The epigenetic reprogramming taking place in primordial germ cells has been the object of number of previous experiments. Whereas the majority of publications concerned the onset of new, sex specific, imprints, our interest was focused on the poor characterised process of demethylation / imprint erasure. Based on the published data describing the hypomethylation and the absence of imprints in the primordial germ cells at 12.5-13.5 dpc (Kafri, et al. (1992)), we decided to focus our analysis mainly on the primordial germ cells of the earlier developmental stages (11.5 and 12.5 dpc), the stages where we expected the process of imprint erasure to take place.
The samples of 11.5 dpc and 12.5 dpc primordial germ cells were isolated from the genital ridges using the immunoaffinity purification in combination with the magnetic bead sorting system (see Material and Methods). Shortly, whole genital ridges of mouse embryos were trypsinised in order to produce a single cell suspension. Primordial germ cells were subsequently isolated using monoclonal TG1 antibody (Gomperts, et al. (1994)) in combination with the secondary anti-mouse antibody coupled to magnetic beads (MiniMacs sorting system, Miltenyi Biotec – see Fig. 10). TG1 antibody recognise the germ line specific SSEA1 antigen (SSEA1 – Stage Specific Embryonic Antigen 1 - is a trisaccharide of the form galactose [β1-4]N-acetylglucosamine[α1-3]fucose , Fox, et al. (1981)).
|Fig. 10: Scheme of the magnetic bead-based cell separation|
Whereas the samples of 11.5 dpc and 12.5 dpc primordial germ cells were collected using the immunoaffinity purification, the method did not approve to be useful in the case of 13.5 dpc cells. The expression of the SSEA1 antigen, which is the target for the TG1 antibody, diminishes after 12.5 dpc; thus it loses the properties of a suitable selection marker. To isolate the primordial germ cells of later as well as earlier stages of development, we took the advantage of a Oct4-GFP transgenic mouse with the germ line specific GFP expression (see Fig. 11), which was created at Wellcome/CRC Institute, Cambridge, UK (for details see Material and methods).
|Fig. 11: GFP expression in 13.5 dpc gonads of Oct4-GFP transgenic embryos|
|Note the sexual dimorphism of embryonic gonads at this developmental stage.|
Since PGCs are characterised by high levels of alkaline phosphatase activity (Ginsburg, et al. (1990)) each of our PGC preparations was checked for the purity using the alkaline phosphatase staining (see Fig. 12, and Material and Methods). The purity of the samples always exceeded 95%.
|Fig. 12: Single cell suspension of MiniMacs sorted PGCs (12.5 dpc) stained for tissue non-specific alkaline phosphatase.|
|The positive cells are stained in brown.|
The genital ridges of the 12.5 dpc embryos show sex specific morphology (see Fig. 13), thus making the sex determination of PGC samples simple. However, at 11.5 dpc the genital ridges of female and male are still indistinguishable. As a consequence, in majority of our experiments the 11.5 dpc germ cells were used as a mixed gender population. In limited number of control experiments the sex of collected 11.5 dpc samples was determined by the PCR approach (amplification of Ube1 genes, Ube1X and Ube1Y are located on X and Y chromosomes, respectively - for details see Material and Methods).
|Fig. 13: Sex specific morphology of the mouse embryonic genital ridges (Hogan et al.,1994)|
To assess the methylation status of primordial germ cells of different developmental stages we took the advantage of bisulphite genomic sequencing (see 1.7 Molecular techniques used for DNA methylation studies andHajkova et al., 2002). The method is based on the chemical reaction of single stranded DNA with sodium bisulphite under acidic pH and following desulphonation in highly alkalic conditions (see Fig. 14). Consequently, the single stranded cytosin residues are converted through several reaction intermediates into uracil residues, whereas 5’methyl-cytosin residues remain unconverted. The bisulphite modified DNA strands are in the following steps amplified (PCR), cloned and sequenced. In the PCR reaction the uracil residues (originally non-methylated cytosines) are amplified as thymines, i.e. the cytosines found in the final sequence correspond to the positions of methylated cytosines in original DNA molecules (see Fig. 15). Among advantages of this method belong: a) low requirements for the amount of starting material (a crucial parameter when working with embryonic material - as few as several dozens cells are sufficient for the analysis); b) the possibility to analyse all the cytosine residues within the region defined by the PCR primers (to the contrary, using the methods based on methyl-sensitive restriction it is possible to analyse only a limited number of CpG sites) and c) the possibility to reveal the pattern of single DNA molecules, which is important when studying the dynamic processes such as imprint establishment or erasure.
|Fig. 14: The reaction of single stranded cytosine residues with sodium bisulphite|
Chemistry of the reaction steps: I) sulphonation at the position C6 of cytosine, II) irreversible hydrolytic deamination at the position C4 generating 6-sulphonate-uracil and III) subsequent desulphonation under alkaline conditions. Note that methylation at the position C5 impedes sulphonation at the C6 position (step 1).
|Fig. 15: The examples of bisulphite treated sequences.|
|The example of a non-methylated clone is shown above (cytosines are converted to thymines). The example of a methylated clone is given in the lower part of the figure (methylated cytosines resisted the conversion).|
In order to investigate the reprogramming (especially the erasure) of imprints we decided to follow the methylation status of well described differentially methylated regions (DMRs; sequences carrying sex specific methylation) connected to the imprinted genes. The DMRs of the following imprinted genes were included into our analysis: peg3, lit1, Snrpn (DMR1) as examples of maternally methylated regions; and igf2, h19, and Snrpn (DMR2) representing paternally methylated sequences (see Table 2)
Lit1 (long QT intronic transcript 1, GenBank acc.# AJ271885, mouse distal chromosome 7) was originally identified as an antisense orientation transcript within the imprinted KVLQT1 gene (Lee, et al. (1999)). The lit1 itself shows also imprinted character, the transcription being active only on a paternal chromosome. The expression of both imprinted genes seems to be regulated from a pronounced lit1 CpG island located within the intron 10 of KVLQT1, which was proposed to function as an additional imprinting centre in the Beckwith-Wiedemann cluster (Maher and Reik (2000,Smilinich, et al. (1999); Engemann, et al. (2000)).
