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Non-random, individual-specific methylation profiles are present at the sixth CTCF binding site in the human H19/IGF2 imprinting control region.

Tost J, Jammes H, Dupont JM, Buffat C, Robert B, Mignot TM, Mondon F, Carbonne B, Siméoni U, Grangé G, Kerjean A, Ferré F, Gut IG, Vaiman D - Nucleic Acids Res. (2006)

Bottom Line: Expression of imprinted genes is classically associated with differential methylation of specific CpG-rich DNA regions (DMRs).This epigenetic polymorphism was confined to the sixth CTCF binding site while a unique median-methylated profile was found at the third CTCF binding site as well as in the H19 promoter.The existence of three individual-specific epigenotypes in the H19 DMR in a non-pathological situation means it is important to reconsider the diagnostic value and functional importance of the sixth CTCF binding site.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire d'Epigénétique, Centre National de Génotypage, 2 rue Gaston Crémieux, 91000 Evry, France. tost@cng.fr

ABSTRACT
Expression of imprinted genes is classically associated with differential methylation of specific CpG-rich DNA regions (DMRs). The H19/IGF2 locus is considered a paradigm for epigenetic regulation. In mice, as in humans, the essential H19 DMR--target of the CTCF insulator--is located between the two genes. Here, we performed a pyrosequencing-based quantitative analysis of its CpG methylation in normal human tissues. The quantitative analysis of the methylation level in the H19 DMR revealed three unexpected discrete, individual-specific methylation states. This epigenetic polymorphism was confined to the sixth CTCF binding site while a unique median-methylated profile was found at the third CTCF binding site as well as in the H19 promoter. Monoallelic expression of H19 and IGF2 was maintained independently of the methylation status at the sixth CTCF binding site and the IGF2 DMR2 displayed a median-methylated profile in all individuals and tissues analyzed. Interestingly, the methylation profile was genetically transmitted. Transgenerational inheritance of the H19 methylation profile was compatible with a simple model involving one gene with three alleles. The existence of three individual-specific epigenotypes in the H19 DMR in a non-pathological situation means it is important to reconsider the diagnostic value and functional importance of the sixth CTCF binding site.

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Profiles of IGF2 DMR methylation obtained by pyrosequencing. The degree of methylation (%) was analyzed for two differentially methylated regions, 17 CpGs in DMR2a and 9 CpGs in DMR2b. Analysis was performed on maternal blood cell DNA samples (A, Blood 1 n = 17), on placenta samples at early term of gestation (B, n = 6) and at full term (C, n = 16).
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fig6: Profiles of IGF2 DMR methylation obtained by pyrosequencing. The degree of methylation (%) was analyzed for two differentially methylated regions, 17 CpGs in DMR2a and 9 CpGs in DMR2b. Analysis was performed on maternal blood cell DNA samples (A, Blood 1 n = 17), on placenta samples at early term of gestation (B, n = 6) and at full term (C, n = 16).

Mentions: In mice, the H19 DMR is needed on the maternal allele to protect the Igf2 DMR1 and 2 from methylation (7). To assess a putative link of the methylation level at the sixth CTCF site of the H19 DMR with the methylation level in the IGF2 DMR2 we analyzed the methylation status of the IGF2 DMR2 by pyrosequencing. Two regions encompassing 17 CpGs (278 bp, IGF2 DMR2a) and 9 CpGs (195 bp, IGF2 DMR2b), separated by 265 bp and located in exons 8 and 9 of IGF2 (Figure 6) were quantitatively screened for individual-specific differential methylation. A highly specific and reproducible profile was observed for the DMR, irrespective of the sample used (placentas or maternal blood cells). Strong variability of methylation was observed between consecutive CpGs. The average proportion of methylated molecules in the first region (IGF2 DMR2a) was 62.9 ± 7.3% in lymphocytes (maternal blood cells, MBC), 47.5 ± 4.9% in early gestation placenta (ETP) and 41.1 ± 5.4% in term placenta (FTP). The methylation of placenta samples was significantly lower than in adult lymphocytes (P < 0.0001). Similarly, the IGF2 DMR2b was hypo-methylated in placenta samples: 35.0 ± 8.9% in ETP, 34.0 ± 3.6% in FTP and 57.4 ± 3.9% in maternal blood cells, respectively (Figure 6). Again the difference between [FTP-ETP] and MBC methylation levels was highly significant (P < 0.0001). By contrast, the methylation status of both DMR2a and 2b were never significantly different between early gestation and full term placentas. The comparative analysis of the H19 and IGF2 DMRs from the same samples revealed that their methylation levels were not correlated in humans.


