Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 Aug;17(8):4322–4329. doi: 10.1128/mcb.17.8.4322

A 5' 2-kilobase-pair region of the imprinted mouse H19 gene exhibits exclusive paternal methylation throughout development.

K D Tremblay 1, K L Duran 1, M S Bartolomei 1
PMCID: PMC232285  PMID: 9234689

Abstract

The imprinted mouse H19 gene is hypermethylated on the inactive paternal allele in somatic tissues and sperm. Previous observations from a limited analysis have suggested that methylation of a few CpG dinucleotides in the region upstream from the start of transcription may be the mark that confers parental identity to the H19 alleles. Here we exploit bisulfite mutagenesis coupled with genomic sequencing to derive the methylation status of 68 CpGs that reside in a 4-kb region 5' to the start of transcription. This method reveals a 2-kb region positioned between 2 and 4 kb upstream from the start of transcription that is strikingly differentially methylated in midgestation embryos. At least 12 of the cytosine residues in this region are exclusively methylated on the paternal allele in blastocysts. In contrast, a 350-bp promoter-proximal region is less differentially methylated in midgestation embryos and, like most of the genome, is largely devoid of methylation on both alleles in blastocysts. We also demonstrate exclusive expression of the maternal H19 allele in the embryos that exhibit paternal methylation of the upstream 2-kb region. These data suggest that the 2-kb differentially methylated region acts as a key regulatory domain for imprinted H19 expression.