The lit1 CpG island carries the germline methylation mark; the region was shown to be heavily methylated in oocytes, whereas it appears to be non-methylated in sperm (Engemann, et al. (2000)). Our bisulphite analysis was directed to the 3’part of the CpG island; the amplified part of the CpG island (524bp) contains 43 CpG sites (Fig. 16).
|Fig. 16: Bisulphite analysis of Lit1 CpG island|
|The upper part of the figure shows the results of the bisulphite analysis. Each line and circle represent a unique clone and single CpG position, respectively. The filled circles stand for the methylated CpGs, the open circles represent non-methylated positions. The product of bisulphite specific PCR is shown on the gel left: M - 1kb ladder (Fermentas), 1- PGC sample, 2 - water control for bisulphite treatment, 3 - water control for PCR amplification.|
The mixed gender 11.5 dpc primordial germ cells display approximately 1:1 distribution of completely methylated vs. completely non-methylated clones which corresponds to the pattern seen in zygote and ES cells (Engemann, et al. (2000)) and expected in somatic cells. Thus, at 11.5 dpc the PGCs still maintain the methylation imprint. However, at 12.5 dpc only completely unmethylated clones are present in samples of both genders (the data represent the combination of two independent bisulphite treatments and several PCR amplifications). These results might bring about the following conclusions: a) PGCs contain normal imprint at 11.5 dpc which excludes the theory that the precursors for the germ cells are separated early during the embryogenesis and never gain somatic type of methylation; b) reprogramming of the imprint does not seem to be a gradual process of imprint loss and reestablishment, but it might be initiated as a rapid and complete erasure of a full somatic type of imprint.
Another example of a maternally imprinted gene is Peg3. Peg3 (paternally expressed gene 3, GenBank acc.# AF105262; mouse proximal chromosome 7) encodes a zinc finger protein that is expressed only from the paternal allele in embryos and adult brain (Kuroiwa, et al. (1996,Li, et al. (2000)). The protein has been shown to be involved in TNF-NFkappaB signal transduction pathway (Relaix, et al. (1998)) and in regulation of maternal behaviour (Li, et al. (1999)). The gene consists of nine exons spanning 26kb; the 5`region is rich in repeated sequences and contains a CpG island. This region has been proven to carry a differentially methylated mark: the gene is preferentially methylated on the inactive maternal allele, as shown by comparing embryos with paternal and maternal duplication of proximal chromosome 7 (Li, et al. (2000)).
Our methylation analysis was focused on this CpG rich region; namely the 1st exon, which is a part of the above-mentioned CpG island. The amplified region is 422bp long and contains 24 CpG dinucleotides (Fig. 17).
|Fig. 17: Bisulphite analysis of Peg3 gene.|
|The results of the bisulphite analysis including the scheme of the genomic organisation of Peg3 gene are depicted in the upper part of the figure. Each line represents a unique bisulphite clone. Filled and open circles represent methylated and non-methylated CpG dinucleotides, respectively. The gel with the products of bisulphite PCR is shown left: M - 1kb marker (MBI Fermentas), 1,2 - examples of PGC samples, 3 - water control for the bisulphite treatment, 4 - water control for PCR amplification.|
The mixed gender samples of 11.5 dpc primordial germ cells were used in the previous experiments. To exclude the possibility that there is a sex specific difference at the onset of epigenetic reprogramming we isolated the PGCs separately from the individual 11.5 dpc embryos, that were later sexed using a PCR approach (see Material and Methods). The presented bisulphite data argue that there is no difference in the timing of major demodification event in female and male germlines.
The 11.5 dpc samples show methylation of a distribution explainable by the presence of a normal imprint (maternal allele methylated, paternal allele not methylated; i.e. methylated vs. non-methylated clones in a 1:1 ratio). However, only one day later in the development the same tested region appears to be completely demethylated (at least two independent bisulphite treatments for sample type) in PGCs of both female and male.
The results on the 11.5 dpc samples uncover an interesting phenomenon: not all the methylated DNA molecules appear to be methylated completely (the normal bisulphite pattern observed in somatic cells consists of either completely methylated or completely non-methylated DNA strands). Interestingly, the partially methylated DNA strands do not show the stochastic type of methylation, but rather patchy pattern of modification. This observation might shed more light on kinetics (or possible mechanism) of the demethylation process; the responsible enzymatic machinery is likely to work in a processive manner. Additionally, the presence of partially demethylated DNA strands ad 11.5 dpc suggests, that 11.5 dpc might be the critical time point when the reprogramming starts.
Lit1 and Peg3 represent the imprinted genes carrying maternal methylation imprint, to gain more complex insight into the problematic of imprint reprogramming we focused on the paternally methylated genes in the following experiments.
Insulin-like growth factor 2 (GenBank acc.# U71085, mouse distal chromosome 7) is a typical candidate of an imprinted gene characterised by paternal methylation. Igf2 begins to be transcribed shortly after implantation first in extraembryonic tissues, and then throughout mesodermal and endodermal tissues in postimplantation embryos (Lee, et al. (1990)). The gene encodes a single polypeptide involved in signalling through the IGF/INS pathway, thus having implications for embryonic growth (foetuses lacking IGF-II are growth retarded (DeChiara, et al. (1991)), overepression causes an increase of size at birth (Sun, et al. (1997); Eggenschwiler, et al. (1997)).
Imprinting of the Igf2 gene has been the subject for number of publications. Using the knock-out technology it was shown that the transcription of the gene is active only on the paternal allele (DeChiara, et al. (1991)). There have been three differentially methylated regions identified within the gene; methylation of those regions is tightly connected with Igf2 expression. DMR0 (positioned upstream of placenta specific promoter 1) shows differential methylation only in placenta, where the maternal allele is preferentially methylated (Moore, et al. (1997)). DMR1 (located upstream from the promoter 1) is not differentially methylated in germ cells, but becomes so soon after fertilisation (Sasaki, et al. (1992); Shemer, et al. (1996)). Preferentially methylated is the paternal transcriptionally active allele. The best-characterised DMR is the DMR2, which lies in 3’part of the coding region. DMR2 is differentially methylated in germ cells, and loses its methylation in early preimplantation embryo, which then becomes re-established later on (Oswald, et al. (2000); Feil, et al. (1994)).
