Molecular Human Reproduction, Vol. 8, No. 7, 630-635,
July 2002
© 2002 European Society of Human Reproduction and Embryology
Embryology |
Epigenetic marks at BRCA1 and p53 coding sequences in early human embryogenesis
1 Laboratoire de Génétique, UMR 5641 CNRS, 2 Laboratoire de Biologie de la Reproduction et du Développement, UCBL1, 8 avenue Rockefeller and 3 INSERM U453, Centre Leon Berard, 69373 Lyon Cedex 08, France
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Abstract |
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In the vertebrate genome, methylation of deoxycytosine residues of CpGs dinucleotide has been associated with transcriptional silencing of genes, parental imprinting, X-inactivation and chromatin remodelling. In human somatic tissues, the 5' end of the BRCA1 CpG island is methylated, whereas this region is unmethylated in mature germ cells and early embryos. In gametes, as in somatic tissues, the CpG sites in the coding region are methylated. We took advantage of this bimodal distribution as a model to analyse the epigenetic reprogramming of coding regions during early human embryogenesis using the bisulphite-based genomic sequencing method. During preimplantation divisions, exon 11 of BRCA1 was slowly demethylated and retained
30% of its methylated residues at the blastocyst stage. Moreover, the change in the distribution of methylated residues was not restricted to the BRCA1 gene, since for another gene, p53, a relatively high level of methylation (50%) of exon 4 was observed in blastocysts. Taken together, these data suggest that a significant part of the methylated residues of coding sequences might be conserved during preimplantation development. bisulphite sequencing/CpG island/DNA methylation/human embryogenesis/single copy gene
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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In mammals, variations of the DNA methylation patterns have been described in adult somatic cells during normal (differentiation and ageing) and cancer processes. However, analysis of mouse models strongly suggests that the extensive remodelling of this epigenetic mark is a characteristic of gametogenesis and early embryogenesis in mammals. DNA methylation levels are high in mature gametes and the patterns are progressively erased during preimplantation cleavages (Monk et al., 1987
; Sanford et al., 1987; Howlett and Reik, 1991; Shemer et al., 1991; Kafri et al., 1992; Rougier et al., 1998; Warnecke and Clarck, 1999). After implantation, a global de-novo methylation would serve to reset the tissue-specific methylation patterns which are crucial for organogenesis and differentiation (Jaenisch, 1997).
The mechanism of genome-wide demethylation remains unclear since not all CpG sites in the genome are subject, at the same time, to this wave of demethylation. In preimplantation mouse embryos, it has been demonstrated that active demethylation of the paternal genome operates over a wide range of DNA sequences at the 1-cell stage (Mayer et al., 2000; Oswald et al., 2000). However, repeated sequences are progressively demethylated until the blastocyst stage, whereas L1 sequences and the 
-actin promoter retain a relatively high methylation level until the morula stage and are demethylated at the blastocyst stage (Howlett and Reik, 1991; Warnecke and Clark, 1999). Despite this global DNA demethylation, not only several repeated sequences, the intracisternal A particle proviruses (Walsh et al., 1998) and centromeric regions (Rougier et al., 1998), but also some imprinted regions (Olek and Walter, 1997; Tremblay et al., 1997; Ferguson-Smith and Surani, 2001; review) retain a high level of methylation through the blastocyst stage.
However, although the genome-wide demethylation seems to be a general event in mammals, the timing of this event differs among species. In bovine embryos, immunostaining of interphase nuclei with antibodies against 5-methyl-cytosine indicates a demethylation of the paternal genome at the 1-cell stage (Bourc'his et al., 2001; Dean et al., 2001) as observed in the mouse (Rougier et al., 1998; Mayer et al., 2000; Oswald et al., 2000), and both genomes are then progressively demethylated until the 8-cell stage. However, in bovine embryos, remethylation occurs at the 16-cell stage and is maintained through the blastocyst stage (Dean et al., 2001; Fairburn et al., 2002; review). In mouse embryos, remethylation is also observed but only from the blastocyst stage (Santos et al., 2002). It has been also hypothesized that the demethylation of some sequences continues in the germ line, leading to an epigenetic mark of the differences between the gametes according to their parental origin (Goto and Monk, 1998).