Full Text

The Full Text of this article is available as a PDF (1.5 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barlow D. P. Gametic imprinting in mammals. Science. 1995 Dec 8;270(5242):1610–1613. doi: 10.1126/science.270.5242.1610. [DOI] [PubMed] [Google Scholar]
  2. Bartolomei M. S., Webber A. L., Brunkow M. E., Tilghman S. M. Epigenetic mechanisms underlying the imprinting of the mouse H19 gene. Genes Dev. 1993 Sep;7(9):1663–1673. doi: 10.1101/gad.7.9.1663. [DOI] [PubMed] [Google Scholar]
  3. Bartolomei M. S., Zemel S., Tilghman S. M. Parental imprinting of the mouse H19 gene. Nature. 1991 May 9;351(6322):153–155. doi: 10.1038/351153a0. [DOI] [PubMed] [Google Scholar]
  4. Brandeis M., Kafri T., Ariel M., Chaillet J. R., McCarrey J., Razin A., Cedar H. The ontogeny of allele-specific methylation associated with imprinted genes in the mouse. EMBO J. 1993 Sep;12(9):3669–3677. doi: 10.1002/j.1460-2075.1993.tb06041.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brannan C. I., Dees E. C., Ingram R. S., Tilghman S. M. The product of the H19 gene may function as an RNA. Mol Cell Biol. 1990 Jan;10(1):28–36. doi: 10.1128/mcb.10.1.28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DeChiara T. M., Efstratiadis A., Robertson E. J. A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting. Nature. 1990 May 3;345(6270):78–80. doi: 10.1038/345078a0. [DOI] [PubMed] [Google Scholar]
  7. DeChiara T. M., Robertson E. J., Efstratiadis A. Parental imprinting of the mouse insulin-like growth factor II gene. Cell. 1991 Feb 22;64(4):849–859. doi: 10.1016/0092-8674(91)90513-x. [DOI] [PubMed] [Google Scholar]
  8. Efstratiadis A. Parental imprinting of autosomal mammalian genes. Curr Opin Genet Dev. 1994 Apr;4(2):265–280. doi: 10.1016/s0959-437x(05)80054-1. [DOI] [PubMed] [Google Scholar]
  9. Elson D. A., Bartolomei M. S. A 5' differentially methylated sequence and the 3'-flanking region are necessary for H19 transgene imprinting. Mol Cell Biol. 1997 Jan;17(1):309–317. doi: 10.1128/mcb.17.1.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Feil R., Charlton J., Bird A. P., Walter J., Reik W. Methylation analysis on individual chromosomes: improved protocol for bisulphite genomic sequencing. Nucleic Acids Res. 1994 Feb 25;22(4):695–696. doi: 10.1093/nar/22.4.695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ferguson-Smith A. C., Sasaki H., Cattanach B. M., Surani M. A. Parental-origin-specific epigenetic modification of the mouse H19 gene. Nature. 1993 Apr 22;362(6422):751–755. doi: 10.1038/362751a0. [DOI] [PubMed] [Google Scholar]
  12. Frommer M., McDonald L. E., Millar D. S., Collis C. M., Watt F., Grigg G. W., Molloy P. L., Paul C. L. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1827–1831. doi: 10.1073/pnas.89.5.1827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jinno Y., Sengoku K., Nakao M., Tamate K., Miyamoto T., Matsuzaka T., Sutcliffe J. S., Anan T., Takuma N., Nishiwaki K. Mouse/human sequence divergence in a region with a paternal-specific methylation imprint at the human H19 locus. Hum Mol Genet. 1996 Aug;5(8):1155–1161. doi: 10.1093/hmg/5.8.1155. [DOI] [PubMed] [Google Scholar]
  14. Leighton P. A., Ingram R. S., Eggenschwiler J., Efstratiadis A., Tilghman S. M. Disruption of imprinting caused by deletion of the H19 gene region in mice. Nature. 1995 May 4;375(6526):34–39. doi: 10.1038/375034a0. [DOI] [PubMed] [Google Scholar]
  15. Li E., Beard C., Jaenisch R. Role for DNA methylation in genomic imprinting. Nature. 1993 Nov 25;366(6453):362–365. doi: 10.1038/366362a0. [DOI] [PubMed] [Google Scholar]
  16. Li E., Bestor T. H., Jaenisch R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell. 1992 Jun 12;69(6):915–926. doi: 10.1016/0092-8674(92)90611-f. [DOI] [PubMed] [Google Scholar]
  17. McGrath J., Solter D. Completion of mouse embryogenesis requires both the maternal and paternal genomes. Cell. 1984 May;37(1):179–183. doi: 10.1016/0092-8674(84)90313-1. [DOI] [PubMed] [Google Scholar]
  18. Monk M., Boubelik M., Lehnert S. Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development. 1987 Mar;99(3):371–382. doi: 10.1242/dev.99.3.371. [DOI] [PubMed] [Google Scholar]
  19. Neumann B., Kubicka P., Barlow D. P. Characteristics of imprinted genes. Nat Genet. 1995 Jan;9(1):12–13. doi: 10.1038/ng0195-12. [DOI] [PubMed] [Google Scholar]
  20. Poirier F., Chan C. T., Timmons P. M., Robertson E. J., Evans M. J., Rigby P. W. The murine H19 gene is activated during embryonic stem cell differentiation in vitro and at the time of implantation in the developing embryo. Development. 1991 Dec;113(4):1105–1114. doi: 10.1242/dev.113.4.1105. [DOI] [PubMed] [Google Scholar]
  21. Razin A., Cedar H. DNA methylation and genomic imprinting. Cell. 1994 May 20;77(4):473–476. doi: 10.1016/0092-8674(94)90208-9. [DOI] [PubMed] [Google Scholar]
  22. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  23. Riggs A. D., Pfeifer G. P. X-chromosome inactivation and cell memory. Trends Genet. 1992 May;8(5):169–174. doi: 10.1016/0168-9525(92)90219-t. [DOI] [PubMed] [Google Scholar]
  24. Sanford J. P., Clark H. J., Chapman V. M., Rossant J. Differences in DNA methylation during oogenesis and spermatogenesis and their persistence during early embryogenesis in the mouse. Genes Dev. 1987 Dec;1(10):1039–1046. doi: 10.1101/gad.1.10.1039. [DOI] [PubMed] [Google Scholar]
  25. Sasaki H., Ferguson-Smith A. C., Shum A. S., Barton S. C., Surani M. A. Temporal and spatial regulation of H19 imprinting in normal and uniparental mouse embryos. Development. 1995 Dec;121(12):4195–4202. doi: 10.1242/dev.121.12.4195. [DOI] [PubMed] [Google Scholar]
  26. Shemer R., Birger Y., Dean W. L., Reik W., Riggs A. D., Razin A. Dynamic methylation adjustment and counting as part of imprinting mechanisms. Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6371–6376. doi: 10.1073/pnas.93.13.6371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Solter D. Differential imprinting and expression of maternal and paternal genomes. Annu Rev Genet. 1988;22:127–146. doi: 10.1146/annurev.ge.22.120188.001015. [DOI] [PubMed] [Google Scholar]
  28. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  29. Stöger R., Kubicka P., Liu C. G., Kafri T., Razin A., Cedar H., Barlow D. P. Maternal-specific methylation of the imprinted mouse Igf2r locus identifies the expressed locus as carrying the imprinting signal. Cell. 1993 Apr 9;73(1):61–71. doi: 10.1016/0092-8674(93)90160-r. [DOI] [PubMed] [Google Scholar]
  30. Surani M. A., Barton S. C., Norris M. L. Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis. Nature. 1984 Apr 5;308(5959):548–550. doi: 10.1038/308548a0. [DOI] [PubMed] [Google Scholar]
  31. Szabó P. E., Mann J. R. Allele-specific expression and total expression levels of imprinted genes during early mouse development: implications for imprinting mechanisms. Genes Dev. 1995 Dec 15;9(24):3097–3108. doi: 10.1101/gad.9.24.3097. [DOI] [PubMed] [Google Scholar]
  32. Tremblay K. D., Saam J. R., Ingram R. S., Tilghman S. M., Bartolomei M. S. A paternal-specific methylation imprint marks the alleles of the mouse H19 gene. Nat Genet. 1995 Apr;9(4):407–413. doi: 10.1038/ng0495-407. [DOI] [PubMed] [Google Scholar]
  33. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yeivin A., Razin A. Gene methylation patterns and expression. EXS. 1993;64:523–568. doi: 10.1007/978-3-0348-9118-9_24. [DOI] [PubMed] [Google Scholar]
  35. Zhang Y., Shields T., Crenshaw T., Hao Y., Moulton T., Tycko B. Imprinting of human H19: allele-specific CpG methylation, loss of the active allele in Wilms tumor, and potential for somatic allele switching. Am J Hum Genet. 1993 Jul;53(1):113–124. [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES