Human Molecular Genetics, 2002, Vol. 11, No. 1 77-86
© 2002 Oxford University Press
Epigenetic analysis of the Dlk1–Gtl2 imprinted domain on mouse chromosome 12: implications for imprinting control from comparison with Igf2–H19
Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK, 1Laboratory of Developmental Genetics and Imprinting, The Babraham Institute, Cambridge CB2 4AT, UK and 2MRC Mammalian Genetics Unit, Didcot, Oxon, UK
Received September 13, 2001; Revised and Accepted November 2, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Dlk1 and Gtl2 are reciprocally imprinted genes located 80 kb apart on mouse chromosome 12. Similarities between this domain and that of the well characterized Igf2–H19 locus have been previously noted. Comparative genomic and epigenetic analysis of these two domains might help identify allele-specific epigenetic regulatory elements and common features involved in aspects of imprinting control. Here we describe a detailed methylation analysis of the Dlk1–Gtl2 domain on both parental alleles in the mouse. Like the Igf2–H19 domain, areas of differential methylation are hypermethylated on the paternal allele and hypomethylated on the maternal allele. Three differentially methylated regions (DMRs), each with different epigenetic characteristics, have been identified. One DMR is intergenic, contains tandem repeats and is the only region that inherits a paternal methylation mark from the germline. An intronic DMR contains a conserved putative CTCF-binding domain. All three DMRs have both unique and common features compared to those identified in the Igf2–H19 domain.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Genomic imprinting is a process that causes genes to be expressed according to their parental origin. DNA methylation is an epigenetic modification that plays an important role in the monoallelic behaviour of the majority of imprinted genes. Many imprinted genes are clustered suggesting that they reside in domains that are epigenetically modified to result in co-ordinate regulation of more than one imprinted gene (1). This cis-acting regulation can act at short range and over long distances. The best-studied example of short-range co-ordinate regulation of imprinted genes is the Igf2–H19 locus on mouse chromosome 7. This is characterized by a series of cis-acting elements that regulate expression of the maternal H19 allele and the paternal Igf2 allele and mediate interactions between common sets of enhancers located downstream of H19 and promoters and silencers specific to the genes themselves (2,3). An important feature of this regulation is an intergenic differentially methylated insulator element 2–4 kb upstream of H19, carrying a germline methylation mark and several binding sites for the zinc finger protein, CTCF. Binding of CTCF to this region on the unmethylated maternal allele is proposed to insulate the downstream enhancers from the Igf2 promoter on the maternal allele hence facilitating H19 expression on that chromosome. The methylated paternal insulator is unable to bind CTCF and the enhancers are proposed to act at the unmethylated Igf2 promoter rather than the methylated H19 paternal promoter (4–7). Recently, another pair of imprinted genes has been identified that exhibit many of the features of the Igf2–H19 locus. This locus, on mouse chromosome 12, contains the Dlk1 and Gtl2 genes (8–10). The region is conserved on human chromosome 14 (11,12).
Dlk1 has homology to the Notch-Delta family of developmentally regulated signalling molecules that contain EGF repeats (13). In the mouse this gene is expressed from the paternal allele, has a completely unmethylated CpG island promoter and an exonic differentially methylated region (DMR) which is hypomethylated on the maternal allele and partially methylated on the paternal allele (8). The gene is associated with a similarly imprinted downstream transcript that may be a cleavage product of a longer Dlk1 transcript. However, unlike Dlk1, the associated transcript lacks a conserved open reading frame (ORF) (14).