The decision to choose DMR2 from the above mentioned Igf2 DMRs for our analysis was based on the following facts: a) DMR2 displays considerable parent-of-origin specific methylation. (As we were not able to distinguish maternal and paternal alleles in our experimental set-up, we had to focus on regions with pronounced difference in sex specific methylation.) b) Our laboratory described in detail the methylation dynamics of DMR2 during embryogenesis (Oswald, et al. (2000); Hajkova - unpublished data). Thus there was a number of comparative data available giving us the idea about the level of methylation to expect in somatic cells of embryo.
The amplified part of DMR2 is 731bp long and spans 30 CpG sites (Fig. 18).
|Fig. 18: Bisulphite analysis of Igf2 DMR2|
|The results of several bisulphite treatments are depicted above. Each line represents a unique bisulphite clone (open and filled circles correspond to non-methylated and methylated CpGs, respectively). Despite the number of bisulphite experiments, it was not possible to detect any methylation in 11.5dpc PGC samples (for more see the text). The photography left shows the gel with the products of bisulphite PCR amplification: M - 1kb ladder (MBI Fermentas), 1 - PGC sample, 2 - water control for the bisulphite treatment, 3 - water control for the PCR amplification.|
Despite performing 3 independent bisulphite treatments we were not able to detect any methylation in 11.5 dpc PGCs. (Each separate line in a figure represents a unique clone based on single nucleotide polymorphism concerning the unconverted cytosines.) This result was rather unexpected as we know, that the methylation imprint is present in this region already in the earlier stages of embryogenesis (see Fig. 19). Absence of methylation is also surprising in the light of the previous experiments: lit1 and peg3 (as well as other genes – see the following chapters) keep the methylation imprints in primordial germ cells up to 12.5 dpc. Thus the primordial germ cells pass during the development a stage when they posses normal methylation imprints present in somatic cells. This taken together suggests that the DMR2 gets probably demethylated earlier during the PGC development.
|Fig. 19: Allele specific methylation of the Igf2 DMR2 in the embryonic tissues of 8.5 dpc embryos.|
|The figure shows the results of the bisulphite analysis: individual lines represent unique bisulphite clones, methylated CpGs are shown as filled circles, non-methylated are represented by open circles. The alleles were distinguished based on the sequence polymorphism. Note the presence of the allele specific methylation already at this early embryonic stage.|
The bisulphite treatments performed on 12.5 dpc samples of both genders revealed no methylation in the tested DMR either. This is in agreement with the previous results showing demethylated status of the tested genes at 12.5 dpc.
The possibility that we were not able to detect methylation in the tested region because of the methodical problems (bias in bisulphite-PCR, bias in cloning etc.) was excluded by the fact, that identical primers, PCR and cloning conditions were previously tested for the bias and used in number of experiments performed in our laboratory (Oswald, et al. (2000); Hajkova – unpublished results).
The methylation changes occurring in Igf2 DMR2 differ from the changes detected in previous experiments on peg3 and lit1. The demethylation step obviously occurs earlier, so that the DMR region stays unmodified throughout the time window tested. The demethylated status at 12.5 dpc is, however, in agreement with the above-described experiments showing that the imprints of PGCs are erased at 12.5 dpc.
The next example of a widely studied imprinted gene with paternal methylation is H19 (GenBank acc.# AF049091, mouse distal chromosome 7). This gene which encodes RNA of unknown function is highly expressed from the maternal allele in embryonic tissues of endodermal and mesodermal origin (Brannan, et al. (1990); Poirier, et al. (1991)). The mechanism of H19 imprinting is thought to involve paternal-specific methylation of the 5`flank of the gene (Bartolomei, et al. (1993); Ferguson-Smith, et al. (1993)). As required for a gametic imprinting mark, methylation is found in sperm but not in oocytes and is maintained throughout embryogenesis in all tissues (Olek and Walter (1997); Tremblay, et al. (1995); Tremblay, et al. (1997)).
First we decided to analyse the proximal (5’part) of the H19 DMR (see Fig. 20). The analysed region corresponds to the part of the DMR amplified by (Olek and Walter (1997)) using F9R9 primer combination. The sequence was shown to carry methylation imprint in gametes (sperm completely methylated, oocytes non-methylated) as well as in somatic cells of the embryo.
The amplified fragment is 553bp long and comprises 19 CpG sites.
|Fig. 20: Bisulphite analysis of the distal part of upstream H19 DMR.|
|The results of the bisulphite analysis are depicted above. The lines represent unique clones, filled circles stand for methylated CpG positions, the open circles represent non-methylated CpGs. The gel photograph (left) shows the product of the bisulphite PCR amplification. M - 1kb ladder (MBI Fermentas), 1 - PGC sample, 2 - water control for the bisulphite treatment, 3 - water control for the PCR amplification.|
The bisulphite analysis of the H19 DMR was technically challenging. Despite extensive optimisation of PCR condition, the rate of successful amplification appeared to be low with frequent appearance of multiple bands. Additional problems appeared during the cloning procedure - the number of obtained clones was low; additionally, some clones turned out to be not completely converted. It is interesting to note, that incomplete bisulphite conversion (patchy pattern) was observed solely when analysing the H19 DMR.
Analysis of methylation pattern of upstream H19 DMR revealed pattern similar to that of Igf2 DMR2. The bisulphite experiments were carried out several times, nevertheless, there was no methylation imprint detectable in the samples of 11.5 dpc neither 12.5 dpc PGCs (the group of 11.5 dpc bisulphite clones showing the same single methylated site at 5’end indicate a possible problem of clonality in one of the bisulphite treatments). The obtained results are raising the following questions: a) the paternally methylated imprinted genes might behave differently (i.e. the imprint erasure of those genes occurs earlier ev. faster) b) the phenomena could be specifically connected to the H19/Igf2 locus (the genes are located within the same chromosomal region 80 kb apart from each other) c) our bisulphite analysis might not target the real imprinting mark; the regions we investigated could represent the regions of secondary level in “imprinting hierarchy”, which could eventually display less of the imprint maintenance.
Recently a new investigation on the methylation status of H19 DMR in primordial germ cells was published by (Ueda, et al. (2000)). Using a methylation sensitive PCR the authors identified 2 CpG positions where the methylation seems to be present still (or already?) at 13.5 dpc (the earliest time point included into the analysis). According to the analysis the HhaI sites #5 and #7 show significant methylation, whereas the rest of the tested region seems to be non-methylated. The results were subsequently confirmed by bisulphite analysis on the region surrounding the HhaI site #7. In male 13.5 dpc germ cells the region shows about 30% methylation. This observation might suggest that the real “imprinting centre” lies in the vicinity of the described HhaI sites.