In humans, defects in biological methylation and chromatin remodelling contribute to several pathologies and birth defects and the description of the physiology of this epigenetic modification is a major task in understanding the molecular mechanism of these diseases (El-Osta and Wolffe, 2000; review). In this report, we analyse the methylation of the breast cancer predisposition gene BRCA1 during early human embryogenesis. The involvement of BRCA1 in breast cancer predisposition is now well established (Ponder, 2001) and it appears to play an important role in early embryogenesis. Homozygous disruption of Brca1 in transgenic mice results in embryonic lethality (Deng and Scott, 2000), and during spermatogenesis the human BRCA1 protein is localized to the axial elements of developing synaptonemal complexes (Scully et al., 1997). Although the exact functions of BRCA1 are still under investigation, its association with recombination/repair proteins (Scully and Livingston, 2000) indicates that this gene might participate in mechanisms preserving genome integrity. In human somatic tissues, the CpG island located at the 5' end of the BRCA1 gene is methylated, whereas this region is unmethylated in mature germ cells (Magdinier et al., 2000). In gametes, as in somatic tissues, the CpG sites in the coding region are methylated. We took advantage of this bimodal distribution as a model to analyse the epigenetic reprogramming of coding regions during early human embryogenesis using the bisulphite-based genomic sequencing method.
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Materials and methods |
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Preparation of samples
Sperm were purified from semen samples by Percoll gradient centrifugation. The purity of the preparation was controlled by microscopy (Magdinier et al., 2000
). Human oocytes that had failed to fertilize 3 days after in-vitro insemination were collected from the Assisted Conception Unit (E.Herriot Hospital, Lyon, France). In order to remove the follicular cells linked to the zona pellucida, oocytes were treated by enzymatic digestion with hyaluronidase (150 units, type VIII hyaluronidase; Sigma, L'Isle d'Abeau, France) to discard contaminating somatic cells. A digestion by Trypsin was then performed to remove the zona pellucida and the remaining somatic cells. Oocytes were rinsed several times in phosphate-buffered saline 1x and stored in liquid nitrogen until use. The cumulus cells were collected after hyaluronidase digestion for further studies. Supernumerary human embryos obtained after IVF that were considered to be unsuitable for transfer and could not be frozen were collected with the couple's consent and staged (EHH, Assisted Conception Unit) and selected samples were purified by enzymatic digestion as described for the oocytes. All the purification steps were performed under microscopy. The research programme was licensed by the Commission Nationale de Médecine et Biologie de la Reproduction (Ministère de l'Emploi et de la Solidarité).
In-vivo derived mouse blastocysts were obtained from 6-week-old female mice (OF1; IFFA CREDO, Lyon, France) mated with OF1 males. Female mice were superovulated by i.p. of 5 IU of pregnant mare's serum (Folligon, Intervet, France) and 48 h later with 5 IU HCG (Chorulon, Intervet, France). Blastocysts were collected by flushing the uterus 3.5 days post-coitum and purified as described for the oocytes.
DNA extraction
DNA was extracted from frozen samples and cells by standard procedures. Briefly, to prepare decondensed DNA from sperm, samples were resuspended in 10 mmol/l Tris–0.1 mol/l EDTA buffer and digested with proteinase K (300 µg/ml final concentration) in the presence of 1-CELL and 0.001% (v/v) ß-2 mercaptoethanol. When DNA was analysed from a small number of cells (oocytes or preimplantation embryos), 2 µg of pGEM-T plasmid (Promega, Lyon, France) was added as carrier to the samples, in a final volume of 100 µl of 50 mmol/l Tris–50 mmol/l EDTA buffer containing 0.25% sodium dodecyl sulphate (SDS) and 14 µg/ml of proteinase K. The mixture was incubated at 55°C for 2 h, then the samples were processed as described in the `bisulphite modification' section.