Gtl2 is also developmentally regulated and shares many of the features of H19 (8,9,15). The gene is expressed from the maternal allele, it lacks a conserved ORF and, like H19, has a CpG-rich promoter that is unmethylated on the maternal allele and becomes hypermethylated on the paternal allele after fertilization. However, previous work has also identified some differences between the two loci. In particular, conserved intergenic consensus sequences for CTCF binding have not been found in Dlk1–Gtl2 in a three mammalian species sequence comparison. Rather, a single highly conserved putative CTCF-binding site has been identified in the first intron of Gtl2 (14). Although the conservation suggests a function for this site, its location and genomic environment indicate that this may be different to the insulator function found at Igf2–H19. Nonetheless, studies by Schmidt et al. (9) have shown that in the absence of DNA methylation, Dlk1 becomes repressed and the silent allele of Gtl2 becomes activated just as for Igf2–H19, suggesting regulatory parallels between the imprinting of the two domains.
We have previously conducted a comparative sequence analysis of the Dlk1–Gtl2 domain in three mammalian species and identified genomic features that are highly conserved (14). Twenty highly conserved elements were identified; seven corresponding to Dlk1 exons including the exonic DMR, and the 5' portion of the Gtl2 gene. Here we have conducted a methylation analysis of the Dlk1–Gtl2 region on both parental chromosomes. This analysis used DNA isolated from embryos with uniparental duplications of chromosome 12 (PatDp12 and MatDp12, and mUPD12 and pUPD12). Together, the sequence and epigenetic analysis has allowed the determination of the relationship between conserved elements and the DMRs and further elucidated common features between Dlk1–Gtl2 and Igf2–H19. Furthermore, we have identified a single intergenic DMR (IG-DMR) carrying a germline imprint suggesting a key role in domain-associated imprinting control.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Methylation profile of the Dlk1–Gtl2 region
Using probes isolated from a sublibrary of the Dlk1–Gtl2-containing BAC103N10 (14) and its genomic sequence commencing 7.5 kb upstream of Dlk1 and extending into the eighth intron of Gtl2, we generated an allele-specific methylation profile of the whole domain. Methylation-sensitive HhaI and HpaII restriction enzyme sites were mapped and the region systematically analysed for allele-specific methylation using Southern blot analysis of DNA isolated from embryos with uniparental duplications of mouse chromosome 12. A schematic representation of the results is shown in Figure 1 which summarizes the methylation status of the maternal chromosome, the paternal chromosome and that of sperm DNA. Three DMRs were identified; the known Dlk1 DMR and Gtl2 DMR and a third DMR located
13 kb upstream of the Gtl2 promoter. These three DMRs are described in detail below. No further DMRs were identified by Southern blot analysis. Indeed, most of the intergenic region is hypermethylated on both parental alleles, with the exception of an area located 35 kb upstream of Gtl2 that is hypomethylated on both alleles. This region encompasses three highly conserved elements (nucleotides 61827–61939, nucleotides 62810–62990 and nucleotides 66345–66479; GenBank accession no. AJ320506) (14). Interestingly, a well conserved, relatively hypomethylated area located mid-way between Igf2 and H19 has recently been shown to contain tissue-specific regulatory elements associated with Igf2 expression though these are not critical for its imprinting (16–19).
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Bisulfite mutagenesis at a putative CTCF-binding site located in the Gtl2 DMR
The previously identified Gtl2 promoter-specific differential methylation starts
1.5 kb upstream of the Gtl2 promoter and extends into the first exon and first intron. Thereafter, both alleles are partially methylated equivalently. This DMR overlaps with a single putative consensus CTCF-binding sequence (for sequence see 11), conserved between three mammalian species and located in the first intron in the mouse (14). Although the promoter-specific differential methylation occurs after fertilization, we were interested in examining the precise methylation status of the area of homology to the putative CTCF-binding site and determining whether this carried a germline imprint. This region contains 12 CpG dinucleotides (Fig. 2, a–l) in 284 bp. DNA isolated from PatDp12 and MatDp12 embryos and sperm was subjected to bisulfite mutagenesis, then amplified across the region and individual PCR products were cloned and sequenced. Data is presented in Figure 2. In embryos, all 12 CpGs on the paternal allele were methylated with individual sites ranging from 40 to 100% methylated. In contrast, the maternal allele was less methylated with levels at individual sites ranging from 3.6 to 53.6%. The site embedded in the putative CTCF-binding sequence was 17.9% methylated on the maternal allele and 56.7% methylated on the paternal allele. In sperm, the region was hypomethylated with a methylation range of 5–35% and a methylation level of 5% at the putative CTCF-binding sequence. These epigenetic characteristics are different from the CTCF-binding domain at Igf2–H19 in which the maternal allele is fully unmethylated and the paternal allele is completely methylated in sperm and in embryos (20).
