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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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Genomic and transcriptional organization at the Inpp5f locus. (A) Exonic structure of Inpp5f, Inpp5f_v2 and Inpp5f_v3. Exons are black rectangles and splice patterns are indicated. The RT-PCR products for the allele-specific assays in Figure 2 are indicated below each transcript. The positions of two CpG islands are shown at the bottom, and the region enlarged in panel B is indicated by a thick horizontal line. (B) Genomic features within Inpp5f intron fifteen. The ORF within the first exon of Inpp5f_v2 is a retrotransposed duplicate of the X-linked Tmem114a gene. The two internal promoters are represented by arrows. The region upstream of Inpp5f_v3 is rich in repeats, which are examined further in Figure 3. Amplicon 1 and Amplicon 2 denote the regions within which DNA methylation is examined in Figure 4.
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Figure 1: Genomic and transcriptional organization at the Inpp5f locus. (A) Exonic structure of Inpp5f, Inpp5f_v2 and Inpp5f_v3. Exons are black rectangles and splice patterns are indicated. The RT-PCR products for the allele-specific assays in Figure 2 are indicated below each transcript. The positions of two CpG islands are shown at the bottom, and the region enlarged in panel B is indicated by a thick horizontal line. (B) Genomic features within Inpp5f intron fifteen. The ORF within the first exon of Inpp5f_v2 is a retrotransposed duplicate of the X-linked Tmem114a gene. The two internal promoters are represented by arrows. The region upstream of Inpp5f_v3 is rich in repeats, which are examined further in Figure 3. Amplicon 1 and Amplicon 2 denote the regions within which DNA methylation is examined in Figure 4.

Mentions: Recent evidence suggests that a number of mammalian imprinted domains were formed by the retrotransposition of X-linked genes into autosomal regions, generating sequences that undergo methylation in the maternal germ line (9,10). In order to gain insights into the regulation of imprinting at this class of imprinted domain, we chose to study the murine Inpp5f_v2 locus, searching for additional imprinted transcripts and putative regulatory elements. Inpp5f_v2 is a paternally expressed transcript variant of the murine Inpp5f gene and is transcribed at high levels in the developing nervous system (21). The unique first exon of the imprinted isoform contains a retrotransposed ORF originating from the X-linked Tmem114a gene, which is spliced onto five downstream exons of the non-imprinted Inpp5f gene (Figure 1A and B). A CpG island overlapping the retrotransposed sequence undergoes sexually dimorphic patterns of DNA methylation during gametogenesis, with maternal allele-specific methylation established during oogenesis (10). EST evidence suggests the presence of an additional promoter ∼1 kb upstream of Inpp5f_v2, generating a transcript variant of Inpp5f that has not previously been described (Inpp5f_v3). RT-PCR assays demonstrate that Inpp5f_v3 contains a unique first exon which, like the first exon of Inpp5f_v2, is spliced onto the five downstream exons of Inpp5f (Figure 1A). The unique first exon of Inpp5f_v3 lacks putative translational start codons. However, a number of in-frame start codons occur within the five exons that are shared with Inpp5f, potentially giving rise to C-terminal Inpp5f polypeptides.Figure 1.


Allele-specific demethylation at an imprinted mammalian promoter.

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

Genomic and transcriptional organization at the Inpp5f locus. (A) Exonic structure of Inpp5f, Inpp5f_v2 and Inpp5f_v3. Exons are black rectangles and splice patterns are indicated. The RT-PCR products for the allele-specific assays in Figure 2 are indicated below each transcript. The positions of two CpG islands are shown at the bottom, and the region enlarged in panel B is indicated by a thick horizontal line. (B) Genomic features within Inpp5f intron fifteen. The ORF within the first exon of Inpp5f_v2 is a retrotransposed duplicate of the X-linked Tmem114a gene. The two internal promoters are represented by arrows. The region upstream of Inpp5f_v3 is rich in repeats, which are examined further in Figure 3. Amplicon 1 and Amplicon 2 denote the regions within which DNA methylation is examined in Figure 4.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
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Figure 1: Genomic and transcriptional organization at the Inpp5f locus. (A) Exonic structure of Inpp5f, Inpp5f_v2 and Inpp5f_v3. Exons are black rectangles and splice patterns are indicated. The RT-PCR products for the allele-specific assays in Figure 2 are indicated below each transcript. The positions of two CpG islands are shown at the bottom, and the region enlarged in panel B is indicated by a thick horizontal line. (B) Genomic features within Inpp5f intron fifteen. The ORF within the first exon of Inpp5f_v2 is a retrotransposed duplicate of the X-linked Tmem114a gene. The two internal promoters are represented by arrows. The region upstream of Inpp5f_v3 is rich in repeats, which are examined further in Figure 3. Amplicon 1 and Amplicon 2 denote the regions within which DNA methylation is examined in Figure 4.
Mentions: Recent evidence suggests that a number of mammalian imprinted domains were formed by the retrotransposition of X-linked genes into autosomal regions, generating sequences that undergo methylation in the maternal germ line (9,10). In order to gain insights into the regulation of imprinting at this class of imprinted domain, we chose to study the murine Inpp5f_v2 locus, searching for additional imprinted transcripts and putative regulatory elements. Inpp5f_v2 is a paternally expressed transcript variant of the murine Inpp5f gene and is transcribed at high levels in the developing nervous system (21). The unique first exon of the imprinted isoform contains a retrotransposed ORF originating from the X-linked Tmem114a gene, which is spliced onto five downstream exons of the non-imprinted Inpp5f gene (Figure 1A and B). A CpG island overlapping the retrotransposed sequence undergoes sexually dimorphic patterns of DNA methylation during gametogenesis, with maternal allele-specific methylation established during oogenesis (10). EST evidence suggests the presence of an additional promoter ∼1 kb upstream of Inpp5f_v2, generating a transcript variant of Inpp5f that has not previously been described (Inpp5f_v3). RT-PCR assays demonstrate that Inpp5f_v3 contains a unique first exon which, like the first exon of Inpp5f_v2, is spliced onto the five downstream exons of Inpp5f (Figure 1A). The unique first exon of Inpp5f_v3 lacks putative translational start codons. However, a number of in-frame start codons occur within the five exons that are shared with Inpp5f, potentially giving rise to C-terminal Inpp5f polypeptides.Figure 1.

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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