Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (35)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Lefebvre, L.
Right arrow Articles by Surani, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lefebvre, L.
Right arrow Articles by Surani, M. A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Human Molecular Genetics Pages 1907-1915

Genomic structure and parent-of-origin-specific methylation of Peg1
Introduction
Results
   Structure and mapping of the Peg1 locus
   The 5'-portion of Peg1 contains a CpG island
   Partial methylation of the CpG island in post-implantation embryos
   Partial methylation precedes Peg1 activation
   Parental-specific methylation of the Peg1 promoter region
   Gametic signals: sperm and parthenogenetic embryos
Discussion
   Gene structure
   Tandem repeats
   Parental-specific methylation
Materials And Methods
   Isolation and characterization of the Peg1 locus
   Embryos and ES cells
   Methylation analysis
Acknowledgements
References

Genomic structure and parent-of-origin-specific methylation of Peg1

Genomic structure and parent-of-origin-specific methylation of Peg1 Louis Lefebvre*, Stéphane Viville+, Sheila C. Barton, Fumitoshi Ishino1 and M. Azim Surani

Wellcome/CRC Institute of Cancer and Developmental Biology and Physiological Laboratory, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK and 1Gene Research Center, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-226, Japan

Received June 2, 1997; Revised and Accepted July 22, 1997

We previously identified Peg1/Mest as a novel paternally expressed gene in the developing mouse embryo. The human PEG1 gene was recently assigned to 7q32 and shown to be imprinted and paternally expressed. Therefore, PEG1 deficiency could participate in the aetiology of pre- and post-natal growth retardation associated with maternal uniparental disomy 7 in humans. We have now initiated the characterization of the Peg1 locus in order to identify and dissect cis-acting elements implicated in its imprinted monoallelic expression. The genomic structure of Peg1 as well as the DNA sequence of the 5'-end of the gene, including 2.4 kb of promoter sequences and covering the first 2 exons, have been determined. Important sequence elements, such as a CpG island spanning exon 1 and direct repeats, are identified and discussed. To address the role of epigenetic modifications in the imprinting of Peg1, a methylation analysis of the Peg1 gene is presented. Partially methylated cytosine residues in 13.5 d.p.c. embryos and undifferentiated ES cells were identified. Using embryos carrying a targetted mutation at the Peg1 locus, we show that this partial promoter methylation pattern reflects a strict parent-of-origin-specific differential methylation: the expressed paternal allele is unmethylated, whereas the silenced maternal allele is fully methylated at the CpG sites studied. That the gametes carry the epigenetic information necessary to lay down this allele-specific methylation pattern is suggested by analysis of DNA isolated from sperm and parthenogenetic embryos.

INTRODUCTION

The mammalian genome is now known to contain a number of genes subjected to an unusual transcriptional regulatory mechanism directing expression of a single allele based on its parental origin and coordinate silencing of the other allele. The molecular mechanism of this epigenetic phenomenon, termed genomic imprinting, is currently under investigation, the scope of which will be increased by identification of new imprinted genes in the mouse and in humans (1 ). Appropriate expression of imprinted genes is essential for normal development in the mouse and imprinting defects are associated with developmental abnormalities and carcinogenesis in human (2 ,3 ). Identification and characterization of putative cis-acting imprinting elements responsible for parental-specific monoallelic expression of these genes is currently an active area of research.

Based on the available data, it is difficult to explain the functional or evolutionary pressure leading to imprinting of specific genes and why a particular gene should be maternally as opposed to paternally expressed. However, the increasing list of imprinted genes being characterized helped to reveal intriguing structural similarities conserved amongst these monoallelically expressed/silenced genes (1 ,4 ,5 ). First, it was noted that imprinted genes tend to comprise compact transcriptional units with smaller introns (6 ,7 ). Second, modification of CpG residues within imprinted loci by DNA methylation has been extensively studied and the emerging picture suggests that imprinted genes are often associated with characteristic parental-specific methylation patterns: a 5' CpG-rich region covering or close to the first exon is exclusively methylated on the silenced allele, whereas downstream CpG sites, located 3' of the transcription start site within the transcribed sequences, are often methylated on the expressed allele (1 ,4 ). For example, in the mouse the maternally expressed genes H19 (8 ,9 ) and Igf2/Mpr (10 ) as well as the paternally expressed gene U2afbp-rs (11 ) all contain a CpG island which is specifically methylated on the silenced allele only, and an intronic region is methylated on the expressed allele of Igf2/Mpr (10 ). Several lines of evidence suggest that the sequences carrying these differential methylation patterns are important in the initial marking and subsequent expression of imprinted genes (12 ,13 ).

Using a general screen for paternally expressed genes by cDNA subtraction between normal and parthenogenetic mouse embryos, we previously reported the characterization of Peg1 cDNA, the first known imprinted gene expressed from mouse proximal chromosome 6 (14 ). Genetic studies using chromosomal translocations showed that this domain is subjected to parent-of-origin effects, with maternal uniparental disomy for proximal 6 leading to early embryonic lethality (15 ). Peg1 was independently isolated and named Mest, based on its predominant mesoderm-specific embryonic expression pattern (16 ). Recently, the human PEG1 homologue was mapped to 7q32 and analysis of cysts and moles enriched in cells of uniparental origin suggested that the human gene is also imprinted, with the paternal allele being expressed (17 ). This result is particularly interesting in the light of the correlation between maternal uniparental disomy for chromosome 7 (or only 7q) and cases of short stature and intrauterine growth retardation, suggesting that a paternally expressed imprinted gene, such as PEG1, is involved in the aetiology of these conditions (18 -21 ). The function of Peg1 is still unknown, although the predicted Peg1 open reading frame codes for a polypeptide showing sequence similarity with the [alpha]/[beta]-hydrolase fold, a structural motif conserved in several enzymes hydrolysing various substrates (14 ).

In this study we present a first characterization of the mouse Peg1 locus, including the full gene structure and DNA sequence of the 5'-portion of the gene. We identified a CpG island covering exon 1 of Peg1 and showed that this element is partially methylated in post-implantation embryos. Using a mutant Peg1 allele integrated by gene targetting into ES cells we show that this region of the locus is differentially methylated, with the expressed paternal allele unmethylated and the silenced maternal allele fully methylated. Analysis of DNA isolated from sperm and parthenogenetic embryos suggests that the gametes carry or can direct this differential methylation pattern.

RESULTS

Using a Peg1 genomic probe obtained by PCR with two primers designed from the Peg1 cDNA sequence (probe 4, Fig. 1 A) we isolated and mapped by restriction enzyme digestions two overlapping clones from a mouse 129/Sv genomic library constructed in the [lambda]KO vector (22 ). These clones overlap by ~2.5 kb to form a contig covering a genomic region of 17.4 kb and containing the complete Peg1 gene, as determined from the published sequence of the 2543 nt Peg1/Mest cDNA (GenBank accession no. D16262; 16 ). Figure 1 A shows a schematic representation of the Peg1 locus with a restriction map determined from the two genomic clones pPeg1A and pPeg1C.