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Differential methylation of the Dlk1 DMR is tissue specific but does not correlate with expression
A single DMR was previously identified in Dlk1 (8) and is a CpG island (14). The use of methylation-sensitive restriction enzymes identified three sites that were completely unmethylated on the maternal allele and
50% methylated on the paternal allele in embryos and sperm (site u in Fig. 3B). This feature of partial methylation associated with the active allele is reminiscent of the allele-specific methylation features of the Igf2 DMR2 which has been proposed to act as a methylation-associated activation domain (21). To extend the Dlk1 analysis further and to determine whether methylation of the paternal allele is associated with tissues in which Dlk1 is expressed, we conducted bisulfite mutagenesis of DNA isolated from E18.5 tissues. Northern blot analysis of E18.5 total RNA probed with an exon 1–2 probe showed Dlk1 expression is highest in lung and muscle, low in liver and undetectable in other tissues examined using this technique (Fig. 3A). This expression pattern is similar to that observed by in situ hybridization at E13.5 (22). Methylation analysis of 24 CpGs over 413 bp in DNA from lung, muscle, liver, kidney and brain, showed a different methylation profile for each tissue. In most cases, the paternal allele was more methylated than the maternal allele (Fig. 3B) with no obvious correlation between the level of methylation and the level of expression. However, within tissues, some individual sites did consistently exhibit differential methylation. For example, in muscle, site h is completely unmethylated on the maternal allele and fully methylated on the paternal allele whereas the reverse is true for site c. This pattern is not observed in other tissues where other sites show allele-specific methylation to varying extents. These findings suggest that allele-specific methylation is not regulating expression.
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Identification of a novel IG-DMR carrying a germline imprint
Using this systematic approach, an area extending 10 kb upstream of the Gtl2 promoter was found to be devoid of allele-specific methylation. This is in contrast to the upstream region of H19 where differential methylation extends several kilobases upstream of the promoter. However, a novel 8 kb long IG-DMR was identified associated with a CpG island located
15 kb upstream of Gtl2 and 70 kb downstream of the Dlk1 promoter. This IG-DMR is unmethylated on the maternal allele and hypermethylated on the paternal allele (Figs 1 and 4B–D). The region is also methylated in sperm suggesting that the methylation on the paternal allele is a germline imprint. The IG-DMR can be subdivided into three domains; a 3 kb 5' area showing a progressive increase in methylation on the paternal allele in embryos and sperm (Fig. 1), a 1.5 kb middle portion (identified by probe M3 in Fig. 4) that is fully methylated on both alleles and a 3' adjacent 4.5 kb differentially methylated area that also contains a series of tandem repeats at its 5' end (shown by probes M4 and M5 in Fig. 4) (14). Similar repeats have been identified in analogous positions in human and sheep (14). Initially, we observed the region immediately 3' to the repeats to be a DMR by Southern analysis (Fig. 4). Subsequently, bisulfite sequencing analysis was conducted to determine whether the differential methylation was present in the tandem repeats. Thirty-three CpG dinucleotides were analysed encompassing the seven tandem repeats (Fig. 4A and E). The results show that the paternal allele is almost completely methylated and the maternal allele hypomethylated. The fully methylated pattern on the paternal allele was also evident in sperm (Fig. 4E). To confirm the germline specificity of the IG-DMR, bisulfite mutagenesis and sequencing was conducted on DNA isolated from oocytes. Six CpGs (located in M5) shown to be differentially methylated in embryos and fully methylated in sperm, were found to be completely unmethylated in unfertilized eggs (Fig. 4A and F). Although this region has allele-specific methylation patterns like those seen for the H19 upstream DMR, there is no apparent homology to CTCF-binding sites in this IG-DMR (14).