The region analysed by (Ueda, et al. (2000)) does not, overlap with the region analysed in our study, but is located more downstream in direction to the H19 promoter (see Fig. 21).
Our analysis of this downstream region (445bp, 18 CpG sites) revealed the following:
|Fig. 21: Bisulphite analysis of the proximal part of H19 upstream DMR.|
|The results of the bisulphite treatments are depicted above. Each line represents a unique bisulphite clone, the filled circles stand for the methylated CpG positions, open circles represent the non-methylated CpGs. The example of a result of the bisulphite PCR amplification is shown left. M - 1kb ladder (MBI Fermentas), 1 - PGC sample, 2- water control for the bisulphite treatment.|
For our analysis of epigenetic reprogramming it was important to know if there is any difference in the initial processes taking place in female and male PGCs. As it was discussed already before, all the 12.5 dpc samples used in our experiments were sex sorted. The determination of sex is more laborious in the case of 11.5 dpc genital ridges (for details concerning the sexing procedure see Material and Methods). As a consequence, the sexed samples were used only in limited number of control experiments. In the case of downstream part of H19 DMR (as in the case of peg3 gene) we carried out the bisulphite experiments on sexed 11.5 dpc samples. As obvious from the result, also in this case we could not detect any significant difference between the 11.5 dpc primordial germ cells of female and male.
The bisulphite treatments of 11.5 dpc samples revealed fully methylated as well as completely non-methylated clones. The presence of methylated clones indicates that the methylation imprint is probably still maintained at this time point (the uneven distribution of methylated vs. non-methylated clones is possibly due to the low number of obtained clones or the bias in the bisulphite PCR). Similarly to peg3 11.5 dpc results, some of the clones show patchy pattern of methylation. As discussed above (see 3.1.7 Methylation changes in peg3) this finding supports the idea, that 11.5 dpc is the critical time point in the scenario of germ cell reprogramming.
To the contrary, the tested region showed no methylation in the 12.5 dpc samples, or only the remnants of it (see Fig. 21). This finding corresponds to the results of previous experiments: the imprints are maintained up to 11.5 dpc and diminish at 12.5 dpc.
The results of the downstream H19 DMR analysis are interesting in the light of previous experiments performed on the upstream part of the DMR. Taken together with the observation of (Ueda, et al. (2000)) the data indicate that the sequence in the vicinity of HhaI site #7 might function as an imprinting “core” element. Interestingly, the same part of the H19 DMR is conserved between human, mouse and rat (Frevel, et al. (1999)) and was shown to contain CTCF binding site (see Fig. 22) (Bell and Felsenfeld (2000); Hark, et al. (2000)), which is involved in the regulation of whole Igf2/H19 cluster (methylation of the site blocks the binding of CTCF thus allowing the enhancers to access the Igf2 promoter – see Fig. 22 and Fig. 23). Thus the region tested in our bisulphite experiments may serve as a primary imprint for the whole Igf2/H19 cluster (which might also explain the absence of methylation imprint in the upstream part of H19 DMR and in Igf2 DMR2 at 11.5 dpc)
|Fig. 22: Proposed model of CTCF function|
|(taken from Bell et al. 2000). On the maternally inherited chromosome CTCF binds to unmethylated CTCF binding sites (two in each of the nuclease hypersensitive regions - shaded boxes). The resulting insulator prevents activation of the maternal Igf2 allele by the H19 enhancer. On the paternally inherited chromosome the CTCF sites are methylated, thus preventing CTCF binding. Absence of insulator activity enables activation of Igf2 by the H19 enhancer. (+/- corresponds to the transcriptional activity).|
|Fig. 23: Conserved CTCF sites within the H19 DMR|
|(taken from Bell et al.,2000). Comparison of the sequence of the mouse, rat and human H19 DMR regions shows the conservation of the CTCF binding site (for comparison see the b-globin CTCF binding site). Species-specific identities are shown in grey, crossspecies sequence conservation is depicted in black. The sequence of the mouse strand used in our experiments corresponds to m4. The arrows indicate the CpGs included into our bisulphite analysis (the number corresponds to the position of CpG in the bisulphite dot diagram). Note that the methylation detected in 12.5dpc samples was located explicitly outside the labeled CpG sites.|
Whereas the previous chapters concerned the reprogramming of imprinted genes carrying either a maternal (lit1, peg3) or a paternal (Igf2, H19) methylation mark, the following paragraphs deal with a unique example of a gene carrying both parental imprints.
Snrpn (small nuclear ribonucleoprotein polypeptide N, mouse central chromosome 7, GenBank acc.# AF063659) is an imprinted gene situated in the centre of the chromosomal domain involved in two neurogenetic disorders, Prader-Willi syndrome (PWS) of Angelman syndrome (AS), respectively. The gene is paternally expressed (Leff, et al. (1992);Glenn, et al. (1996)) primarily in brain and heart (Gerrelli, et al. (1991)), coding for a protein (Smn) that is thought to be involved in splicing (Steitz, et al. (1988)).
The Snrpn gene was included into our analysis because of its unique imprinting features: the promoter and the 1st exon were shown to be maternally methylated, whereas the 3’end of the gene is paternally modified (Shemer, et al. (1997)). Thus analysing the reprogramming process in both regions, we were able to perform a direct comparison of behaviour of a maternal and a paternal imprint within the same chromosomal locus.
First we analysed the maternal imprint in the 5’part of the Snrpn gene:
Maternal methylation at the 5’end of the gene (DMR1) spans from the promoter region over the 1st exon to the 1st intron (Shemer, et al. (1997);El-Maarri, et al. (2001)) and was shown to correlate inversely with the Snrpn expression (the gene is expressed from the paternal non-methylated allele). The imprint is present in gametes: the region being completely methylated in oocytes, whereas non-methylated in sperm; as well as in embryonic and adult tissues (Shemer, et al. (1997); El-Maarri, et al. (2001)).