Bisulphite modification
The sodium bisulphite modification method followed by the sequencing of PCR products was used to determine the CpG methylation pattern. Sodium bisulphite converts unmethylated cytosines to uraciles while the methylated cytosines remain unmodified. In the resulting modified DNA, uraciles are replicated as thymines during PCR amplification. The sodium bisulphite reaction was carried out on 4 µg of DNA (3 µg of carrier DNA and 1 µg of human genomic DNA or 2 µg of carrier and the mixture in 100 µl). Alkali-denatured DNA was incubated in 3 mol/l NaHSO3 and 5 mmol/l hydroquinone for 16 h at 50°C in a final volume of 500 µl. Modified DNA was purified using the Wizard DNA Clean-up System (Promega) and eluted into 50 µl of sterile water. Modification was completed by 0.3 mol/l NaOH and DNA was precipitated by 0.5 mol/l ammonium acetate pH 4.6 and resuspended in water. DNA fragments were amplified by a nested PCR
A 258 bp region (position –1643 to –1358, containing 24 CpG sites) in the 5' BRCA1 CpG island was amplified as described (Magdinier et al., 2000).
Two regions within exon 11 of BRCA1 were amplified. The two first primers of set A have been previously described (Rodenhiser et al., 1996). Primers of set B were designed to amplify a 350 bp sequence from position 34387–34734 in exon 11 (Accession number L78833). The first round of PCR amplification was accomplished in 100 µl of a buffer containing 10 mmol/l Tris–HCl (pH 8.3), 3 mmol/l MgCl2, 50 mmol/l KCl, 0.1 mg/ml gelatin, 100 µmol/l of each of the four deoxyribonucleoside triphosphates, 0.25 µmol/l of the primers and 0.6 units of Taq DNA polymerase (Roche Diagnostics, Meylan, France) after 35 cycles in a thermocycler (1 min denaturation at 94°C, 2 min annealing at 55°C and 3 min extension at 72°C). An aliquot of the first amplification was reamplified with internal primers in the same conditions. PCR products were first analysed by digestion with restriction enzymes, then PCR products were cloned into a pGEM-T vector (Promega) and random clones were analysed by automatic sequencing (Eurogentec, Seraing, Belgium) to determine the proportion of methylated (CpG) or unmethylated (TpG) sites. The sensitivity of PCR amplification after bisulphite modification was monitored by mixing different proportions of unmethylated plasmid DNA containing the BRCA1 sequence (0–100%) and in-vitro methylated DNA (100–0%). The amount of PCR products cleaved by specific enzymatic digestion is directly related to the percentage of methylated or unmethylated DNA used in the co-amplification assay.
Exon 4 of the p53 gene and exon 11 of the mouse Brca1 gene were amplified using the HotstarTaq DNA polymerase (Qiagen, Courtaboeuf, France) in the buffer recommended by the supplier. The primers used in the PCR reactions can be seen in Table I.
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Results |
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Kinetics of demethylation of the BRCA1 coding sequence in human preimplantation embryos
The breast cancer susceptibility gene BRCA1 exhibits a complex methylation pattern. Southern blot analysis and bisulphite genomic sequencing indicated that the CpG island at the 5' end of BRCA1 is regionally methylated (position –1750 to –700) in all somatic tissues analysed and fully demethylated in mature oocytes and sperm. In contrast exon 11, which represents
60% of the BRCA1 coding sequences, is methylated in both somatic tissues and sperm (Magdinier et al., 1998, 2000).