Figure 1. Genomic structure of the mouse Peg1 gene. (A) Schematic representation (top) of the two overlapping clones (pPeg1A and pPeg1C) isolated by screening a 129/Sv [lambda]KO genomic library with probe 4. Vertical lines indicate the position of restriction sites labelled on the gene map. Structure of the Peg1 locus (middle) as determined by restriction analyses and sequencing of these two clones. Exons, shown by solid black boxes, cover the published Peg1/Mest cDNA sequence. The CpG island is highlighted above exon 1 and the arrowhead shows the location of the G-rich repeats within intron 1. Restriction enzymes are as follows: E, EcoRI; Sm, SmaI; X, XhoI; Xb, XbaI. Genomic fragments used as probes in this study are represented as open boxes (bottom). (B) Details of the genomic structure of Peg1. The size of each exon and intron (expressed in bp) was determined from DNA sequence data for the locus, except for introns 2 and 6 (asterisk), the sizes of which were estimated from restriction analyses of clone pPeg1A. Also shown are the position of each exon on the published Peg1/Mest cDNA sequence as well as the structure of the 5' and 3' splice junctions for each intron. Exon sequences are shown in capital letters.

Structure and mapping of the Peg1 locus

Sequencing primers were designed from the Peg1 cDNA sequence and used on the two genomic clones to determine the exon-intron structure of the Peg1 gene (Fig. 1 A). The transcribed sequence is divided into 12 exons, with the last exon containing >55% of the cDNA sequence (1406 bases, including 1288 bases of 3'-untranslated sequence). Based on this genomic organization, the primary Peg1 transcript is predicted to be at least 10.4 kb in length. The conserved 1005 bp Peg1 open reading frame (16 ) spans the 12 exons identified, with translation initiating within exon 1 (ATG at cDNA position 248) and terminating in exon 12 (TAA at cDNA position 1253). Figure 1 B presents a detailed account of the exon-intron structure of the Peg1 gene as determined from our analysis, with the DNA sequences at the splice junctions between coding and intervening sequences and their respective sizes. All the introns analysed show the consensus 5' donor and 3' acceptor sequences GT...AG.

The 5'-portion of Peg1 contains a CpG island

We determined the DNA sequence of a 5.4 kb genomic fragment covering the promoter region of the Peg1 gene, from the XbaI site at map position 4.28 (Fig. 1 A), and the 5'-portion of the gene, up to a BglII site following exon 2 (Fig. 3 A; GenBank accession no. AF017994). Analysis of the DNA sequence revealed two interesting features with regard to potential cis-acting elements involved in the imprinted expression of Peg1. First, a G-rich intronic sequence of 122 bp was identified 87 bp 5' of the beginning of exon 2 (arrowhead, Fig. 1 A). This sequence element is comprised of 24 tandem repeats of the consensus pentamer unit 5'-(A/T)GGGG-3', including 14 perfect copies (eight copies of 5'-AGGGG-3' and six copies of 5'-TGGGG-3') and eight copies with a single mismatch when compared with the consensus repeat unit. No other significant repetitive structure was identified within this genomic segment.

Second, a region of 550 bp comprising 157 bp of 5' flanking sequences, exon 1 and 120 bp of intron 1 was identified as being a CpG island (nt 510-1059, Fig. 2 ). This DNA segment exhibits a high G+C content (69.5 %) and a high frequency of CpG dinucleotides (CpG observed/expected = 0.83), parameters which are well within the proposed definition for a CpG island (23 ). As a consequence of this primary structure, this region of the Peg1 gene is particularly rich in recognition sites for methylation-sensitive restriction enzymes, such as SacII, BssHII and HpaII (Fig. 3 A). It also contains four Sp1 sites (GC box, GGGCGG) and a TATA-like AT-rich element at position -41 relative to the transcription start site (Fig. 2 B). The 3' boundary of this CpG island, located within intron 1 (position 1059, Fig. 2 A), is defined by a region showing a lower G+C content. However, the frequency of CpG dinucleotides remains elevated well beyond this boundary, such that a larger GC-rich sequence (nt 510-1544, Fig. 2 A), extending further into intron 1, still exhibits a high GC content (58.8 %) and a high CpG frequency (obs/exp = 0.84). Only the smaller element, revealed by the analysis presented in Figure 2 , will be referred to as the Peg1 CpG island below.


Figure 2. Analysis of the Peg1 CpG island. (A) CpG plot of a 1925 bp PstI fragment from the 5'-end of Peg1 (nt 1765-3689 from our genomic sequence; GenBank accession no. AF017994). The plot shows the observed/expected CpG ratio (black line) and the (G+C) percentage (dashed grey line) in the sequence containing exon 1 (black box; nt 667-939). A 550 bp region, from nt 510 to 1059 [-157 to +393 relative to the transcription start site (arrow)], marked above the plot, has the characteristics of a CpG island (% C+G = 69.5%, CpG obs/exp = 0.83; see 22). (B) DNA sequence of the Peg1 CpG island, as defined in (A). Exon 1 is shown in upper case letters. The arrow marks the transcription start site and the beginning of the Peg1 open reading frame is shown in bold. This region contains four GC boxes (underlined) and a putative TATA box at -41 (box).


Figure 3. Partial methylation of the CpG island of Peg1 in 13.5 d.p.c. embryos and in embryonic stem cells. (A) Structure of the 5'-region of Peg1, showing the 5.4 kb XbaI-BglII (Bg) fragment sequenced. This fragment contains the transcription start site (arrow), exons 1 and 2 (black boxes), the CpG island (highlighted above) and the G-rich tandem repeats (arrowheads). Also shown are the positions of recognition sites for the methylation-sensitive restriction enzymes BssHII (B), SacII (S), SmaI (Sm) and XhoI (X), as well as genomic probes 2 and 3 (open boxes). The numerical subscripts 1 and 2 are used to distinguish two identical sites. Vertical lines below the diagram show the location of HpaII/MspI sites. (B) Autoradiogram of the Southern blot analysis of genomic DNA purified from 13.5 d.p.c. C57BL/6 embryos, digested with XbaI plus one of four methylation-sensitive enzymes and probed with the 3.0 kb XbaI fragment (probe 2, Fig. 1A) after electrophoresis and blotting. The approximate size of detected fragments, based on the migration of a molecular weight marker (not shown) and mapping of clone pPeg1C, are shown in kilobases. The lane marked - was only digested with XbaI. (C) Methylation analysis of intron 1. Embryonic DNA was analyzed as described in (B), following an EcoRI primary digestion and using intron 1 probe 3. Abbreviations are as described above and also include EcoRI (E), MspI (M) and HpaII (Hp). (D) Methylation analysis outside the CpG island. The blot used in (B) was reprobed with fragments from the 5'-promoter 3.6 kb XbaI fragment (probe 1) and exons 9 and 10 (probe 5; see Fig. 1A). (E) As in (B) but using genomic DNA isolated from undifferentiated ES cells.

Partial methylation of the CpG island in post-implantation embryos

The epigenetic modification of DNA by methylation of cytosine residues currently represents an important candidate if not for the gametic imprint itself, at least for maintenance of the differential transcriptional activity of the alleles of imprinted genes. In the light of the DNA sequence analysis presented above, we determined whether the CpG island of the Peg1 gene carries a parental-specific methylation pattern in expressing tissues. To address this question we first studied the methylation status of different CpG residues, using methylation-sensitive restriction enzymes, in DNA isolated from C57BL/6 embryos collected at 13.5 d.p.c. This material was chosen since it was previously shown that: (i) Peg1 is transcribed at high levels in post-implantation embryos following gastrulation (14 ,16 ); (ii) only the paternal allele is expressed during embryonic development (14 ; L.Lefebvre, unpublished data).

More than 2.4 kb of Peg1 promoter sequences, exon 1 and part of intron 1 are present on a 3.0 kb XbaI fragment of the Peg1 gene (Fig. 3 A). This fragment spans the CpG island identified above and also contains recognition sites for the methylation-sensitive restriction enzymes BssHII (two sites), XhoI, SmaI and SacII (two sites). A Southern blot analysis of this region, using probe 2 (Fig. 1 A), shows that several CpG residues in the 5'-end of Peg1 are partially methylated in 13.5 d.p.c. embryos (Fig. 3 B). The distal XhoI site is the only one which appears to be hypomethylated in this sample (Fig. 3 B, lane X), with a very low level of residual methylation. However, the most distal BssHII site (B1) is too close to the XbaI site to allow accurate determination of its methylation status (see below). On the other hand, the exonic BssHII site (B2) is clearly partially methylated, as shown by the fully protected 3.0 kb and the unmethylated 2.5 kb bands (Fig. 3 B, lane B). Digestion with SacII (lane S) gives a protected band of 3.0 kb, as well as a lower molecular weight band (2.17 kb) corresponding to the fully digested product (Fig. 3 B, lane S). The 3.0 kb XbaI fragment contains two recognition sites for SacII. Since we do not detect the fragments predicted from digestion at only one of these two sites (only at S1, 2.17 + 0.87 kb; only at S2, 2.55 kb), we conclude that the two SacII sites are partially methylated, but are always both methylated or both unmethylated on the same DNA molecule. Analysis of the single SmaI site (Sm1) also shows a clear partial methylation pattern, with both the full-length (methylated) and digested (unmethylated) patterns being detected (Fig. 3 B, lane Sm).

As mentioned above, the CpG-rich region of Peg1 extends downstream of the intronic XbaI site (position 1278 on the plot in Fig. 2 A). To analyse the methylation status of CpG residues present in this 5'-portion of intron 1 we studied five methylation-sensitive restriction sites, a single SmaI site (Sm2) and four HpaII/MspI sites, within a 1.5 kb EcoRI fragment using probe 3. These five sites are also partially methylated in our embryonic DNA sample. Following SmaI digestion both a protected 1.5 kb band and a digested 1.3 kb product are detected (Fig. 3 C, lane Sm). Since probe 3 hybridizes to the 1.3 kb region common to both digestion products, the fact that both SmaI fragments give a signal of similar intensity suggests that close to 50% of the DNA molecules are methylated at this site. Similarly, for the HpaII sites, one of which overlaps with the SmaI site, both a 1.5 kb methylated band and smaller digestion products are detected (Fig. 3 C, lane Hp). That the small fragments correspond to digestion at unmethylated sites is shown by the pattern seen following digestion with the HpaII isoschizomer MspI, which is methylation insensitive (Fig. 3 C, lane M). As for the two SacII sites, since we only detect a fully protected 1.5 kb fragment and products of complete (MspI-like) digestion, but no intermediate size bands, the four HpaII sites studied are either all methylated or all unmethylated on a given DNA molecule.

In order to establish if the partial methylation pattern observed around the CpG island extends to the entire Peg1 locus, we studied the methylation status of two other sites mapped in Figure 1 A: a XhoI site in the distal promoter region (probe 1) and a XhoI site within exon 8 (probe 5). The results show that whereas the promoter-distal XhoI site is partially methylated (with a low residual methylation comparable with that of the downstream XhoI site; Fig. 3 B), the 3' exonic XhoI site is fully unmethylated (Fig. 3 D, probes 1 and 5 respectively). This observation suggests that CpG residues located more than 2.0 kb upstream of the transcription start site of Peg1 do not show a strict parental-specific methylation pattern. Therefore, we focused our analysis on the more proximal sites.

Partial methylation precedes Peg1 activation

Peg1 transcripts are first detected in mesodermal derivatives of 7.0 d.p.c. post-gastrulation embryos (16 ). To determine whether the partial methylation patterns detected in 13.5 d.p.c. embryos are already present at a developmental time point preceding activation of Peg1, we studied the methylation status of the CpG island in DNA isolated from undifferentiated embryonic stem (ES) cells. These cells are derived from the inner cell mass (ICM) of blastocyst stage embryos (4.0 d.p.c.) and retain the developmental potential of ICM cells after culture in vitro. The analysis presented in Figure 3 E shows that undifferentiated ES cells present a Peg1 promoter methylation profile indistinguishable from that seen in 13.5 d.p.c. embryos, described above. Whereas the SmaI, BssHII (B2) and SacII (S1 and S2) sites are partially methylated, the distal XhoI site is hypomethylated. In this particular analysis a faint 2.6 kb band seen upon BssHII digestion suggests that a small proportion of the DNA molecules unmethylated at the downstream site (site B2, 2.5 kb band) are also methylated at the distal site B1. Although the analysis on DNA from 13.5 d.p.c. embryos presented above did not allow observation of this partial methylation at site B1, a similar result was obtained during analysis of polymorphic embryonic material (see below), suggesting that this pattern is not unique to ES cell DNA.

Parental-specific methylation of the Peg1 promoter region

To address whether the partial methylation patterns described above reflect a parental-specific marking of the two Peg1 alleles, we took advantage of a HindIII polymorphism introduced at the Peg1 locus by gene targetting in ES cells. The details of the construction and analysis of this mutant allele will be described elsewhere (L.Lefebvre and M.A.Surani, manuscript in preparation). The structural features of the wild-type (wt) and mutant (knock-out, KO) alleles relevant to this study are presented in Figure 4 A. Whereas in both alleles the promoter region and CpG island remain intact, in the mutant allele the body of the gene is disrupted by a reporter construct (IRES-[beta]geo) inserted within intron 2. As a consequence of this rearrangement a new HindIII site, absent in the wild-type allele, is inserted 1.4 kb downstream of exon 2, such that the CpG island of the wild-type and mutant alleles are present on HindIII fragments of 13.2 and 10.6 kb respectively. Therefore, this HindIII polymorphism allows us to distinguish between the two alleles by Southern blot analysis and to establish the methylation status of each allele independently in heterozygotes.