The following results were obtained when analysing the region described in (El-Maarri, et al. (2001)); the amplified sequence lies within the Snrpn exon 1 region, is 289bp long and contains 14 CpG positions (Fig. 24).
|Fig. 24: Bisulphite analysis of Snrpn DMR1.|
|The results of the bisulphite treatment are shown right Individual line represent unique bisulphite clones, the filled and open circles stand for the methylated and non-methylated CpG positions, respectively. The result of one of the bisulphite PCRs is shown in the upper right part of the figure. M - 1kb ladder (MBI Fermentas), 1 - PGC sample, 2 - water control for the bisulphite treatment, 3 - water control for the bisulphite PCR.|
The results of the Snrpn DMR1 bisulphite analysis document the presence of methylated clones in 11.5 dpc primordial germ cells. The distribution of methylated vs. non-methylated clones (approximately 1:1) corresponds to the pattern observed in zygote and somatic cells (El-Maarri, unpublished results) and is characteristic for a DMR of an imprinted gene. As obvious from the experiments, between 11.5 and 12.5 dpc the Snrpn DMR1 methylation pattern changes from the fully established imprint to a completely erased status (as observed with other genes in previous experiments). The continuous methylation was detected only in one of the 12.5 dpc female clones and in two of the male 12.5 dpc clones (see Fig. 24).
Similarly to the phenomena observed in peg3 and in the downstream part of the H19 DMR, there is a tendency to detect a patchy methylation rather than a random one. Such an observation could be explained by a processive action of the demethylation machinery. The sporadic occurrence of patchy methylated clones in 12.5 dpc samples (as well as the presence of such clones in 11.5 dpc samples when analysing other genes) brings about not only the hint of the character of possible demodification mechanism; it also defines a time window of its action.
The second region analysed within the Snrpn gene is a preferentially paternally methylated DMR2 situated at the 3’end of the gene (see Fig. 25). The region has been identified by restriction mapping of the P1 clone containing the complete mouse Snrpn gene (Shemer, et al. (1997)). The complete genomic sequence of the gene has, however, not been published yet. In order to perform the bisulphite analysis in the DMR2 region, the clones had to be fished out from the database of the Sanger Centre Mouse Sequencing Project and of the Celera Corp. based on the homology with the Snrpn cDNA. Those clones were subsequently aligned into a contig with the help of the Lasergene - MegAlign software. Using this approach it was possible to reconstitute the part of the genomic Snrpn sequence spanning from exon 5 to exon 10. The following sequence analysis revealed that this part of the Snrpn gene is very poor in respect to CpG dinucleotides. The only fragment with slightly higher density of CpG sites (5 CpG positions within approximately 400bp) is localised within the intron 8 as a part of a Line1 repetitive element. Being the only suitable candidate, this part of the genomic sequence was chosen as a target for our bisulphite analysis.
The amplified fragment (578bp) contains 5 CpG positions (Fig. 26).
|Fig. 25: Genomic organisation of mouse Snrpn gene.|
|The barrows represent the Snrpn exons 1-10. CpG positions are shown as vertical lines, the region analysed by bisulphite sequencing is depicted in red.|
|Fig. 26: Bisulphite analysis of Snrpn DMR2.|
|The results of the bisulphite treatments are depicted in the upper part of the figure. Each line represents a unique bisulphite clone, methylated CpG positions are represented by filled circles; non-methylated CpG by open circles. The example of the bisulphite PCR amplification is shown left: M - 1kb ladder (MBI Fermentas), 1,2 - PGC samples, 3 - water control for the bisulphite treatment, 4 - water control for the bisulphite PCR. The unspecific PCR products frequently appeared despite extensive optimization of the PCR conditions. The primers for the bisulphite PCR were designed outside the Line 1 repetitive element; nevertheless, the fact that the target for the amplification was a repetitive element might explain the observed low specificity of amplification.|
Bisulphite analysis performed on 11.5 dpc samples documents that also the Snrpn DMR2 is still differentially methylated at this stage of PGC development. The samples of both genders revealed the presence of methylated and non-methylated clones in approx. ratio 1:1, which characterises the imprinted pattern. It should be pointed out, that the Snrpn DMR2 was the 3rd control region, where the 11.5 dpc primordial germ cells of female and male were analysed separately (for the other examples: peg3 and the downstream part of H19 DMR see the previous chapters; for the details concerning the sexing procedure see Material and Methods). As even in this case we could not detect any difference between the 11.5 dpc germ cells of either sex, we accepted as proven that at the initial stage of PGCs reprogramming the cells of both genders follow the identical scenario.
Due to technical problems connected with the Snrpn DMR2 amplification and cloning (caused possibly by the fact that the tested region was a part of a repetitive element) the number of obtained bisulphite clones was very low. Despite number of trials we were not able to obtain any specific PCR fragment from the male 12.5 dpc sample; in the case of female 12.5 dpc cells the analysis yielded only 4 specific clones.
At the same time when the experiments described in this thesis were carried out, the parallel investigation of the reprogramming of repetitive elements in the same PGC samples was being performed in the co-operation with the group of Dr. W. Reik in the Babraham Institute, Babraham, UK. The methylation analysis of Line1 and IAP repetitive elements showed that those sequences undergo gradual loss of modification rather than a fast demethylation event (N.Lane – unpublished observation, Hajkova et.al - submitted). In the light of those findings the presence of a fully methylated clone in female 12.5 dpc cells may indicate, that the tested DMR as being a part of a Line1 repeat follows the scenario of repetitive elements, i.e. the gradual loss of methylation.
The imprinted status of the Snrpn DMR2 has been up to now elucidated solely on the basis of the methylation-sensitive restriction analysis of 4 HhaI sites (Shemer, et al. (1997)). Thus, our experiments represent the first detailed bisulphite based methylation study of the region. The data confirm the existence of a differential methylation within the region, which was in the samples of PGCs more pronounced than in the original report – Shemer etal., 1997 described the presence of partial methylation on a maternal allele and complete methylation on a paternal allele.
In the previously described experiments we elucidated the methylation changes occurring within the differentially methylated regions of imprinted genes. Though approximately 40 examples have been identified up to date, the imprinted genes still represent a very specialised part of the genome. In order to gain more complex understanding about the PGC reprogramming, the examples of non-imprinted single copy genes had to be included into our analysis.
The skeletal α-actin (GenBank acc.# M12347) is an example of a single copy gene characterised by tissue specific expression. The gene is not transcribed in the early undifferentiated embryo, expression corresponds with the appearance of differentiated muscle tissue following implantation (Sassoon, et al. (1988,Taylor and Piko (1990)).