Non-random, individual-specific methylation profiles are present at the sixth CTCF binding site in the human H19/IGF2 imprinting control region.

Tost J, Jammes H, Dupont JM, Buffat C, Robert B, Mignot TM, Mondon F, Carbonne B, Siméoni U, Grangé G, Kerjean A, Ferré F, Gut IG, Vaiman D - Nucleic Acids Res. (2006)

Profiles of IGF2 DMR methylation obtained by pyrosequencing. The degree of methylation (%) was analyzed for two differentially methylated regions, 17 CpGs in DMR2a and 9 CpGs in DMR2b. Analysis was performed on maternal blood cell DNA samples (A, Blood 1 n = 17), on placenta samples at early term of gestation (B, n = 6) and at full term (C, n = 16).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1636469&req=5

fig6: Profiles of IGF2 DMR methylation obtained by pyrosequencing. The degree of methylation (%) was analyzed for two differentially methylated regions, 17 CpGs in DMR2a and 9 CpGs in DMR2b. Analysis was performed on maternal blood cell DNA samples (A, Blood 1 n = 17), on placenta samples at early term of gestation (B, n = 6) and at full term (C, n = 16).
Mentions: In mice, the H19 DMR is needed on the maternal allele to protect the Igf2 DMR1 and 2 from methylation (7). To assess a putative link of the methylation level at the sixth CTCF site of the H19 DMR with the methylation level in the IGF2 DMR2 we analyzed the methylation status of the IGF2 DMR2 by pyrosequencing. Two regions encompassing 17 CpGs (278 bp, IGF2 DMR2a) and 9 CpGs (195 bp, IGF2 DMR2b), separated by 265 bp and located in exons 8 and 9 of IGF2 (Figure 6) were quantitatively screened for individual-specific differential methylation. A highly specific and reproducible profile was observed for the DMR, irrespective of the sample used (placentas or maternal blood cells). Strong variability of methylation was observed between consecutive CpGs. The average proportion of methylated molecules in the first region (IGF2 DMR2a) was 62.9 ± 7.3% in lymphocytes (maternal blood cells, MBC), 47.5 ± 4.9% in early gestation placenta (ETP) and 41.1 ± 5.4% in term placenta (FTP). The methylation of placenta samples was significantly lower than in adult lymphocytes (P < 0.0001). Similarly, the IGF2 DMR2b was hypo-methylated in placenta samples: 35.0 ± 8.9% in ETP, 34.0 ± 3.6% in FTP and 57.4 ± 3.9% in maternal blood cells, respectively (Figure 6). Again the difference between [FTP-ETP] and MBC methylation levels was highly significant (P < 0.0001). By contrast, the methylation status of both DMR2a and 2b were never significantly different between early gestation and full term placentas. The comparative analysis of the H19 and IGF2 DMRs from the same samples revealed that their methylation levels were not correlated in humans.

Bottom Line: Expression of imprinted genes is classically associated with differential methylation of specific CpG-rich DNA regions (DMRs).This epigenetic polymorphism was confined to the sixth CTCF binding site while a unique median-methylated profile was found at the third CTCF binding site as well as in the H19 promoter.The existence of three individual-specific epigenotypes in the H19 DMR in a non-pathological situation means it is important to reconsider the diagnostic value and functional importance of the sixth CTCF binding site.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire d'Epigénétique, Centre National de Génotypage, 2 rue Gaston Crémieux, 91000 Evry, France. tost@cng.fr

ABSTRACT
Expression of imprinted genes is classically associated with differential methylation of specific CpG-rich DNA regions (DMRs). The H19/IGF2 locus is considered a paradigm for epigenetic regulation. In mice, as in humans, the essential H19 DMR--target of the CTCF insulator--is located between the two genes. Here, we performed a pyrosequencing-based quantitative analysis of its CpG methylation in normal human tissues. The quantitative analysis of the methylation level in the H19 DMR revealed three unexpected discrete, individual-specific methylation states. This epigenetic polymorphism was confined to the sixth CTCF binding site while a unique median-methylated profile was found at the third CTCF binding site as well as in the H19 promoter. Monoallelic expression of H19 and IGF2 was maintained independently of the methylation status at the sixth CTCF binding site and the IGF2 DMR2 displayed a median-methylated profile in all individuals and tissues analyzed. Interestingly, the methylation profile was genetically transmitted. Transgenerational inheritance of the H19 methylation profile was compatible with a simple model involving one gene with three alleles. The existence of three individual-specific epigenotypes in the H19 DMR in a non-pathological situation means it is important to reconsider the diagnostic value and functional importance of the sixth CTCF binding site.

Show MeSH
Related in: MedlinePlus