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Allele-specific demethylation at an imprinted mammalian promoter.

Wood AJ, Bourc'his D, Bestor TH, Oakey RJ - Nucleic Acids Res. (2007)

Bottom Line: Methylation is then lost specifically on the paternally derived allele during the latter stages of embryonic development, resulting in imprinted transcriptional activation on the demethylated allele.Methylation analyses in embryos lacking maternal methylation imprints suggest that the primary imprinting mark resides within an intronic CpG island approximately 1 kb downstream of the Inpp5f_v3 transcriptional start site.These data support the hypothesis that SINEs can influence gene expression by attracting de novo methylation during development, a property likely to explain their exclusion from other imprinted promoters.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical and Molecular Genetics, King's College London, Guy's Hospital, London, SE1 9RT, UK, INSERM U741, Institut Jacques Monod, 2 Place Jussieu, 75251 Paris, CEDEX 05, France.

ABSTRACT
A screen for imprinted genes on mouse Chromosome 7 recently identified Inpp5f_v2, a paternally expressed retrogene lying within an intron of Inpp5f. Here, we identify a novel paternally expressed variant of the Inpp5f gene (Inpp5f_v3) that shows a number of unusual features. Inpp5f_v3 initiates from a CpG-rich repeat region adjoining two B1 elements, despite previous reports that SINEs are generally excluded from imprinted promoters. Accordingly, we find that the Inpp5f_v3 promoter acquires methylation around the time of implantation, when many repeat families undergo de novo epigenetic silencing. Methylation is then lost specifically on the paternally derived allele during the latter stages of embryonic development, resulting in imprinted transcriptional activation on the demethylated allele. Methylation analyses in embryos lacking maternal methylation imprints suggest that the primary imprinting mark resides within an intronic CpG island approximately 1 kb downstream of the Inpp5f_v3 transcriptional start site. These data support the hypothesis that SINEs can influence gene expression by attracting de novo methylation during development, a property likely to explain their exclusion from other imprinted promoters.

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Allele-specific CpG methylation at the two imprinted promoter regions shown in Figure 1B. At embryonic stages, the parental origin of each strand was determined on the basis of SNPs between the B6 and cast alleles. Germ cell material was from purebred B6 adults. Horizontal lines represent individual strands of DNA and circles depict CpG dinucleotides. Closed circles are methylated CpGs, open circles are unmethylated. Strands derived from the same PCR amplification are connected by brace symbols to the right of the figure. The germ cell data for Inpp5f_v2 have been published previously (10). (A) Gametes, (B) 16 cell morulae, (C) E8.5 whole embryo and (D) E13.5 head.
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Figure 4: Allele-specific CpG methylation at the two imprinted promoter regions shown in Figure 1B. At embryonic stages, the parental origin of each strand was determined on the basis of SNPs between the B6 and cast alleles. Germ cell material was from purebred B6 adults. Horizontal lines represent individual strands of DNA and circles depict CpG dinucleotides. Closed circles are methylated CpGs, open circles are unmethylated. Strands derived from the same PCR amplification are connected by brace symbols to the right of the figure. The germ cell data for Inpp5f_v2 have been published previously (10). (A) Gametes, (B) 16 cell morulae, (C) E8.5 whole embryo and (D) E13.5 head.

Mentions: Both imprinted promoters are associated with clusters of CpG dinucleotides, for which the methylation profile was assessed by the sequencing of bisulphite-modified genomic DNA from C57BL/6J germ cells or F1 inter-subspecific hybrid embryos. Maternal germ-line methylation at the Inpp5f_v2 promoter has been reported previously (10), and the Inpp5f_v3 promoter is also methylated in oocytes but unmethylated in sperm (Figure 4A). Allele-specific methylation is maintained at both regions at the 16 cell morula stage (Figure 4B). By E8.5, the Inpp5f_v3 promoter has undergone de novo methylation on the paternally derived allele, and so allele-specific methylation differences are lost (Figure 4C). In contrast, the CpG island overlapping the retrotransposed ORF maintains maternal allele-specific methylation at this stage and throughout somatic development [Figure 4C and D, and (21)], suggesting that the primary imprinting mark lies within this region. In genomic DNA extracted from E13.5 head, the paternally derived Inpp5f_v3 promoter has undergone loss of methylation relative to E8.5 (Fisher's Exact P < 0.01; Figure 4D). No significant differences in methylation levels are observed on maternally derived alleles between E8.5 and E13.5 (Fisher's Exact P = 0.76), which are hypermethylated at both stages (Figure 4C and D). The allele-specific demethylation at CpG's surrounding the Inpp5f_v3 promoter shows a temporal correlation with the onset of expression (Figure 2C).Figure 4.