Figure 4. Parental-specific methylation of the Peg1 promoter in 13.5 d.p.c. embryos. (A) Schematic representations of the wild-type (wt) and mutant (KO) Peg1 alleles showing the location of the polymorphic downstream HindIII sites relative to the positions of Peg1 exons (black boxes) and methylation-sensitive sites (as described in Fig. 3). The targeting construct introduces an IRES-[beta]geo reporter cassette carrying internal SmaI (Sm3) and HindIII (H) sites. Genomic probes 2 and 3 used in this analysis (open boxes) are positioned below the diagram. (B and C) Southern blot analyses of 13.5 d.p.c. embryonic DNA digested with HindIII and secondary methylation-sensitive enzymes as described in Figure 3, using probe 2 (B) or 3 (C). The genotypes of the embryos analysed are as follows: +/+, homozygous wild-type at Peg1; -/+, heterozygous with maternally inherited mutant allele; +/-, reciprocal heterozygous with paternally inherited mutant allele. Molecular weights are given in kilobases.

Genomic DNA was isolated from 13.5 d.p.c. embryos which were homozygous wild-type at the Peg1 locus (+/+) or heterozygous with the mutant allele inherited maternally (-/+) or paternally (+/-). Using probes 2 and 3 the methylation status of the promoter region in these DNA samples was analysed by Southern analysis, as described above. In the heterozygous embryos the silenced maternal allele of Peg1 is fully methylated in the CpG island and the expressed paternal allele is unmethylated (Fig. 4 B and C). This conclusion is most readily appreciated by focusing on the full-length HindIII fragments generated for each allele in heterozygotes. Whereas a single 13.2 kb band is detected in +/+ embryos, an additional fragment of 10.6 kb corresponding to the mutant allele is seen in both -/+ and +/- heterozygotes (Fig. 4 B and C, lanes marked -). Upon secondary digestion with any of the methylation-sensitive enzymes studied (SacII, BssHII and SmaI) only one of these two fragments is unprotected and digested in the heterozygotes: the paternal wild-type 13.2 kb band in -/+ embryos or the paternal mutant 10.6 kb band in +/- embryos. In itself, this result does not preclude the possibility that the silenced maternal allele is partially unmethylated and weakly digested in our samples. However, analysis of the smaller digestion products generated clearly shows that digestion of the maternal allele is never observed (see below).

Analysis with probe 2 (Fig. 4 B) confirms the strict parental-specific methylation at several sites studied, except perhaps the upstream BssHII site (B1). In the three samples (+/+, -/+, and +/-) digestion with SacII gives a 6.2 kb band corresponding to the promoter H-S1 fragment. This confirms hypomethylation of the paternal S1 site (lanes S). The methylation status of site S2 is unclear, since probe 2 hybridizes only weakly to the S2-H fragment (for example, a faint 4.0 kb band in the +/- sample, lane S). Digestion with BssHII gives a 2.5 kb band in all samples, which corresponds to the promoter B1-B2 fragment (lanes B). Here again, probe 2 hybridizes only weakly to the downstream fragment B2-H (6.6 kb for the wt allele in +/+ and -/+ samples; 3.9 kb for the KO allele in +/- DNA). However, the presence of a faint 6.6 kb band in all samples (H-B2 promoter fragment, clearer in the +/+ sample) is consistent with the presence of a weak level of methylation at the promoter-distal site B1 on an otherwise hypomethylated paternal allele. The alternative interpretation suggesting that the maternal allele might be weakly unmethylated at site B2 is ruled out below. Analysis with SmaI gives the expected 4.9 and 2.4 kb bands (H-Sm1 and Sm1-Sm2 fragments respectively) showing full digestion of the paternal allele and hypomethylation of the two paternally inherited SmaI sites (lanes Sm).

The possibility that the maternally inherited CpG island might be weakly unmethylated was addressed using probe 3, which hybridizes within intron 1 downstream of site Sm2 (Fig. 4 A). Both the wild-type (+/+) and maternal mutant (-/+) embryos show identical patterns, with unmethylated hybridizing bands at 6.6 kb for SacII and BssHII (wt S2-H and B2-H fragments respectively) and at 5.9 kb for SmaI (wt Sm2-H fragment). The smaller fragments expected from digestion of the KO allele (mutant S2-H, B2-H and Sm2-H fragments) are only seen when the mutant allele is paternally inherited (+/- embryos). These correspond to the 4.0 kb band seen for SacII and BssHII and the 3.3 kb band for SmaI (Fig. 4 C, +/- embryo). These results confirm that the maternally inherited Peg1 allele is fully methylated and protected from digestion at the CpG island sites S2, B2 and Sm2 and, from the results presented in the previous section, at sites S1 and Sm1.

As shown in Figure 4 A, the mutant allele also introduces a new SmaI site (Sm3), 440 bp upstream of the polymorphic HindIII site (L.Lefebvre, unpublished data). Digestion at this site is expected to produce a 2.9 kb Sm2-Sm3 fragment hybridizing with probe 3 on the unmethylated paternal allele. Since both the 3.3 kb Sm2-H fragment and the 2.9 kb Sm2-Sm3 fragment are detected upon paternal transmission of the mutant allele (Fig. 4 C, +/- embryo, lane Sm), we conclude that this newly introduced site does not exhibit the strict parental-specific methylation observed for the endogenous sites studied here within the CpG island.

Gametic signals: sperm and parthenogenetic embryos

Since the results presented so far are consistent with a role for DNA methylation not only in the maintenance of allele-specific silencing, but also in the marking or pre-programming of the two Peg1 alleles, we asked whether the mature gametes carry or can independently direct the expected parental methylation of Peg1. Genomic DNA was isolated from sperm collected from the epididymus of adult C57BL/6J males and subjected to the same methylation analysis as described in Figure 3 using promoter probe 2. The four sites studied (Sm1, S1, S2 and B2) are completely unmethylated in sperm DNA, a result consistent with the inheritance of a potentially active paternal allele (Fig. 5 A). In order to assess the potential of the maternally inherited Peg1 allele present in unfertilized oocytes to independently generate the methylation pattern observed in ES cells and embryos we studied the methylation status of the Peg1 promoter region in mid-gestation parthenogenetic embryos. These embryos were obtained from unfertilized (C57BL/6J * CBA)F1 eggs activated in vitro by treatment with ethanol and transferred to pseudopregnant females. Consequently, these embryos develop in the absence of a paternal contribution and reflect the developmental potential of the maternal genome. Our analysis shows that the same promoter sites are all fully methylated in 9.5 d.p.c. parthenogenetic embryos, a pattern reciprocal to that observed in sperm DNA (Fig. 5 B). However, the methylation status of the Peg1 gene in oocytes is still unknown and our results do not exclude the possibility that the observed methylation of the maternal alleles has been acquired during development of the parthenogenetic embryos. Together, these results show that whereas the paternally inherited Peg1 allele is unmethylated and pre-programmed for expression, the maternally inherited genome carries epigenetic information necessary to lay down the extensive methylation observed on the silenced allele in the absence of a paternal contribution.