The methylation pattern of two regions of exon 11 of BRCA1, containing eight CpG sites, was determined in gametes and in preimplantation human embryos (Figure 1A,B). We have shown in a previous report that this method is applicable to a very small amount of DNA and primers and experimental conditions of amplification were chosen for obtaining a quantitative assay of the ratio of methylated versus unmethylated CpG (Magdinier et al., 2000). After DNA extraction and modification, PCR products were cloned and sequenced. In human germ cells, both regions were highly methylated (90–100%) (Figure 1C). As a control experiment, the methylation pattern of the cumulus cells surrounding the oocytes after ovulation was also determined and all the CpG sites were methylated, as expected for non-CpG island sequences in somatic tissues (Figure 1C). At the 2-cell stage, some cloned DNA molecules (46%) exhibited unmethylated CpGs, but fully demethylated clones were not observed and the methylation level (89%) was similar to those observed in gametes (Figure 1C). Then, DNA demethylated slowly at the 8- to 16-cell stage (47% unmethylated CpG) and through the morula stage (60% unmethylated CpGs) (Figure 1C). However, in these samples most of the cloned DNA molecules exhibited a mixed pattern of non-methylated and methylated CpGs, indicating that DNA methylation was still present. As has been described for L1 sequences and the -actin promoter in the mouse (Howlett and Reik, 1991; Warnecke and Clark, 1999), a second reduction in DNA methylation occurred between the morula and blastocyst stages, since in the analysed blastocysts fully demethylated cloned DNA molecules were observed and 68% of the CpGs were unmethylated.
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Methylation patterns of p53 coding sequences in human gametes and blastocysts
The partial conservation of the epigenetic marks at exonic sequences during early human embryogenesis was further investigated for exon 4 of p53 (Figure 2A,B
). In human oocytes and sperm the nine CpGs sites of the p53 exon 4 were heavily methylated (Figure 2C). In order to determine the methylation status of exon 4 in the last steps of preimplantation embryogenesis, 22 cloned PCR fragments, obtained after bisulphite modification of genomic DNA from human blastocysts, were randomly selected and sequenced. As observed for two regions of BRCA1, the analysis of these clones indicated a complex pattern of methylation at the blastocyst stage. A large number of the cloned DNA molecules (45%) contained both methylated and unmethylated CpGs, but fully demethylated (23%) and fully methylated (32%) patterns were also observed (Figure 2C), indicating that part (50%) of the CpGs of p53 exon 4 are still methylated at the blastocyst stage.
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The evolution (Figure 3
) of the methylation status of the BRCA1 and p53 exonic sequences suggested a progressive demethylation during early human embryogenesis; however, a demethylation step followed by a remethylation cannot be excluded. In contrast the BRCA1 5' CpG island seemed to remain unmethylated at the stages analysed (Figure 3).
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Methylation patterns of the Brca1 coding sequence in mouse blastocysts
The analysis of two exonic sequences in human preimplantation embryos indicated that methylation at CpG sites is not fully erased at the blastocyst stage. In order to investigate this phenomenon in embryos not obtained from in-vitro fertilized oocytes, we have determined the methylation status of a coding sequence of in-vivo derived mouse blastocysts. The exonic sequence analysed corresponds to a 297 bp long DNA segment of exon 11 of the mouse Brca1 gene (Figure 4
). The sequencing of 19 randomly selected cloned PCR fragments, obtained after bisulphite modification, indicated a complex pattern of methylation at the blastocyst stage (Figure 4). As observed for the in-vitro fertilized human embryos, partial methylation (47%) was observed in mouse in-vivo derived blastocysts. As far as human and mouse embryos can be compared, these data might suggest that the mosaic DNA methylation patterns observed are not the result of embryo culture.
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Discussion |
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The data presented in this report indicate that exon 11 of BRCA1 and exon 4 of p53 are partially methylated in human preimplantation embryos and suggest an absence of complete reprogramming of coding sequences during human early embryogenesis.