Allele-specific demethylation at an imprinted mammalian promoter.

Wood AJ, Bourc'his D, Bestor TH, Oakey RJ - Nucleic Acids Res. (2007)

Allele-specific CpG methylation at the two imprinted promoter regions shown in Figure 1B. At embryonic stages, the parental origin of each strand was determined on the basis of SNPs between the B6 and cast alleles. Germ cell material was from purebred B6 adults. Horizontal lines represent individual strands of DNA and circles depict CpG dinucleotides. Closed circles are methylated CpGs, open circles are unmethylated. Strands derived from the same PCR amplification are connected by brace symbols to the right of the figure. The germ cell data for Inpp5f_v2 have been published previously (10). (A) Gametes, (B) 16 cell morulae, (C) E8.5 whole embryo and (D) E13.5 head.
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Related In: Results  -  Collection

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Figure 4: Allele-specific CpG methylation at the two imprinted promoter regions shown in Figure 1B. At embryonic stages, the parental origin of each strand was determined on the basis of SNPs between the B6 and cast alleles. Germ cell material was from purebred B6 adults. Horizontal lines represent individual strands of DNA and circles depict CpG dinucleotides. Closed circles are methylated CpGs, open circles are unmethylated. Strands derived from the same PCR amplification are connected by brace symbols to the right of the figure. The germ cell data for Inpp5f_v2 have been published previously (10). (A) Gametes, (B) 16 cell morulae, (C) E8.5 whole embryo and (D) E13.5 head.
Mentions: Both imprinted promoters are associated with clusters of CpG dinucleotides, for which the methylation profile was assessed by the sequencing of bisulphite-modified genomic DNA from C57BL/6J germ cells or F1 inter-subspecific hybrid embryos. Maternal germ-line methylation at the Inpp5f_v2 promoter has been reported previously (10), and the Inpp5f_v3 promoter is also methylated in oocytes but unmethylated in sperm (Figure 4A). Allele-specific methylation is maintained at both regions at the 16 cell morula stage (Figure 4B). By E8.5, the Inpp5f_v3 promoter has undergone de novo methylation on the paternally derived allele, and so allele-specific methylation differences are lost (Figure 4C). In contrast, the CpG island overlapping the retrotransposed ORF maintains maternal allele-specific methylation at this stage and throughout somatic development [Figure 4C and D, and (21)], suggesting that the primary imprinting mark lies within this region. In genomic DNA extracted from E13.5 head, the paternally derived Inpp5f_v3 promoter has undergone loss of methylation relative to E8.5 (Fisher's Exact P < 0.01; Figure 4D). No significant differences in methylation levels are observed on maternally derived alleles between E8.5 and E13.5 (Fisher's Exact P = 0.76), which are hypermethylated at both stages (Figure 4C and D). The allele-specific demethylation at CpG's surrounding the Inpp5f_v3 promoter shows a temporal correlation with the onset of expression (Figure 2C).Figure 4.

Bottom Line: Methylation is then lost specifically on the paternally derived allele during the latter stages of embryonic development, resulting in imprinted transcriptional activation on the demethylated allele.Methylation analyses in embryos lacking maternal methylation imprints suggest that the primary imprinting mark resides within an intronic CpG island approximately 1 kb downstream of the Inpp5f_v3 transcriptional start site.These data support the hypothesis that SINEs can influence gene expression by attracting de novo methylation during development, a property likely to explain their exclusion from other imprinted promoters.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical and Molecular Genetics, King's College London, Guy's Hospital, London, SE1 9RT, UK, INSERM U741, Institut Jacques Monod, 2 Place Jussieu, 75251 Paris, CEDEX 05, France.

ABSTRACT
A screen for imprinted genes on mouse Chromosome 7 recently identified Inpp5f_v2, a paternally expressed retrogene lying within an intron of Inpp5f. Here, we identify a novel paternally expressed variant of the Inpp5f gene (Inpp5f_v3) that shows a number of unusual features. Inpp5f_v3 initiates from a CpG-rich repeat region adjoining two B1 elements, despite previous reports that SINEs are generally excluded from imprinted promoters. Accordingly, we find that the Inpp5f_v3 promoter acquires methylation around the time of implantation, when many repeat families undergo de novo epigenetic silencing. Methylation is then lost specifically on the paternally derived allele during the latter stages of embryonic development, resulting in imprinted transcriptional activation on the demethylated allele. Methylation analyses in embryos lacking maternal methylation imprints suggest that the primary imprinting mark resides within an intronic CpG island approximately 1 kb downstream of the Inpp5f_v3 transcriptional start site. These data support the hypothesis that SINEs can influence gene expression by attracting de novo methylation during development, a property likely to explain their exclusion from other imprinted promoters.

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