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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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(A) Allele-specific expression of Inpp5f transcript variants, assessed by RT-PCR sequencing assays. PCR primers were designed to specifically amplify each transcript (Figure 1A) over regions containing SNPs between two inbred strains of mice: C57BL/6J (B6) and Mus mus castaneus (cast). Allele-specific expression can be determined in F1 hybrids by the relative height of the sequence trace corresponding to each parental allele at the polymorphic site. The strain of the mother is given first in each cross. (B). Tissue-specific expression of Inpp5f_v2 transcript variants, assessed by qualitative RT-PCR. All primers were designed to span introns (Figure 1A), and each reaction was conducted using cDNA prepared in the presence (RT+) or absence (RT–) of reverse transcriptase, to eliminate the possibility of template contamination. cDNA was prepared from 1 μg of RNA extracted from B6 × cast F1 hybrid tissues. Adult material was prepared from animals sacrificed at 6 weeks. In every case, Inpp5f_v2 and Inpp5f_v3 underwent 33 cycles of amplification, whereas Gapdh was amplified for 30 cycles. (C) Developmental expression screen for Inpp5f_v2 and Inpp5f_v3 transcript variants under the same experimental conditions as panel B.
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Figure 2: (A) Allele-specific expression of Inpp5f transcript variants, assessed by RT-PCR sequencing assays. PCR primers were designed to specifically amplify each transcript (Figure 1A) over regions containing SNPs between two inbred strains of mice: C57BL/6J (B6) and Mus mus castaneus (cast). Allele-specific expression can be determined in F1 hybrids by the relative height of the sequence trace corresponding to each parental allele at the polymorphic site. The strain of the mother is given first in each cross. (B). Tissue-specific expression of Inpp5f_v2 transcript variants, assessed by qualitative RT-PCR. All primers were designed to span introns (Figure 1A), and each reaction was conducted using cDNA prepared in the presence (RT+) or absence (RT–) of reverse transcriptase, to eliminate the possibility of template contamination. cDNA was prepared from 1 μg of RNA extracted from B6 × cast F1 hybrid tissues. Adult material was prepared from animals sacrificed at 6 weeks. In every case, Inpp5f_v2 and Inpp5f_v3 underwent 33 cycles of amplification, whereas Gapdh was amplified for 30 cycles. (C) Developmental expression screen for Inpp5f_v2 and Inpp5f_v3 transcript variants under the same experimental conditions as panel B.

Mentions: To assess the allele-specific expression of Inpp5f_v3 transcripts, RT-PCR sequencing assays were performed in neonatal brain cDNA from F1 hybrids of the C57BL/6J and Mus musculus castaneus sub-species. Like Inpp5f_v2 (21), Inpp5f_v3 is expressed exclusively from the paternally derived allele (Figure 2A). These findings were confirmed by the lack of Inpp5f_v3 expression in neonatal brains from mice carrying maternally inherited uniparental duplications for the proximal portion of Chromosome 7 (Figure S1). The 20 exon Inpp5f transcript is expressed from both parental alleles, as previously reported (21).Figure 2.


Allele-specific demethylation at an imprinted mammalian promoter.

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

(A) Allele-specific expression of Inpp5f transcript variants, assessed by RT-PCR sequencing assays. PCR primers were designed to specifically amplify each transcript (Figure 1A) over regions containing SNPs between two inbred strains of mice: C57BL/6J (B6) and Mus mus castaneus (cast). Allele-specific expression can be determined in F1 hybrids by the relative height of the sequence trace corresponding to each parental allele at the polymorphic site. The strain of the mother is given first in each cross. (B). Tissue-specific expression of Inpp5f_v2 transcript variants, assessed by qualitative RT-PCR. All primers were designed to span introns (Figure 1A), and each reaction was conducted using cDNA prepared in the presence (RT+) or absence (RT–) of reverse transcriptase, to eliminate the possibility of template contamination. cDNA was prepared from 1 μg of RNA extracted from B6 × cast F1 hybrid tissues. Adult material was prepared from animals sacrificed at 6 weeks. In every case, Inpp5f_v2 and Inpp5f_v3 underwent 33 cycles of amplification, whereas Gapdh was amplified for 30 cycles. (C) Developmental expression screen for Inpp5f_v2 and Inpp5f_v3 transcript variants under the same experimental conditions as panel B.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2175309&req=5

Figure 2: (A) Allele-specific expression of Inpp5f transcript variants, assessed by RT-PCR sequencing assays. PCR primers were designed to specifically amplify each transcript (Figure 1A) over regions containing SNPs between two inbred strains of mice: C57BL/6J (B6) and Mus mus castaneus (cast). Allele-specific expression can be determined in F1 hybrids by the relative height of the sequence trace corresponding to each parental allele at the polymorphic site. The strain of the mother is given first in each cross. (B). Tissue-specific expression of Inpp5f_v2 transcript variants, assessed by qualitative RT-PCR. All primers were designed to span introns (Figure 1A), and each reaction was conducted using cDNA prepared in the presence (RT+) or absence (RT–) of reverse transcriptase, to eliminate the possibility of template contamination. cDNA was prepared from 1 μg of RNA extracted from B6 × cast F1 hybrid tissues. Adult material was prepared from animals sacrificed at 6 weeks. In every case, Inpp5f_v2 and Inpp5f_v3 underwent 33 cycles of amplification, whereas Gapdh was amplified for 30 cycles. (C) Developmental expression screen for Inpp5f_v2 and Inpp5f_v3 transcript variants under the same experimental conditions as panel B.
Mentions: To assess the allele-specific expression of Inpp5f_v3 transcripts, RT-PCR sequencing assays were performed in neonatal brain cDNA from F1 hybrids of the C57BL/6J and Mus musculus castaneus sub-species. Like Inpp5f_v2 (21), Inpp5f_v3 is expressed exclusively from the paternally derived allele (Figure 2A). These findings were confirmed by the lack of Inpp5f_v3 expression in neonatal brains from mice carrying maternally inherited uniparental duplications for the proximal portion of Chromosome 7 (Figure S1). The 20 exon Inpp5f transcript is expressed from both parental alleles, as previously reported (21).Figure 2.

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.

Show MeSH
Related in: MedlinePlus