Figure 5. Methylation of the Peg1 promoter region in sperm and parthenogenetic embryos. The methylation analysis presented in Figure 3B using probe 2 was repeated on genomic DNA isolated from sperm (A) or 9.5 d.p.c. parthenogenetic embryos (B). The primary digestion was with XbaI. Abbreviations are as described in Figure 3. Molecular weights are given in kilobases.

DISCUSSION

Peg1 mRNA, which may code for an enzyme of the [alpha]/[beta]-hydrolase fold family (14 ), is expressed predominantly in mesodermal derivatives during embryogenesis and only from the paternally inherited allele (14 ,16 ). It is the first imprinted gene to be identified on mouse proximal chromosome 6, a region associated with embryonic lethality in maternal uniparental disomy (15 ). We have initiated characterization of the Peg1 gene and identified structural and epigenetic features shared between Peg1 and other known imprinted genes.

Gene structure

A statistical analysis of 16 imprinted genes from mouse and man revealed that, as a group, they have fewer exons (6.5 versus 12.01 on average) and a much smaller average intron size (729 versus 2396 bp) when compared with a control set of genes (6 ), even when intronless, candidate retroposons such as U2afbp-rs, Mas and Znf-127 are not included in the analysis (7 ,24 ). Peg1, which is not evolutionarily related to any of the genes included in the previous analysis (6 ,24 ), consists of 12 exons, with an average exon size of 212 bp for the 2543 nt cDNA. These parameters are very similar to those previously obtained for the control set of genes (6 ) and support the view that imprinted genes may not contain fewer introns, contrary to the initial proposition (7 ,24 ). On the other hand, the total intronic DNA of Peg1 is very small (~7.9 kb), with an average intron size of ~720 bp, which agrees well with the average obtained for other imprinted genes (729 +- 220 bp), supporting the conclusion that imprinted genes may have been selected for a smaller intronic content (6 ).

Tandem repeats

A 122 bp region located downstream of the CpG island of Peg1, within intron 1 (Fig. 3 A), consists of several repeats of the G-rich consensus pentamer 5'-(A/T)GGGG-3'. Several imprinted mouse and human genes, including Igf2r, IGF2R, Igf2, U2afbp-rs and SNRPN were found to contain direct repeats within differentially methylated CpG-rich regions (25 ). Elements rich in tandem repeats were also identified at the imprinted loci Kip2 (26 ) and H19 (27 ). The consensus repeat unit present in the upstream region of H19 [5'-(G)GGGGTATA-3'] shows a G-rich motif reminiscent of that identified here at Peg1 and can also be described as interspersed repetitions of the consensus pentamers 5'-(A/T)GGGG-3' and 5'-ATAGC-3'. Similarly, the Xist cDNA, which is paternally expressed in extra-embryonic tissues, contains a 182 bp sequence consisting of multiple copies of a 6 bp repeat unit, which on the antisense strand is 5'-(A/T)GGGGC-3' (28 ).

The RSVIgmyc transgene imprints in a position-independent fashion (29 ). The region required for its methylation imprinting lies in the IgA C[alpha] and S[alpha] fragment of the construct. In their endogenous location these sequences do not confer methylation or expression imprinting (29 ), but mediate DNA rearrangements associated with immunoglobulin class switching. The switch sequences are composed of simple repetitions of pentameric unit sequences, such as 5'-TGAGC-3' and 5'-TGGGG-3', which is similar to the Peg1 repeat unit (30 ). Although a small fragment of RSVIgmyc containing these repeats (206 bp) was shown to be required for methylation imprinting of the transgene, a larger construct fails to imprint a lacZ reporter and extensive repetitive arrays (several kb) fail to imprint the endogenous locus (29 ,30 ). These results emphasize the importance of the context these elements find themselves in and possibly the requirement for coordinate action of neighbouring sequence elements. Interestingly, all the deletion variants of the RSVIgmyc transgene which do show parental-specific methylation contain not only part of the repetitive C[alpha] or S[alpha] sequences, but also CG-rich fragments of pBR322 or c-myc (29 ). It should be noted that in the mutant Peg1 allele used in our study, which was shown to exhibit a clear parental-specific methylation pattern, the repeat sequences present 5' of exon 2 have not been deleted (Fig. 4 A).

Parental-specific methylation

Peg1 contains a typical CpG island of 550 bp (Fig. 2 ). However, unlike the CpG islands of normal, biallelically expressed genes (23 ,31 ), the CpG island of Peg1 is unmethylated exclusively on the expressed paternal allele, but fully methylated on the silenced maternal allele. We found that the region of Peg1 showing parental-specific methylation extends >1 kb upstream of the CpG island, to include the SmaI site Sm1 (Fig. 3 A), and at least 880 bp downstream of it, to include the HpaII site located 1 kb into intron 1, therefore covering ~2.4 kb of genomic DNA. This differential methylation is restricted to the 5'-end of the gene, since we showed that the XhoI site within exon 8 is unmethylated on both alleles. This finding is somewhat unexpected, since CpG dinucleotides found outside CpG islands are usually methylated in the mouse genome. As proposed previously to explain the survival of CpGs outside islands (31 ), this particular site may have been maintained because of its role in the Peg1 coding region. Indeed, deamination of a 5-methylcytosine present within this XhoI site could lead to a C -> T transition, mutating the Arg215 codon CGA to the stop codon TGA.

Since CpG islands are usually fully unmethylated for normal autosomal genes, even in non-expressing tissues, it seems that monoallelically expressed genes, including autosomal imprinted genes and X-linked genes in females, belong to a special class of genes which utilize extensive CpG island methylation as an allele-specific silencing mechanism. The finding of a CpG island showing parental-specific methylation at a new imprinted locus suggests that this common feature of imprinted genes is functionally important for maintenance of their characteristic monoallelic expression. Several independent lines of evidence support this conclusion. First, analyses of the maternally expressed H19 gene have shown that the CpG island and several other 3' and 5' CpGs are specifically methylated on the silenced paternal allele (8 ,27 ,32 ). In mutant embryos deficient for DNA methyltransferase both alleles of H19 are unmethylated and expressed (33 ). Second, small deletions removing a differentially methylated CpG island at the human SNRPN locus alter both the methylation and parental-specific expression of several transcriptional units within a 200 kb region (12 ).

Together with our study, these results support the notion that differential methylation of CpG islands is utilized at imprinted loci to maintain their parent-of-origin-specific expression. The question of whether or not these same CpG residues actually play a direct role in gametic marking of the two alleles of imprinted genes has received considerable attention. Definitive experimental evidence supporting a role for CpG methylation as the primary imprint is still lacking, but analyses of imprinted genes such as H19 and Igf2r have identified specific CpG residues which are differentially methylated in the gametes and throughout embryogenesis (10 ,27 ). The candidate sites for the primary imprint lie outside the differentially methylated CpG island. Identification of differentially methylated sites 5' and 3' of the Peg1 CpG island raise the possibility that this feature may also be conserved at this imprinted locus. The role of these sites, as well as the intronic repeats, can now be directly addressed by transgenic and/or gene replacement approaches.