To assess the methylation changes occurring within this gene we analysed the 5` region covering the 3`part of the promoter and proximal part of the 1st exon. Our choice was based on the following facts: a) 5’region of the gene contains a CpG island, which makes it a suitable candidate for a bisulphite analysis b) this region has been used in the previously published studies describing the methylation changes occurring during the embryogenesis Warnecke and Clark (1999);Oswald, et al. (2000)), thus we were provided with satisfactory amount of comparative data.
The analysed region is 266bp long and comprises 11 CpG dinucleotides (Fig. 27).
|Fig. 27: Bisulphite analysis of a-actin.|
|The right part of the figure shows genomic organisation of a-actin gene (the position of the region chosen for the bisulphite analysis is shown in red) and the results of bisulphite treatments Individual lines represent unique bisulphite clones. Methylated and non-methylated CpG positions are represented by filled and open circles, respectively. The example result of the bisulphite PCR is shown left M - 100 bp ladder (MBI Fermentas), 1 - PGC sample, 2 - water control for the bisulphite treatment, 3 - water control for the bisulphite PCR.|
As obvious from the dot diagrams the 5`region of α-actin is still methylated at 11.5 dpc (mixed gender samples). The level of methylation at distinct CpG sites varies between 7.1 and 42.9%, which corresponds to the level found in adult tissues (0-30% in heart and skeletal muscle) expressing the gene (Warnecke and Clark (1999)). Similarly to the behaviour of imprinted genes, the region appears to be completely non-methylated in both female and male 12.5 dpc PGCs (the results based on two independent bisulphite treatments and several amplifications).
As the second candidate non-imprinted gene we chose the alkaline myosin light chain (GenBank acc.# X12972). Similarly to the α—actin the MylC belongs among the tissue specific genes, expression limited to ventricular myocardium and slow skeletal muscle (Barton, et al. (1985)). Methylation features of the gene have been extensively studied (Walsh and Bestor (1999, Oswald, 2000 #2)) – the 5`region being found heavily methylated in oocytes and sperm and partially methylated in somatic tissues.
To follow the methylation changes we analysed 9 CpG sites positioned within the promoter region (Fig. 28).
|Fig. 28: Bisulphite analysis of mylC.|
|The results of the bisulphite treatments are shown in the upper part of the figure.including the schematic drawing of the mylC genomic orgainisation. The region analysed by the bisulphite genomic sequencing is depicted as a red box. Each line represents a unique bisulphite clone. Methylated and non-methylated CpG positions are represented by filled and open circles, respectively. The figure left shows the result of the bisulphite PCR. M - 100bp ladder (MBI Fermentas), 1,2 - PGC sample, 3 - water control for the bisulphite treatment, 4 - water control for the bisulphite PCR.|
The methylation changes of the mylC promoter region follow the scenario observed with the other genes. The 11.5 dpc samples show at the tested CpG sites methylation between 20 and 66.7 %, which is in the range found in somatic tissues (Walsh and Bestor (1999)). After 11.5 dpc the methylation level decreases dramatically (similar to behaviour of other tested genes); there was no methylated site detected in the 12.5 dpc primordial germ cells of either sex.
The control experiments carried out on the non-imprinted genes confirmed the previous results obtained on imprinted genes. The promoter regions of α-actin and mylC carry at 11.5 dpc the methylation comparable to that detected in somatic tissues. In the 12.5 dpc PGC samples, both regions, however, appeared to be completely non-methylated. These findings are in full agreement with the observation made on the imprinted genes - The majority of the tested regions (the only exceptions were Igf2 DMR2 and upstream part of the H19 DMR) appeared to be methylated in 11.5 dpc primordial germ cells. Whereas all the tested regions were found completely non-methylated in 12.5 dpc PGCs of both genders.
These findings thus implicate a presence of a phenomenon, which is not related solely to the imprinted genes, but concerns the whole genome of the primordial germ cells. This reprogramming starts in the PGCs of both genders around 11.5 dpc, the main erasure step being finished at 12.5 dpc i.e. within only 24 hours. Our data also exclude the hypothesis of a gradual change from the maternal to the paternal (or vice versa) imprinting pattern. The reprogramming obviously happens in two distinct steps – the initial imprint erasure and the following imprint re-establishment, where only the latter is sex specific.
After their entry into the genital ridges around 10.5 dpc, the primordial germ cells keep on slowing down the life cycle until 13.5 dpc, when the female PGCs enter the meiotic and the male PGCs the mitotic arrest, respectively (Tam and Snow (1981); McLaren (2000)). The results presented in previous chapters of this thesis describe in detail the methylation changes taking place in PGCs of 11.5 dpc and 12.5 dpc embryos, i.e. at stages, when the PGCs still undergo proliferation. However, to gain a complete overview of the process of methylation erasure it was necessary to follow the PGC development beyond the critical time up to the point of the sex-specific differentiation.
The 13.5 dpc PGCs were isolated from the Oct4-GFP transgenic embryos (see Material and Methods). The FACS sorting of the cell suspension prepared from the GFP positive 13.5 dpc genital ridges revealed surprisingly high variability in the size of positive cells (see Fig. 29a and 29b). As mentioned above, the primordial germ cells are supposed to undergo meiotic/mitotic arrest at 13.5 dpc. The germ cells of the genital ridges are, however, not synchronised, so at 13.5 dpc the “arrested” germ cells represent still only a fraction of the cell population. As we believed that those cells might be characterised by a different cell size, we decided to separate the GFP positive cells into two distinct fractions: the fraction of “large” cells and the fraction of “small” cells. Those fractions were treated in the subsequent experiments separately.
