).
Identification of CpG islands for analysis
Promoter regions (up to 10 kb upstream of the putative transcription start site) and transcribed regions of selected genes were analysed for the presence of CpG islands (observed/expected CpG dinucleotide ratio of > 0.60, C+G content > 50%, length > 200 bp) using Emboss on EBI website.
PCR primers used for Sequenom analysis
Product size (bp)
Tissues were ground up in liquid nitrogen to a powder to ensure homogeneity for DNA sampling. Between 20 and 100 mg of tissue was then used for DNA extraction using either phenol/chloroform  or a DNeasy kit following the manufacturer's protocol (Qiagen, Austin, TX). DNA concentration and purity was measured using the Nanodrop spectrophotometer (Thermo Scientific, DE, USA)
Analysis of DNA methylation
DNA samples were analyzed using the methods described [38, 52, 53]. Briefly, 1 μg DNA was bisulfite treated using the EZ-96 DNA Methylation gold kit (Zymo, CA, USA) to produce methylation-dependent sequence variations of C to T and regions of interest were amplified using T7 tagged PCR primers. PCR conditions were: 200 nM of forward and reverse primers, 200 μM of each dNTP, 1× Qiagen HotStar buffer, 0.2 U Qiagen HotStar Taq polymerase and 2 μl bisulfite converted DNA per reaction in a total volume of 10 μl. PCR cycling conditions were: 94°C 15 min followed by 45 cycles of 94°C, 20 sec; 56°C, 30 sec; 72°C, 1 min with a final extension at 72°C for 3 min. PCR products were analyzed by agarose gel electrophoresis to confirm successful amplification. In vitro amplification and transcription was performed on the reverse strand using 2 μl of PCR product using T7 DNA and RNA polymerases and a simultaneous U specific cleavage by RNAse A. Approximately 20 nl of each sample was spotted onto Sequenom MassARRAY chips and subject to mass spectrometry. The efficiency of bisulfite conversion was determined by assessing the quality of the raw data. Incomplete bisulfite conversion generates mass peaks at a mass/charge ratio of 16, 32, 48, etc. greater than the expected peaks, in addition to the expected peaks. Such data were infrequent and excluded from the analyses.
Spectra were analyzed using proprietary peak picking and signal-to-noise ratio calculations. The relative methylation of the CpG sites was then calculated (EpiTYPER, Sequenom, CA, USA) by dividing the peak intensity (area under the peak) of the fragment representing the original methylated DNA, by the sum of the intensities of the peaks representing both methylated and non-methylated DNA. Mean DNA methylation levels for each fragment were compared using the least significant differences calculated from the analysis of variance across the three treatment groups in each tissue examined. The mean methylation level across the region contained in the amplicon was also calculated for each gene and compared pair-wise between treatment groups for each tissue using the t-test. Results are presented as mean +/- standard error of the mean (S.E.M).
DNA methylation levels at CpG sites in the amplicons
Each CpG site or groups of sites which could be analyzed by Sequenom MassARRAY are arranged in the order that they appear in the DNA sequence, 5' to 3' on the x-axis. Where there are more than one CpG sites in a fragment, the numbered CpG sites are grouped together in one position on the x-axis and the proportion of methylation refers to the most methylated site (Sequenom EpiTYPER 1 software). The y-axis represents the proportion of methylation at specific CpG sites in the region analyzed. The error bars represent the SEM and arrows indicate CpG sites where there is significant difference (P < 0.05) among the treatment groups. a) adrenal, b) kidney and c) liver tissues, d) average methylation in the genomic region represented by the amplicon; columns with different labels are significantly different from each other (P < 0.05). AI: AI controls; SCNT: SCNT samples from apparently viable foetuses; SCNT-hydrops: SCNT samples from foetuses terminated because of hydrops.
The authors would like to thank David Wells and cloning group for producing the SCNT embryos, Martin Berg for animal care and treatment, Tim Manley for the Sequenom MassARRAY sample analysis, Darryl Irwin from Sequenom for excellent technical support, Ning Li and Anita Ledgard for assisting with tissue collections, Neil Cox for statistical assistance, and Pauline Hunt for graphics assistance. This work was supported by a grant from the New Zealand Foundation for Research, Science, and Technology (C10X0303).
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