MATERIALS AND METHODS

Isolation and characterization of the Peg1 locus

A 686 bp Peg1 genomic fragment (probe 4, Fig. 1 A) was generated by PCR amplification of DBA mouse genomic DNA using two oligonucleotide primers designed from the Peg1 cDNA sequence (cDNA nt 427-446, 5'-AGATTCTGTCGGTGTGGTCG-3'; nt 693-673, 5'-CCTGCTGTCTCACGATTTGG-3'). This fragment was used as a probe to screen a genomic library in the vector [lambda]KO, containing size-selected Sau3A fragments (16-20 kb) of genomic DNA isolated from 129/Sv ES cells (22 ). After two rounds of screening, four positive clones were identified, excised as plasmid clones (pPeg1A-pPeg1D) in vivo by infection of the cre recombinase-expressing Escherichia coli strain BNN132 (34 ) and analysed by restriction enzyme digestions. Clones B and D both contained inserts present within clone A and were not analysed further. The clones pPeg1A (8.70 kb insert) and pPeg1C (11.15 kb insert) clearly contained sequences homologous to probe 4 and showed evidence of overlap, based on their respective EcoRI restriction maps (Fig. 1 A).

Sequencing reactions were performed on double-stranded plasmid DNA templates, first with primers designed from the Peg1 cDNA sequence, then with oligonucleotides complementary to intronic sequences on clones pPeg1A and pPeg1C or on specific subclones derived from them. For the promoter region several overlapping templates were obtained by subcloning the 3.0 kb XbaI fragment of pPeg1C containing exon 1 and generating deletion variants by restriction enzyme digestions.

Embryos and ES cells

Post-implantation embryos (13.5 d.p.c.) were obtained from natural matings between C57BL/6J mice. Undifferentiated R1 ES cells, isolated from 129/Sv blastocysts (35 ), were cultured in standard ES cell medium supplemented with LIF in the absence of feeder cells. The derivation and analysis of mutant ES cells and embryos carrying a targeted mutation at the Peg1 locus will be described elsewhere. In the mutant allele of Peg1 the genomic sequence from the EcoRI site in intron 2 to the middle of exon 9 (see Fig. 1 A) has been replaced with an IRES-[beta]geo cassette (36 ), while the flanking sequences remain unchanged (see Fig. 4 A). The targeted allele was originally derived on the 129/Sv inbred background, whereas the mutant embryos recovered for the methylation analysis were obtained as F2 progeny from an intercross between F1 heterozygotes on the C57BL/6J * 129/Sv mixed background. Parthenogenetic embryos were obtained by ethanol activation of unfertilized (C57BL/6J * CBA/Ca)F1 oocytes as described (37 ) and recovered on day 9.5 of gestation after uterine transfer to pseudopregnant females (14 ).

Methylation analysis

Genomic DNA from ES cells and embryos was purified as follows. Cells or embryos were treated for 16 h with proteinase K solution (500 [mu]g/ml) in DNA lysis buffer (50 mM Tris-HCl, pH 8.0, 100 mM EDTA, 0.5% SDS) at 65oC. The crude lysates were cleared of cellular debris by centrifugation for 5 min at 12 000 r.p.m. and extracted with phenol and phenol:chloroform (1:1) solutions. The genomic DNA was recovered by precipitation with 95% ethanol in 0.3 M sodium acetate, pH 6.0, washed with 70% ethanol and resuspended in TE, pH 8.0. Mature sperm was collected from the epididymus of adult C57BL/6 males and sperm DNA was isolated essentially as described above, except that 5 mM [beta]-mercaptoethanol was added to the lysis buffer. Digestions of 5-10 [mu]g genomic DNA were carried out in two consecutive reactions of 12-16 h each, first with the primary restriction enzyme (XbaI, EcoRI or HindIII) and second with the methylation-sensitive enzymes. Southern blotting was performed by alkaline transfer onto Hybond N+ (Amersham). DNA fragments used as probes are shown in Figure 1 A, below the map of the Peg1 locus. Probe 1 is a 1.3 kb XbaI-XhoI fragment isolated from the genomic clone pPeg1C. Probe 2 is the 3.0 kb XbaI fragment isolated from pPeg1C and was used for all the analyses of the promoter region. Probe 3 was generated by PCR amplification of a 1.3 kb region of intron 1, using two sequencing primers (5'-GTGTTGGCCACGGCTATAAG-3' and 5'-CAAGTCTTGGGAGCAGATTA-3') directly on pPeg1C DNA. Probe 5 is a 0.98 kb XhoI-XbaI fragment isolated from the genomic clone pPeg1A and containing exons 9 and 10. These DNA probes were labelled randomly by incorporation of [32P]dCTP using High Prime (Boehringer Mannheim). Hybridizations were performed at 65oC in Church's buffer (38 ) and washes at 65oC in 0.5% SDS, 2* SSC, then 0.2* SSC.

ACKNOWLEDGEMENTS

The authors would like to thank T.Boehm for the [lambda]KO library. This work was supported by a grant from the Wellcome Trust to M.A.S. L.L. was a Research Fellow of the National Cancer Institute of Canada, supported with funds provided by the Terry Fox Run, and S.V. was supported by a Fellowship from EMBO.

REFERENCES

1 Ainscough,J.F.-X. and Surani,M.A. (1996) Organization and control of imprinted genes: the common features. In Russo,V.E.A., Martienssen,R.A. and Riggs,A.D. (eds), Epigenetic Mechanisms of Gene Regulation. CSHL Press, New York, NY, pp. 173-194.

2 Surani,M.A. (1991) Genomic imprinting: developmental significance and molecular mechanism. Curr. Opin. Genet. Dev., 1, 241-246. MEDLINE Abstract

3 Feinberg,A. (1993) Genomic imprinting and gene activation in cancer. Nature Genet., 4, 110-113. MEDLINE Abstract

4 Shemer,R. and Razin,A. (1996) Establishment of imprinted methylation patterns during development. In Russo,V.E.A., Martienssen,R.A. and Riggs,A.D. (eds), Epigenetic Mechanisms of Gene Regulation. CSHL Press, New York, NY, pp. 215-229.