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The recently identified pair of imprinted genes, Dlk1 and Gtl2, located within an imprinted cluster on mouse chromosome 12 has remarkable similarities to the well characterized Igf2–H19 domain on mouse chromosome 7. These similarities include the coding (Igf2/Dlk1) and non-coding nature (H19/Gtl2) of the two pairs of transcripts (8–10), the approximate distance between them, the allele-specific methylation profiles at the H19/Gtl2 promoters (8), their behaviour in the Dnmt1–/– mutant (9), the presence of the exonic DMR in Dlk1 and Igf2 (8), and the tissue-specific co-expression during development of the genes within each pair (8,9). However, differences have also been noted. These differences include the absence of conserved putative CTCF-binding sites in the Dlk1–Gtl2 intergenic region and the presence of a single conserved putative CTCF-binding site within an intron of Gtl2 (14). Gene and sequence analysis indicate that the two pairs do not represent a locus duplication. Taken together, these observations suggest that whereas aspects of imprinting control may be the same, it is expected that other elements involved in transcriptional control may be specific to each pair. This relationship allows a comparative functional analysis of the two domains and has the potential to contribute to an understanding of the genomics of imprinting through the characterization of shared and unique elements involved in imprinting control.
The allele-specific methylation analysis conducted over 120 kb of the Dlk1–Gtl2 domain has identified three DMRs. Each DMR exhibits different methylation characteristics; however, like for the Igf2–H19 locus, those on the paternal chromosome are hypermethylated and on the maternal chromosome, hypomethylated.
The Dlk1 and Gtl2 DMRs
The Gtl2 DMR is located at the Gtl2 promoter and extends into the 5' portion of the gene. This differential methylation is absent in sperm and therefore is a post-fertilization event. These epigenetic characteristics are identical to those seen in the H19 promoter and gene (23). The allele-specific methylation extends into the first intron of the gene and encompasses a single conserved putative CTCF-binding consensus sequence. This region is hypermethylated on the paternal allele and hypomethylated on the maternal allele and in sperm. This is different from the CTCF-binding sites upstream of H19 which are fully methylated in sperm and on the paternal allele and completely unmethylated on the maternal allele. Further experiments will determine whether this Gtl2 site can bind CTCF, whether it plays a role in allele-specific chromatin conformation and if it contributes to the regulation of Gtl2. However, its partial methylation status on both parental alleles and its intronic location, suggests that this region may not play a key role in domain-wide imprinting. Our data suggest this conserved region is probably functioning in a manner different to the cluster of CTCF-binding sites in Igf2–H19.
In Igf2, three DMRs have been identified. DMR0 is associated with a placenta-specific promoter that is methylated on the silent maternal allele (24). DMR1 is located between the placental and first fetal promoter of Igf2 and is preferentially methylated on the active paternal allele (25). DMR1 appears to be a methylation-sensitive tissue-specific repressor element for Igf2 (26,27). DMR2, of unknown function, is located in the last exon of Igf2 and shows tissue-specific methylation that is more pronounced on the paternal allele (21). Sequences with methylation properties similar to Igf2 DMR0 and DMR1 have not been found in embryonic DNA up to 7.5 kb upstream of Dlk1. However, the Dlk DMR characterized herein, is similar to Igf2 DMR2 in two respects. First, it is located in exactly the same relative position in the last exon of the gene and, secondly, it exhibits a similar methylation profile, with the methylated allele being the active one. Though, in general, the Dlk DMR is hypermethylated on the paternal allele, individual CpGs within this DMR show a tissue-specific mosaic pattern of differential methylation. This has also been shown for Igf2 DMR2 where methylation is reported to correlate with expression. This is predominantly supported by data showing that in the brain where Igf2 is not expressed, the methylation levels are low and the allelic differences less pronounced compared to the liver where allelic differences are more pronounced and expression is high (21). Nonetheless, it still remains to be shown that DMR2 methylation plays a role in Igf2 regulation. Interestingly, Dlk DMR exhibits similar patterns in methylation at the above mentioned tissues. However, in this case, methylation does not correlate with expression. These tissue-specific differences suggest that allele-specific methylation differences at Dlk DMR may not have a role to play in transcriptional control.