|Fig. 29a: FACS sorting of the 13.5 dpc primordial germ cells (female samples).|
|The isolated genital ridges of 13.5 dp embryos were trypsinised to yield single cell suspension. The cells were subsequently used for FACS sorting- first based on the GFP positive signal (right). Note that the variability in GFP expression is rather low. The positive cells, however, appear to vary in their size (see left part of the figure). To examine the possible differences (for more see the text) the cell size was chosen as the second sorting parameter.|
|Fig. 29b: FACS sorting of the 13.5dpc primordial germ cells (male samples).|
|Single cell suspension of 13.5dpc embnryonic genital ridges was FACS sorted according to the GFP signal and cell size (for details see the previous figure). The results of the FACS sorting procedure did not reveal any significant differences between male and female 13.5dpc primordial germ cells.|
The methylation analysis of 13.5 dpc germ cells brought the following results:
|Fig. 30a: Bisulphite analysis of the 13.5 dpc primordial germ cells.|
|The figure shows the results of bisulphite analysis of the Igf2 DMR2 and the proximal part of the upstream H19 DMR. In both tested regions no methylation is detectable neither in female nor in male PGCs. As there was no significant difference between the fractions of large (L) and small (S) cells the dot diagrams for the other genes are shown as a summary (see fig. Xb).|
|Fig. 30b: Bisulphite analysis of the 13.5 dpc primordial germ cells.|
|The figure shows the overview of the results of the bisulphite treatments. The graphs combine the results of 13.5 dpc germ cells of both genders and both isolated fractions of large and small cells. Higher methylation levels shown in the case of peg3 and a-actin are likely to be caused by very low numbers of unique bisulphite clones.|
As shown by the results of bisulphite analysis, all the tested DMR regions were found non-methylated in 13.5 dpc primordial germ cells. These results are in agreement with the observation published by Kafri, et al. (1992) and Brandeis, et al. (1993). In their report Kafri and colleagues used the methylation sensitive PCR to describe the demethylated status of non-imprinted ApoAI and globin genes in 13.5 dpc germ cells. The same method was used in the study of Brandeis et al. to assess the methylation status of the imprinted genes (Igf2, Igf2r and H19 included into the analysis). The data of both indicate the complete absence of methylation in the tested regions in 13.5 dpc germ cells.
Recently, two independent investigations of the methylation in the H19 upstream region have been published (Ueda, et al. (2000); Davis, et al. (1999)). Although both authors described the presence of low level of methylation in the 3’ part of the H19 DMR, we were not able to confirm the observation. Different results might have been caused by cross-contamination of PGC samples with somatic cells (interestingly, in Davis et al. the male 13.5 dpc samples showing higher level of methylation were of lower purity - 93 and 89% versus 96% for female samples), or possibly by using the different mouse strain.
It should be noted that we did not detect any difference between the fractions of “large” and “small” cells neither between the cells of different gender. Taken together, the data implicate that the primordial germ cells after the initial step of imprint erasure do not change their methylation status upon their entry into the mitotic/meiotic arrest.
Having analysed the methylation status of primordial germ cells between 11.5 and 13.5 dpc, our interest turned towards the earlier stages of PGC development. Our previous analysis showed that the vast majority of the DMRs carries the methylation imprint still at 11.5 dpc. The DMR2 of Igf2 gene appeared to be one of the exceptions being apparently non-methylated at that stage. Based on the bisulphite analysis performed on the 8.5 dpc embryonic cells (for details see 3.1.3 Methylation changes in Igf2) it is probable that the DMR2 methylation imprint is present at the earlier stages of PGC development and is subsequently erased earlier than the methylation marks of other DMRs. Such an option opens, though, a general question concerning the existence and extent of imprints in migrating and early post-migratory primordial germ cells.
The hints to the answer might be found in the earlier report of (Tam, et al. (1994)). The authors are describing in detail the process and timing of X chromosome inactivation (and re-activation) taking place in early migratory and post-migratory PGCs. According to their investigation the X inactivation occurs in most of the PGCs during the time of migration through mesentery (i.e. before entering the genital ridge). It is very likely that the parental imprints inherited from the germ line also become properly established at this time, if not earlier. However, up to now, no data supporting this hypothesis has been published.
We took the advantage of our model system (the transgenic mouse with the germ line specific GFP expression) and isolated the early post-migratory germ cells from 10.5 dpc old mouse embryos to investigate their imprinting status.
|Fig. 31: Bisulhite analysis of Igf2 DMR2 in 10.5 dpc primordial germ cells.|
|The methylated CpG positions are represented by filled circles, the non-methylated CpGs by open circles.|
The bisulphite analysis of early post-migratory germ cells (at 10.5 dpc the PGCs have just reached the genital ridge) confirmed the presence of methylation in the Igf2 DMR2 region (Fig.31). As we were not able to distinguish paternal alleles in our system and the methylation in this region shows never a “black-white” pattern; it is difficult to make any statements concerning the extent of imprint at this stage. However, the more pronounced methylation in the 5’ region of DMR2 corresponds to the pattern seen in early embryos (8.5 dpc).
|Fig. 32: Bisulphite analysis of 10.5dpc primordial germ cells.|
|Methylated CpG are shown as filled circles, non-methylated CpGs as open circles. Each line represents a unique bisulphite clone.|
Due to the very limited amount of 10.5 dpc samples it was not possible to perform the analysis on all the previously described DMRs. The next two tested regions, Snrpn and the downstream part of H19 DMR, both confirmed the presence of methylation in DMRs of imprinted genes at this early stage of PGC development (Fig. 32). However, the pattern observed identically in both regions at 10.5 dpc is not identical with the fully established imprinting pattern seen in PGCs only one day later. The methylation analysis of 11.5 dpc primordial germ cells revealed the presence of completely methylated and completely non-methylated clones (“black-white” pattern) whereas there were only completely non-methylated and partially methylated molecules, present in 10.5 dpc samples. Thus, although some methylation imprint is present in primordial germ cells at the time when they reach the genital ridge, the imprint is obviously not complete (or not so pronounced as observed in somatic cells in later stages of embryogenesis). This fact might, though, be not germ line specific, as the somatic type of imprints is probably only being established at this stage of embryogenesis.
The previously described experiments analysed in detail the reprogramming of primordial germ cells after their colonisation of the genital ridge. The results showed that the PGCs do posses imprints at the time when they reach the genital ridge, the imprints are maintained up to 11.5 dpc and get subsequently erased within only one day before 12.5 dpc. This undermethylated status is kept up to 13.5 dpc. It is believed that the whole reprogramming process is strictly germ line specific to ensure the proper erasure of parent-of-origin specific imprints and possible epimutations and to enable the setting of new, sex specific, modifications. To pronounce the unique character of the germ lineage in comparison with the surrounding cells we decided to assay the cells of the genital ridge stroma for their imprinting/methylation status (Fig. 33).
|Fig. 33: Methylation pattern of 12.5dpc somatic and primordial germ cells.|
|The figure shows the results of bisulphite treatments carried on the somatic cells of the genital ridges compared to the results obtained on the primordial germ cells. The somatic cells were obtained as a flow-through fraction from the immunoaffinity purification procedure of PGCs (for details see Materials and Methods).|
Despite the dramatic changes taking place in the primordial germ cells, the surrounding somatic cells maintain their somatic methylation status. In the tested 12.5 dpc somatic cells we detected the presence of imprints as well as methylation in the promoters of tissue specific non-imprinted genes. The unequal distribution of methylated vs. non-methylated clones in the case of lit1 and H19 could be explained by the fact, that those results are based on a single bisulphite treatment. More bisulphite analyses would be necessary to elucidate whether this uneven distribution is real.
