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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) Sequence of the region upstream of and including Inpp5f_v3 exon one in the B6 genome. Five distinct classes of repeat, as defined by RepeatMasker (www.repeatmasker.org), are shown. DNA methylation at individual CpG dinucleotides (boxed) is assessed in Figure 4. The asterisk denotes a CpG that is polymorphic between B6 and cast, and was thus excluded from methylation analyses. The furthest 5′ point to which ESTs corresponding to Inpp5f_v3 extend is indicated by an arrow. (B) Clustalw alignment showing sequence homology between the inverted B1 SINE repeats upstream of Inpp5f_v3 exon 1 (B1_Mus1 and B1_Mur2) and the consensus sequence for the B1 family [B1_cons; (22)]. CpG dinucleotides in the B1 consensus sequence are boxed. Note that all five consensus CpGs have been lost in the Mus1 and Mur2 sequences shown.
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Figure 3: (A) Sequence of the region upstream of and including Inpp5f_v3 exon one in the B6 genome. Five distinct classes of repeat, as defined by RepeatMasker (www.repeatmasker.org), are shown. DNA methylation at individual CpG dinucleotides (boxed) is assessed in Figure 4. The asterisk denotes a CpG that is polymorphic between B6 and cast, and was thus excluded from methylation analyses. The furthest 5′ point to which ESTs corresponding to Inpp5f_v3 extend is indicated by an arrow. (B) Clustalw alignment showing sequence homology between the inverted B1 SINE repeats upstream of Inpp5f_v3 exon 1 (B1_Mus1 and B1_Mur2) and the consensus sequence for the B1 family [B1_cons; (22)]. CpG dinucleotides in the B1 consensus sequence are boxed. Note that all five consensus CpGs have been lost in the Mus1 and Mur2 sequences shown.

Mentions: EST evidence suggests that Inpp5f_v3 initiates from within a CpG-rich simple repeat ∼150 bp downstream of two B1 SINEs (Figure 3A). Attempts to identify the transcriptional start site by 5' RACE were unsuccessful, likely due to the CpG- and repeat-rich nature of the 5′ region (Figure 3A). RT-PCR using a forward primer upstream of the GT simple repeats but downstream of the B1 SINEs fails to generate a product, whereas products are readily amplified when the forward primer overlaps the 3′ terminus of the CG repeats (data not shown). This suggests that Inpp5f_v3 initiates from within the simple repeat region.Figure 3.


Allele-specific demethylation at an imprinted mammalian promoter.

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

(A) Sequence of the region upstream of and including Inpp5f_v3 exon one in the B6 genome. Five distinct classes of repeat, as defined by RepeatMasker (www.repeatmasker.org), are shown. DNA methylation at individual CpG dinucleotides (boxed) is assessed in Figure 4. The asterisk denotes a CpG that is polymorphic between B6 and cast, and was thus excluded from methylation analyses. The furthest 5′ point to which ESTs corresponding to Inpp5f_v3 extend is indicated by an arrow. (B) Clustalw alignment showing sequence homology between the inverted B1 SINE repeats upstream of Inpp5f_v3 exon 1 (B1_Mus1 and B1_Mur2) and the consensus sequence for the B1 family [B1_cons; (22)]. CpG dinucleotides in the B1 consensus sequence are boxed. Note that all five consensus CpGs have been lost in the Mus1 and Mur2 sequences shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC2175309&req=5

Figure 3: (A) Sequence of the region upstream of and including Inpp5f_v3 exon one in the B6 genome. Five distinct classes of repeat, as defined by RepeatMasker (www.repeatmasker.org), are shown. DNA methylation at individual CpG dinucleotides (boxed) is assessed in Figure 4. The asterisk denotes a CpG that is polymorphic between B6 and cast, and was thus excluded from methylation analyses. The furthest 5′ point to which ESTs corresponding to Inpp5f_v3 extend is indicated by an arrow. (B) Clustalw alignment showing sequence homology between the inverted B1 SINE repeats upstream of Inpp5f_v3 exon 1 (B1_Mus1 and B1_Mur2) and the consensus sequence for the B1 family [B1_cons; (22)]. CpG dinucleotides in the B1 consensus sequence are boxed. Note that all five consensus CpGs have been lost in the Mus1 and Mur2 sequences shown.
Mentions: EST evidence suggests that Inpp5f_v3 initiates from within a CpG-rich simple repeat ∼150 bp downstream of two B1 SINEs (Figure 3A). Attempts to identify the transcriptional start site by 5' RACE were unsuccessful, likely due to the CpG- and repeat-rich nature of the 5′ region (Figure 3A). RT-PCR using a forward primer upstream of the GT simple repeats but downstream of the B1 SINEs fails to generate a product, whereas products are readily amplified when the forward primer overlaps the 3′ terminus of the CG repeats (data not shown). This suggests that Inpp5f_v3 initiates from within the simple repeat region.Figure 3.

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