The Dlk1–Gtl2 IG-DMR
Of particular interest is the identification of an intergenic sequence located in a region 10–15 kb upstream of Gtl2 containing the only allele-specific methylation mark that is inherited from the sperm. This IG-DMR contains a series of tandem repeats and similar repeats were also identified in human and sheep in an analogous position. The precise sequence and number of the repeats differs between the three species; however, a core consensus can be ascertained from the sequence in the three species (14). The differential methylation of these repeats strengthens the hypothesis that such elements are involved in imprinting control. There is no evidence for a transcript associated with the repeat-associated CpG island in mouse (14).
The IG-DMR shares some similarities with, but also has a notable difference to, the intergenic insulator sequence involved in imprinting control at Igf2–H19. In particular, the IG-DMR is devoid of any known consensus CTCF-binding sites indicating that, if it is a methylation sensitive cis-acting regulator, it is unlikely that CTCF is involved. However, as there are several sequences with the potential to bind CTCF (28) this hypothesis awaits direct proof that CTCF does not bind to this DMR. Also, different factors may mediate a cis-acting regulatory function of this DMR. There are similarities to the Igf2–H19 insulator element. These include the DMR methylation profile in embryos and sperm, and their locations upstream of non-coding RNAs that have paternal allele-specific promoter methylation occurring post-fertilization.
Co-linearity of the three DMRs with highly conserved elements in three mammalian species
The three DMRs are located within regions containing highly conserved genomic features identified in a comparative sequence analysis from mouse, human and sheep (Fig. 5). This suggests that these regions may be functional. Areas of conservation that are not DMRs have also been identified. These include an intergenic area of hypomethylation differing from the remainder of the intergenic region that is heavily methylated. Other regions of conservation include repetitive elements in the domain. Although these are not differentially methylated in the fetus, one cannot rule out the possibility of differential methylation in the preimplantation embryo. However, the tandemly repeated region associated with the IG-DMR is one conserved area that is differentially methylated. A role for this region in imprinting control is currently under investigation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mice
Animals with mUPD12 and pUPD12 were generated as described previously by Georgiades et al. (29). Briefly, doubly heterozygous female mice carrying Rb(8.12)5Bnr/Rb(6.12)3Sic on a BALB/C background were mated to Rb(8.12)5Bnr/Rb(4.12)9Bnr males on a C57BL6 background and genotypes of fetuses determined by PCR amplification of tail DNA using strain-specific microsatellite markers for chromosome 12 (D12Nds2 or D12Mit20), chromosome 4 (D4Mit116) and chromosome 8 (D8Mit41). Using this approach UPD12 animals could be easily distinguished from normal littermates, the latter serving as controls for molecular analysis. Animals harbouring PatDp12 and MatDp12 distal uniparental duplications/deficiencies were generated by crossing T(4;12)47H heterozygous males and females on a C3H/He background with T(4;12)47H translocation heterozygote females and males on a 101/129Sv background. Genotypes were confirmed by PCR amplification of fetal tail DNA using D12Nds2 and D4Mit13.