5 John,R.M. and Surani,M.A. (1996) Imprinted genes and regulation of gene expression by epigenetic inheritance. Curr. Opin. Cell Biol., 8, 348-353. MEDLINE Abstract

6 Hurst,L.D., McVean,G. and Moore,T. (1996) Imprinted genes have few and small introns. Nature Genet., 12, 234-237. MEDLINE Abstract

7 McVean,G.T., Hurst,L.D. and Moore,T. (1996) Genomic evolution in mice and men: imprinted genes have little intronic content. BioEssays, 18, 773-775. MEDLINE Abstract

8 Ferguson-Smith,A.C., Sasaki,H., Cattanach,B.M. and Surani,M.A. (1993) Parental-origin-specific epigenetic modification of the mouse H19 gene. Nature, 362, 751-755. MEDLINE Abstract

9 Bartolomei,M.S., Webber,A.L., Bruknow,M.E. and Tilghman,S.M. (1993) Epigenetic mechanisms underlying the imprinting of the mouse H19 gene. Genes Dev., 7, 1663-1673. MEDLINE Abstract

10 Stoger,R., Kubicka,P., Liu,C.-G., Kafri,T., Razin,A., Cedar,H. and Barlow,D.P. (1993) Maternal-specific methylation of the imprinted mouse Igf2r locus identifies the expressed locus as carrying the imprinting signal. Cell, 73, 61-71. MEDLINE Abstract

11 Shibata,H., Yoshino,K., Sunahara,S., Gondo,Y., Katsuki,M., Ueda,T., Kamiya,M., Muramatsu,M., Murakami,Y., Kalcheva,I., Plass,C., Chapman,V.M. and Hayashizaki,Y. (1996) Inactive allele-specific methylation and chromatin structure of the imprinted gene U2af1-rs1 on mouse chromosome 11. Genomics, 35, 248-252. MEDLINE Abstract

12 Sutcliffe,J.S., Nakao,M., Christian,S., Orstavik,K.H., Tommerup,N., Ledbetter,D.H. and Beaudet,A.L. (1994) Deletions of differentially methylated CpG island at the SNRPN gene define a putative imprinting control region Nature Genet., 8, 52-58. MEDLINE Abstract

13 Elson,D.A. and Bartolomei,M.S. (1997) A 5' differentially methylated sequence and the 3'-flanking region are necessary for H19 transgene imprinting. Mol. Cell. Biol., 17, 309-317. MEDLINE Abstract

14 Kaneko-Ishino,T., Kuroiwa,Y., Miyoshi,N., Kohda,T., Suzuki,R., Yokoyama,M., Viville,S., Barton,S.C., Ishino,F. and Surani,M.A. (1995) Peg1/Mest imprinted gene on chromosome 6 identified by cDNA subtraction hybridization. Nature Genet., 11, 52-59. MEDLINE Abstract

15 Beechey,C.V. and Searle,A.G. (1988) Further results on lethal complementation affecting Chr 6. Mouse Newsl., 81, 65.

16 Sado,T., Nakajima,N., Tada,M. and Takagi,N. (1993) A novel mesoderm-specific cDNA isolated from a mouse embryonal carcinoma cell line. Dev. Growth Differentiat., 35, 551-560.

17 Nishita,Y., Yoshida,I., Sado,T. and Takagi,N. (1996) Genomic imprinting and chromosomal localization of the human MEST gene. Genomics, 36, 000-000.

18 Spence,J.E., Perciaccante,R.G., Greig,G.M., Willard,H.F., Ledbetter,D.H., Hejtmancik,J.F., Pollack,M.S., O'Brien,W.E. and Beaudet,A.L. (1988) Uniparental disomy as a mechanism for human genetic disease. Am. J. Hum. Genet., 42, 217-226. MEDLINE Abstract

19 Spotila,L.D., Sereda,L. and Prockop,D.J. (1992) Partial isodisomy for maternal chromosome 7 and short stature in an individual with a mutation at the COL1A2 locus. Am. J. Hum. Genet., 51, 1396-1405. MEDLINE Abstract

20 Eggerding,F.A., Schonberg,S.A., Chehab,F.F., Norton,M.E., Cox,V.A. and Epstein,C.J. (1994) Uniparental isodisomy for paternal 7p and maternal 7q in a child with growth retardation. Am. J. Hum. Genet., 55, 253-265. MEDLINE Abstract

21 Preece,M.A., Price,S.M., Davies,V., Clough,L., Stanier,P., Trembath,R.C. and Moore,G.E. (1997) Maternal uniparental disomy 7 in Silver-Russell syndrome. J. Med. Genet., 34, 6-9. MEDLINE Abstract

22 Nehls,M., Messerle,M., Sirulnik,A., Smith,A.J.H. and Boehm,T. (1994) Two large insert vectors, [lambda]PS and [lambda]KO, facilitate rapid mapping and targeted disruption of mammalian genes. BioTechniques, 17, 770-775. MEDLINE Abstract

23 Bird,A.P. (1986) CpG-rich islands and the function of DNA methylation. Nature, 321, 209-213. MEDLINE Abstract

24 Haig,D. (1996) Do imprinted genes have few and small introns? BioEssays, 18, 351-353. MEDLINE Abstract

25 Neumann,B., Kubicka,P. and Barlow,D.P. (1995) Characteristics of imprinted genes. Nature Genet., 9, 12-13. MEDLINE Abstract

26 Hatada,I. and Mukai,T. (1995) Genomic imprinting of p57KIP2, a cyclin-dependent kinase inhibitor, in mouse. Nature Genet., 11, 204-206. MEDLINE Abstract

27 Tremblay,K.D., Saam,J.R., Ingram,R.S., Tilghman,S.M. and Bartolomei,M.S. (1995) A paternal-specific methylation imprint marks the alleles of the mouse H19 gene. Nature Genet., 9, 407-413. MEDLINE Abstract

28 Brockdorff,N., Ashworth,A., Kay,G.F., McCabe,V.M., Norris,D.P., Cooper,P.J., Swift,S. and Rastan,S. (1992) The product of the mouse Xist gene is a 15 kb inactive X-specific transcript containing no conserved ORF and located in the nucleus. Cell, 71, 515-526. MEDLINE Abstract

29 Chaillet,J.R., Bader,D.S. and Leder,P. (1995) Regulation of genomic imprinting by gametic and embryonic processes. Genes Dev., 9, 1177-1187. MEDLINE Abstract

30 Nikaido,T., Nakai,S. and Honjo,T. (1981) Switch region of immunoglobulin C[mu] gene is composed of simple tandem repetitive sequences. Nature, 292, 845-848. MEDLINE Abstract

31 Antequera,F. and Bird,A.(1993) CpG islands. In Jost,J. and Saluz,H. (eds), DNA Methylation: Molecular Biology and Biological Significance. Birkhauser Verlag, Basel, Switzerland, pp. 169-185.

32 Brandeis,M., Kafri,T., Ariel,M., Chaillet,J.R., McCarrey,J., Razin,A. and Cedar,H. (1993) The ontogeny of allele-specific methylation associated with imprinted genes in the mouse. EMBO J., 12, 3669-3677. MEDLINE Abstract

33 Li,E., Beard,C. and Jaenisch,R. (1993) Role for DNA methylation in genomic imprinting. Nature, 366, 362-365. MEDLINE Abstract

34 Elledge,S.J., Mulligan,J.T., Ramer,S.W., Spottswood,M. and Davis,R.W. (1991) [lambda]YES: a multifunctional cDNA expression vector for the isolation of genes by complementation of yeast and Escherichia coli mutations. Proc. Natl. Acad. Sci. USA, 8, 1731-1735.