The mouse Xist gene is expressed exclusively from the inactive X chromosome and is apparently involved in the initiation of X inactivation (reviewed in Mlynarczyk and Panning (2000)). It has been well documented that the Xist expression is regulated by methylation in the promoter region. In somatic tissues, the 5’end of the silent Xist allele on the active X is known to be fully methylated whereas the expressed allele on the inactive X is unmethylated (Norris, et al. (1994); Allaman-Pillet, et al. (1998)).
The inactivation of the X chromosome occurs during early embryogenesis in a developmentally regulated manner (Monk and Harper (1979)). The inactivation occurs first in the extraembryonic trophectoderm and primitive endoderm lineages. This early inactivation is non-random, with exclusive inactivation of the paternal X chromosome (Takagi and Sasaki (1975);West, et al. (1977)). In the embryonic lineage the random X inactivation occurs around the time of gastrulation. It is known that the random X inactivation occurs during the migration phase of primordial germ cells, which coincides with the time of X inactivation in somatic tissues (Tam, et al. (1994)). However, following the entry of PGCs into the genital ridge the inactive X chromosome is re-activated in the majority of the PGCs by 13.5 dpc (Monk and McLaren (1981,Tam, et al. (1994)).
It should be noted, that in this case the reactivation of the X chromosome, which is normally connected with the methylation of the Xist promoter, occurs at the time when the rest of the genome is apparently undermethylated (see our previous results). To elucidate the methylation processes connected to the X chromosome reactivation we carried out the methylation analysis of the Xist promoter (Fig. 34).
|Fig. 34: Bisulphite analysis of Xist promoter region in male PGCs.|
|The figure shows the results of bisulphite treatments. The methylated and nonmethylated CpG positions are shown as filled and open circles, respectively. The fractions of large and small cells were obtained using FACS sorting (for details see the text). Example of the bisulphite PCR result is shown left. M - 1kb ladder (MBI Fermentas), 1 - PGC sample, 2 - water control for the bisulphite treatment, 3 - water control for the bisulphite PCR.|
In the case of Xist promoter region we focused our interest on the male primordial germ cells. Cells of male individuals contain a single X chromosome that is maintained active, i.e. the Xist gene is not expressed and its promoter region is methylated. According to our results, this is the case for the 11.5 dpc male PGC, where all the clones obtained in our bisulphite analysis indicate full methylation. Similarly to other single copy genes the Xist undergoes full demethylation before 12.5 dpc and is found to be demethylated still at 13.5 dpc. As in the case of the other tested regions we did not detect any difference between the fraction of “small” and “large” 13.5 dpc primordial germ cells. Thus, the Xist gene in male PGCs follows completely the scenario valid for other single copy genes.
Interestingly, the single X chromosome of male germ cells is reported to be active until the onset of meiosis when short period of X inactivation (accompanied by Xist expression) occurs (McCarrey, et al. (1992); McCarrey, et al. (1992); McCarrey and Dilworth (1992)). This implicates that at 13.5 dpc the X chromosome is still active, i.e. Xist not expressed despite the non-methylated status of the promoter region.
Similarly, female 13.5 dpc primordial germ cells (see Fig. 35) with both X chromosomes re-activated (Monk and McLaren (1981,Tam, et al. (1994)) revealed absence of methylation in the Xist promoter region. Thus there must be another mechanism that keeps the Xist gene silent in the absence of DNA methylation. The existence of such a mechanism has been already proposed by McDonald et al. The authors documented the absence of methylation in the Xist promoter region during early mouse embryogenesis, i.e. at the time when the Xist expression is supposed to be imprinted.
|Fig. 35: Bisulphite analysis of Xist promotor in 13.5dpc female PGCs|
|Methylated CpG position are sown as filled circles, non-methylated represented by open circles. The "large" and "small" cells represent the fractions obtained using the FACS sorting (for details see the text).|
|Fig. 36: Immunohistochemical staining of mC.|
|The figure shows nuclei (stained by DAPI in blue) of PGC and stage-matched somatic cells. Foci stained in green (FITC) indicate presence of mC.|
The method of immunofluorescent mC staining (Mayer, et al. (2000)) is based on labelling the denatured DNA of interphase nuclei with anti mC antibodies (Coffigny, et al. (1999)). In our experimental system the technique was used to compare the data obtained from the bisulphite analysis (“single gene” approach) with the global (“whole genome”) approach.
At the first sight there is an obvious difference between the staining pattern of the germ cells and their somatic counterparts (see Fig. 36). Somatic cells of genital ridge stroma show in all the tested stages presence of brightly stained foci. The positive foci are seen only in a fraction of primordial germ cells, the foci are apparently less prominent as those seen in somatic cells. Figure 36Table 3 show that there is a clear tendency of demethylation observed in primordial germ cells between 11.5 and 13.5 dpc, whereas the numbers of positively stained somatic cells remain constant.
However, at 12.5 and 13.5 dpc we could still detect primordial germ cells with positively stained foci despite the results of the bisulphite analysis documenting complete demethylation of the genes. Thus the results of the staining indicate a protracted demethylation process rather than a rapid demethylation. These results may correspond to the observation on methylation of repetitive elements (N. Lane – unpublished results). Repetitive elements undergo during the PGC reprogramming slow and incomplete demethylation, with remarkable percentage of repeats being still methylated at 13.5 dpc.
It is difficult to judge about the nature of the target for the mC antibody staining. The stained foci do apparently not correspond to the heterochromatin as the heterochromatic regions overlap with the areas of brighter DAPI staining. Whether the repetitive elements are the target for the mC immunofluorescent staining remains speculative.
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