For all Southern blot analysis and for the bisulfite sequencing analysis of the Glt2 DMR CTCF region, E13.5 embryos were used. For northern blots and bisulfite sequence analysis of the Dlk1 DMR, E18.5 embryos were used. Sperm DNA was purified from a C3H/He male.
DNA isolation and Southern blot analysis
DNA was isolated by standard techniques (30). Ten micrograms of restriction enzyme-digested DNA was separated on 0.5x TBE gels, transferred to Hybond-N+ (Amersham Pharmacia, Little Chalfont, UK) nylon membranes and pre-hybridized with hybridization buffer (50% formamide, 5x SSPE, 0.5% SDS, 0.5% Bailey’s Irish Cream Liquor, 50 ng/ml heat denatured salmon sperm DNA) at 42°C for at least 2 h. Probes were labelled with the Megaprime DNA labelling system (Amersham Pharmacia) and added to the hybridization buffer. Filters were incubated at 42°C overnight, washed to the stringency of 0.1x SSC/0.1% SDS at 65°C and exposed to X-ray films at –70°C with intensifying screens. DNA fragments used as probes were purified from a sub-library derived from BAC103N10 containing both Gtl2 and Dlk1 (14) that was generated for sequencing purposes by MWG Research.
RNA isolation and northern blot analysis
Tissues were dissected in PBS, dissolved in Solution D (31) and RNA was extracted by the method previously described by Chomczynski and Sacchi (31). For Northern blot analysis, 10 µg of RNA was separated on a formaldehyde gel and blotted on Hybond N+ nylon membrane. Filters were prehybridized, hybridized, washed and exposed to X-ray films as above. A DNA fragment containing exons 1 and 2 of Dlk1 was used as a probe.
Bisulfite sequencing
The method for bisulfite-based cytosine methylation analysis was adapted from Olek et al. (32). DNA was digested with XhoI (for Gtl2 DMR analysis) or HindIII (for Dlk1 DMR). Samples were serially diluted and denatured by boiling, then NaOH was added to a final concentration of 0.4 N. After 15 min incubation at 50°C, DNA was embedded in LMP agarose to form beads at DNA concentrations of 100, 10 and 1 ng/bead. Beads were treated (in the dark) with 5 M sodium bisulfite/10 mM hydroxyquinone on ice for 30 min, incubated at 50°C for 3.5 h and washed with TE, treated with 0.2 N NaOH and washed again with TE and water. One bead was used for semi-nested PCR. Primer sets for Gtl2 DMR were CT-F1 (5'-TGG TTT GGG GGT AGT TTT TTA TTG TAG-3') and CT-R (5'-AAA AAA TAC AAA TAA ATT AAT TAA CAA ATC ACA AA-3') for the first PCR. For the second round of PCR the following primers were used: CT-F2 (5'-ATT TTT AAATGAT GGT TGA TGT GGG TTT-3') and CT-R. Primer sets for Dlk1 DMR were DLKBi2876F (5'-GAT TAG TGA TTT ATA ATT TGT GTT TTG GTT-3') and DLKBi3281R (5'-AAA CTC ACC TAA ATA TAC TAA AAA CAA ATA-3') for the first PCR and DLKBi2926F (5'-GAG ATT AAG TAA GAG GTG GGA AAG GGT-3') and DLKBi3281R for the second PCR. Primers for the IG-DMR first round PCR were IGCF1 (5'-TTA AGG TAT TTT TTA TTG ATA AAA TAA TGT AGT TT-3') and IGCR1 (5'-CCT ACT CTA TAA TAC CCT ATA TAA TTA TAC CAT AA-3') and for the second round PCR IGCF2 (5'-TTA GGA GTT AAG GAA AAG AAA GAA ATA GTA TAG T-3') and IGCR1 or IGCR2 (5'-TAT ACA CAA AAA TAT ATC TAT ATA ACA CCA TAC AA-3'). The first PCR was run in 100 µl in the presence of 2 U BIO-X act Taq polymerase (BioLine, London, UK) 1x manufacturer’s buffer, 3 mM MgCl2, 400 µM dNTPs and 1 µM primers. Second PCR was run in 25 µl in the presence of 0.8 U BIO-X ACT Taq, 1x PCR buffer, 2.5 mM MgCl2, 400 µM dNTPs and 1 µM primers, PCR conditions were five cycles at 94°C for 1 min, 50°C for 2 mins and 72°C for 3 mins followed by 30 cycles of 94°C for 30 s, 50°C for 2 min and 72°C for 1.5 min. PCR products were subcloned using the TOPO TA cloning kit (Invitrogen, Groningen, The Netherlands) and sequencing was performed by the DNA sequencing facility, Department of Genetics, University of Cambridge. The proportion of converted Cs for each bisufite treatment was measured at 95% or greater for non-CpG associated Cs. For the Gtl2 DMR and IG-DMR, bisulfite treatments were conducted twice using different animals; for the Dlk DMR tissue analysis, the experiment was done once due to the difficulty in obtaining pUPD12 late gestation tissues.