35 Nagy,A., Rossant,J., Nagy,R., Abramow-Newerly,W. and Roder,J.C. (1993) Derivation of completely cell culture-derived mice from early passage embryonic stem cells. Proc. Natl. Acad. Sci. USA, 90, 8424-8428. MEDLINE Abstract

36 Mountford,P., Zevnik,B., Düwel,A., Nichols,J., Li,M., Dani,C., Robertson,M., Chambers,I. and Smith,A. (1994) Dicistronic targeting constructs: reporters and modifiers of mammalian gene expression. Proc. Natl. Acad. Sci. USA, 91, 4303-4307. MEDLINE Abstract

37 Barton,S.C., Norris,M.L. and Surani,M.A.(1987) Nuclear transplantation in fertilised and parthenogenetically activated eggs. In Monk,M. (ed.), Mammalian Development: A Practical Approach. IRL Press, Oxford, UK, pp. 235-253.

38 Church,G.M. and Gilbert,W. (1984) Genomic sequencing. Proc. Natl. Acad. Sci. USA, 81, 1991-1995. MEDLINE Abstract

*To whom correspondence should be addressed. Tel: +44 1223 334138; Fax: +44 1223 334182; Email: l.lefebvre@welc.cam.ac.uk
+Present address: Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 rue Laurent Fries BP 163, 67404 Illkirch Cedex, France
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 EndocrinologyHome page
G. P. Finkielstain, P. Forcinito, J. C. K. Lui, K. M. Barnes, R. Marino, S. Makaroun, V. Nguyen, J. E. Lazarus, O. Nilsson, and J. Baron
An Extensive Genetic Program Occurring during Postnatal Growth in Multiple Tissues
Endocrinology, April 1, 2009; 150(4): 1791 - 1800.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
R. Riclet, M. Chendeb, J.-L. Vonesch, D. Koczan, H.-J. Thiesen, R. Losson, and F. Cammas
Disruption of the Interaction between Transcriptional Intermediary Factor 1{beta} and Heterochromatin Protein 1 Leads to a Switch from DNA Hyper- to Hypomethylation and H3K9 to H3K27 Trimethylation on the MEST Promoter Correlating with Gene Reactivation
Mol. Biol. Cell, January 1, 2009; 20(1): 296 - 305.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
L. Nikonova, R. A. Koza, T. Mendoza, P.-M. Chao, J. P. Curley, and L. P. Kozak
Mesoderm-specific transcript is associated with fat mass expansion in response to a positive energy balance
FASEB J, November 1, 2008; 22(11): 3925 - 3937.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
R. Oh, R. Ho, L. Mar, M. Gertsenstein, J. Paderova, J. Hsien, J. A. Squire, M. J. Higgins, A. Nagy, and L. Lefebvre
Epigenetic and Phenotypic Consequences of a Truncation Disrupting the Imprinted Domain on Distal Mouse Chromosome 7
Mol. Cell. Biol., February 1, 2008; 28(3): 1092 - 1103.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
H. Kobayashi, A. Sato, E. Otsu, H. Hiura, C. Tomatsu, T. Utsunomiya, H. Sasaki, N. Yaegashi, and T. Arima
Aberrant DNA methylation of imprinted loci in sperm from oligospermic patients
Hum. Mol. Genet., November 1, 2007; 16(21): 2542 - 2551.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
A. Sato, E. Otsu, H. Negishi, T. Utsunomiya, and T. Arima
Aberrant DNA methylation of imprinted loci in superovulated oocytes
Hum. Reprod., January 1, 2007; 22(1): 26 - 35.
[Abstract] [Full Text] [PDF]

Home page
 GENES CELLSHome page
H. Hiura, Y. Obata, J. Komiyama, M. Shirai, and T. Kono
Oocyte growth-dependent progression of maternal imprinting in mice
Genes Cells, April 1, 2006; 11(4): 353 - 361.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. Imamura, A. Kerjean, T. Heams, J.-J. Kupiec, C. Thenevin, and A. Paldi
Dynamic CpG and Non-CpG Methylation of the Peg1/Mest Gene in the Mouse Oocyte and Preimplantation Embryo
J. Biol. Chem., May 20, 2005; 280(20): 20171 - 20175.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
M. Takahashi, Y. Kamei, and O. Ezaki
Mest/Peg1 imprinted gene enlarges adipocytes and is a marker of adipocyte size
Am J Physiol Endocrinol Metab, January 1, 2005; 288(1): E117 - E124.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
D. Lucifero, M. R.W. Mann, M. S. Bartolomei, and J. M. Trasler
Gene-specific timing and epigenetic memory in oocyte imprinting
Hum. Mol. Genet., April 15, 2004; 13(8): 839 - 849.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
T. Chen, Y. Ueda, J. E. Dodge, Z. Wang, and E. Li
Establishment and Maintenance of Genomic Methylation Patterns in Mouse Embryonic Stem Cells by Dnmt3a and Dnmt3b
Mol. Cell. Biol., August 15, 2003; 23(16): 5594 - 5605.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
K. Hata, M. Okano, H. Lei, and E. Li
Dnmt3L cooperates with the Dnmt3 family of de novo DNA methyltransferases to establish maternal imprints in mice
Development, March 6, 2003; 129(8): 1983 - 1993.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. Li, T. H. Vu, K.-O. Lee, Y. Yang, C. V. Nguyen, H. Q. Bui, Z.-L. Zeng, B. T. Nguyen, J.-F. Hu, S. K. Murphy, et al.
An Imprinted PEG1/MEST Antisense Expressed Predominantly in Human Testis and in Mature Spermatozoa
J. Biol. Chem., April 12, 2002; 277(16): 13518 - 13527.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
A. Kerjean, J.-M. Dupont, C. Vasseur, D. Le Tessier, L. Cuisset, A. Paldi, P. Jouannet, and M. Jeanpierre
Establishment of the paternal methylation imprint of the human H19 and MEST/PEG1 genes during spermatogenesis
Hum. Mol. Genet., September 1, 2000; 9(14): 2183 - 2187.
[Abstract] [Full Text] [PDF]

Home page
 J. Med. Genet.Home page
K. KOSAKI, R. KOSAKI, W. P ROBINSON, W. J CRAIGEN, L. G SHAFFER, S. SATO, and N. MATSUO
Diagnosis of maternal uniparental disomy of chromosome 7 with a methylation specific PCR assay
J. Med. Genet., September 1, 2000; 37(9): 19e - 19.
[Full Text]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (35)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Lefebvre, L.
Right arrow Articles by Surani, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lefebvre, L.
Right arrow Articles by Surani, M. A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?