The evolution of the overall level of methylation of the BRCA1 and p53 sequences during early human embryogenesis is generally in accordance with the model constructed from the mouse (Monk et al., 1987; Sanford et al., 1987; Howlett and Reik, 1991; Shemer et al., 1991; Kafri et al., 1992; Rougier et al., 1998; Warnecke and Clark, 1999), showing a generalized hypomethylation of the genome at the blastocyst stage. In humans, the 5' CpG island of BRCA1 is demethylated in gametes, while the exonic sequences are fully methylated. During the first embryonic cleavages, the kinetics of demethylation of the BRCA1 exon 11 indicated no massive demethylation, although sudden demethylation followed by remethylation between the 2- and 8-cell stages or between the morula and blastocyst stages cannot be excluded. This progressive demethylation after each cell division might favour the hypothesis of a passive loss of methyl groups by absence of remethylation together with a reduced activity of the maintenance of CpG methylation (Carlson et al., 1992; Cardoso and Leonhardt, 1999). In line with this hypothesis, it should be noted that the CpG island at the 5' end of BRCA1 remains unmethylated during early human embryogenesis, also suggesting an absence of remethylation or alternatively that these mechanisms might be targeted to subclasses of sequences. In gametes and in preimplantation embryos, the absence of methylation at this 5' CpG island might be associated with the regulation of BRCAI gene expression. In somatic cells, the methylation of this region is associated with a reduced expression of BRCA1 (Magdinier et al., 2000). In contrast, this 5' region is demethylated in gametes and in preimplantation embryos, and preliminary experiments suggest that BRCAI is highly expressed in these tissues.
At the blastocyst stage, 40% of the clones sequenced are partially methylated and the remaining clones exhibit, in equal proportion, fully methylated patterns and fully unmethylated patterns. The relatively low number of clones analysed, compared with the theoretical number of possible patterns (2n, n = number of CpG, assuming that the methylation of a given CpG is an independent probability), does not allow an exact determination of the probability of a given pattern. Therefore, the methylation percentages are only indications rather than precise estimates. However, bisulphite modifications induce or abolish restriction sites, depending on the methylation status of the starting material. We also performed restriction enzyme analysis of the PCR products for each sample. Despite the fact that only a few CpG sites (at the restriction site of a given enzyme) can be analysed this way, the data obtained also indicated mosaic patterns of methylation.
Although no data about the methylation in human embryonic stem (ES) cells is, to our knowledge, currently available, it has been shown that mouse ES cells exhibit very heterogeneous methylation patterns, which are at least partially conserved after in-vitro differentiation or after transfer to recipient blastocysts (Dean et al., 1998; Humpherys et al., 2001). Mosaic methylation patterns at the Xist locus have also been observed in mouse oocytes and in preimplantation embryos (Goto and Monk, 1998; review).
The demethylation in preimplantation embryos seems to be an essential characteristic of mammalian embryogenesis and it has been recently suggested that developmental anomalies of cloned mammal embryos could be due to alterations in the demethylating process during early embryogenesis (Rideout et al., 2001). It is worthy of note that demethylation during the early steps of embryogenesis is not a general prerequisite for a normal development in vertebrates. In zebrafish, repeated sequences and single copy genes are not demethylated during development (Macleod et al., 1999) and in Xenopus laevis genome-wide changes in DNA methylation are not detected at least until the morula stage (Stancheva and Meehan, 2000). It had been suggested that the absence of demethylation in zebrafish is related to the absence of parental imprinting and chromosome X inactivation in this species, two phenomena associated with DNA methylation. For the coding region of the BRCA1 gene, there is no difference in the distribution of methylated CpG between germ cells and tissues (Magdinier et al., 2000; and this report). If the main role of transient demethylation during preimplantation development is to reset the methylation pattern inherited from parents, some sequences that are not differentially methylated might escape from this process.
Although methylation at exonic sequences is not associated with the control of gene expression, conservation of the epigenetic marks might be involved in the control of genome stability and chromatin remodelling, since DNA methylation is involved in these phenomena.
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Acknowledgements |
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We thank Dr Adrian Bird for helpful comments during the preparation of the manuscript and Dr Jean-Pierre Rouault for assistance in obtaining the mouse embryos used in this work. F.M. is the recipient of a fellowship from the Fondation pour la Recherche Médicale. The present work was supported by the Ligue Nationale contre le Cancer, (Comité de Saône et Loire and Comité du Rhône) and the Association pour la Recherche sur le Cancer.
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Notes |
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4 To whom correspondence should be addressed. E-mail: dante{at}univ-lyon1.fr
* These authors have contributed equally to this work.
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References |
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Submitted on November 26, 2001; accepted on April 16, 2002.
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