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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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Disrupted expression of Inpp5f_v2 and Inpp5f_v3 in Dnmt1 and Dnmt3l mutants. (A) Qualitative RT-PCR expression assay for Inpp5f_v3 in E8 embryos carrying  mutations in Dnmt1. Inpp5f_v3 was amplified for 34 cycles, Gapdh was amplified for 30 cycles. Parallel amplifications were performed using cDNA generated in the presence (+RT) or absence (–RT) of reverse transcriptase. (B) Allele-specific CpG methylation at the two promoter regions in E8.5 embryos derived from Dnmt3l−/– mothers (on a Mus musculus domesticus background) and wild-type cast fathers. (C) Allele-specific RT-PCR sequencing assay for Inpp5f_v2 in E8.5 embryos derived from mothers carrying homozygous  mutations in the Dnmt3l gene, on a Mus domesticus background. The maternal strain or genotype is given first in the hybrid crosses.
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Figure 6: Disrupted expression of Inpp5f_v2 and Inpp5f_v3 in Dnmt1 and Dnmt3l mutants. (A) Qualitative RT-PCR expression assay for Inpp5f_v3 in E8 embryos carrying mutations in Dnmt1. Inpp5f_v3 was amplified for 34 cycles, Gapdh was amplified for 30 cycles. Parallel amplifications were performed using cDNA generated in the presence (+RT) or absence (–RT) of reverse transcriptase. (B) Allele-specific CpG methylation at the two promoter regions in E8.5 embryos derived from Dnmt3l−/– mothers (on a Mus musculus domesticus background) and wild-type cast fathers. (C) Allele-specific RT-PCR sequencing assay for Inpp5f_v2 in E8.5 embryos derived from mothers carrying homozygous mutations in the Dnmt3l gene, on a Mus domesticus background. The maternal strain or genotype is given first in the hybrid crosses.

Mentions: Dnmt1 is responsible for the faithful propagation (or ‘maintenance’) of CpG methylation patterns during DNA replication (24). To establish whether DNA methylation is required for the silencing of Inpp5f_v3 at E8.5, expression was assessed in embryos that were homozygous or heterozygous for mutations in Dnmt1 (16), and stage-matched wt controls. Qualitative RT-PCR showed expression in Dnmt1−/– embryos, with a weak product also generated from Dnmt1+/– cDNA (Figure 6A). No expression was detected in wt E8.5 embryos. These data indicate that all of the trans-acting regulatory factors required for the expression of Inpp5f_v3 are present at E8.5, and that maintenance methylation is sufficient to silence the repeat-rich promoter. Demethylation of the paternal allele later in development (Figures 4D and 5) may therefore be sufficient for imprinted transcriptional activation in neural tissues. However, the lack of Inpp5f_v3 expression in heart tissue (Figure 2B), which also undergoes some degree of paternal allele demethylation (Figure 5B), indicates that neural-specific transcription factors also play a role.Figure 6.


Allele-specific demethylation at an imprinted mammalian promoter.

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

Disrupted expression of Inpp5f_v2 and Inpp5f_v3 in Dnmt1 and Dnmt3l mutants. (A) Qualitative RT-PCR expression assay for Inpp5f_v3 in E8 embryos carrying  mutations in Dnmt1. Inpp5f_v3 was amplified for 34 cycles, Gapdh was amplified for 30 cycles. Parallel amplifications were performed using cDNA generated in the presence (+RT) or absence (–RT) of reverse transcriptase. (B) Allele-specific CpG methylation at the two promoter regions in E8.5 embryos derived from Dnmt3l−/– mothers (on a Mus musculus domesticus background) and wild-type cast fathers. (C) Allele-specific RT-PCR sequencing assay for Inpp5f_v2 in E8.5 embryos derived from mothers carrying homozygous  mutations in the Dnmt3l gene, on a Mus domesticus background. The maternal strain or genotype is given first in the hybrid crosses.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: Disrupted expression of Inpp5f_v2 and Inpp5f_v3 in Dnmt1 and Dnmt3l mutants. (A) Qualitative RT-PCR expression assay for Inpp5f_v3 in E8 embryos carrying mutations in Dnmt1. Inpp5f_v3 was amplified for 34 cycles, Gapdh was amplified for 30 cycles. Parallel amplifications were performed using cDNA generated in the presence (+RT) or absence (–RT) of reverse transcriptase. (B) Allele-specific CpG methylation at the two promoter regions in E8.5 embryos derived from Dnmt3l−/– mothers (on a Mus musculus domesticus background) and wild-type cast fathers. (C) Allele-specific RT-PCR sequencing assay for Inpp5f_v2 in E8.5 embryos derived from mothers carrying homozygous mutations in the Dnmt3l gene, on a Mus domesticus background. The maternal strain or genotype is given first in the hybrid crosses.
Mentions: Dnmt1 is responsible for the faithful propagation (or ‘maintenance’) of CpG methylation patterns during DNA replication (24). To establish whether DNA methylation is required for the silencing of Inpp5f_v3 at E8.5, expression was assessed in embryos that were homozygous or heterozygous for mutations in Dnmt1 (16), and stage-matched wt controls. Qualitative RT-PCR showed expression in Dnmt1−/– embryos, with a weak product also generated from Dnmt1+/– cDNA (Figure 6A). No expression was detected in wt E8.5 embryos. These data indicate that all of the trans-acting regulatory factors required for the expression of Inpp5f_v3 are present at E8.5, and that maintenance methylation is sufficient to silence the repeat-rich promoter. Demethylation of the paternal allele later in development (Figures 4D and 5) may therefore be sufficient for imprinted transcriptional activation in neural tissues. However, the lack of Inpp5f_v3 expression in heart tissue (Figure 2B), which also undergoes some degree of paternal allele demethylation (Figure 5B), indicates that neural-specific transcription factors also play a role.Figure 6.

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