Clonality
For data generated for Gtl2 DMR, the mUPD12 (24 clones) and pUPD12 (30 clones) sequences analysed were all non-clonal and at least 19/20 clones analysed from sperm DNA were non-clonal. All data shown for the IG-DMR (20 clones for each sample) represent unique clones. For the Dlk DMR (10 clones for each sample), all data presented for brain and kidney and pUPD12 muscle and lung represent unique clones. Complete conversion of non-CpG associated Cs made assessment of clonality difficult for the rest of the clones; however, for mUPD12 liver at least 90% were unique, for mUPD12 muscle and lung at least 60 and 50%, respectively, were unique. At least 60% of sequences analysed were non-clonal in pUPD12 liver. For analysis of oocytes, 165 eggs were isolated from superovulated unfertilized (CBA x C57BL6J) F1 females. Eggs (with zonae intact) were incubated in hylauronidase (300 µg/ml) to remove cumulus cells, washed extensively and individually checked for purity. DNA was isolated in the presence of 5 µg carrier yeast tRNA and purified by phenol–chloroform extraction and ethanol precipitation. Bisulfite mutagenesis was carried out as described except the bisulfite treatment time was reduced to 2 h. Treated DNA was amplified using the following primers: M5BF1 (5'-GTT AGT TAG GTG ATA AAT TTT AGA ATA TTG AAT TT-3') and M5BR1 (5'-CCA ACC CCA ATA TAA CTT TCT ACC CTA A-3') for the first PCR and M5BF2 (5'-GTT TTA GGT TTT TAT TTT GAT GTA AGT GGT TT-3') and M5BR2 (5'-CAA AAT AAT CAC CCT AAC CCA ACC TAC TA-3') for the second PCR. PCR conditions were as above except 40 cycles were used for both first and second rounds of amplification. Eleven clones were sequenced and the efficiency of conversion for non-CpG-associated Cs was 89%. However, this was deemed acceptable because there was 100% conversion of CpG-associated Cs. At least 64% of the clones sequenced represented unique clones.
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ACKNOWLEDGEMENTS |
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We thank Professor H.Winking for providing the Robertsonian translocation homozygous stock mice, Carol Rasberry for assistance with the T47H reciprocal translocation stocks, Philippe Arnaud, Annie Lewis and Wolf Reik for advice regarding bisulfite sequencing, MWG Research for making the BAC sublibrary and members of the Ferguson-Smith laboratory for helpful discussions. The work was supported by grants from CRC and MRC. G.K. is an MRC senior fellow.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +44 1223 333 750; Fax: +44 1223 333 786; Email: afsmith@mole.bio.cam.ac.uk Present address: Martina Paulsen, FR 8.2 Genetik, Gebäude 6, Universität des Saarlandes, Postfach 151150, D-66041 Saarbrücken, Germany
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REFERENCES |
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