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A tripartite paternally methylated region within the Gpr1-Zdbf2 imprinted domain on mouse chromosome 1 identified by meDIP-on-chip.

Hiura H, Sugawara A, Ogawa H, John RM, Miyauchi N, Miyanari Y, Horiike T, Li Y, Yaegashi N, Sasaki H, Kono T, Arima T - Nucleic Acids Res. (2010)

Bottom Line: In both humans and mice, the adjacent gene, Gpr1/GPR1, which encodes a G-protein-coupled receptor 1 protein with transmembrane domain, was also imprinted and paternally expressed.The Gpr1-Zdbf2 domain was most similar to the Rasgrf1 domain as both DNA methylation and the actively expressed allele were in cis on the paternal chromosome.This work demonstrates the effectiveness of meDIP-on-chip as a technique for identifying DMRs.

View Article: PubMed Central - PubMed

Affiliation: Innovation of New Biomedical Engineering Center, University of Tohoku, Aoba-ku, Sendai, Japan.

ABSTRACT
The parent-of-origin specific expression of imprinted genes relies on DNA methylation of CpG-dinucleotides at differentially methylated regions (DMRs) during gametogenesis. To date, four paternally methylated DMRs have been identified in screens based on conventional approaches. These DMRs are linked to the imprinted genes H19, Gtl2 (IG-DMR), Rasgrf1 and, most recently, Zdbf2 which encodes zinc finger, DBF-type containing 2. In this study, we applied a novel methylated-DNA immunoprecipitation-on-chip (meDIP-on-chip) method to genomic DNA from mouse parthenogenetic- and androgenetic-derived stem cells and sperm and identified 458 putative DMRs. This included the majority of known DMRs. We further characterized the paternally methylated Zdbf2/ZDBF2 DMR. In mice, this extensive germ line DMR spanned 16 kb and possessed an unusual tripartite structure. Methylation was dependent on DNA methyltransferase 3a (Dnmt3a), similar to H19 DMR and IG-DMR. In both humans and mice, the adjacent gene, Gpr1/GPR1, which encodes a G-protein-coupled receptor 1 protein with transmembrane domain, was also imprinted and paternally expressed. The Gpr1-Zdbf2 domain was most similar to the Rasgrf1 domain as both DNA methylation and the actively expressed allele were in cis on the paternal chromosome. This work demonstrates the effectiveness of meDIP-on-chip as a technique for identifying DMRs.

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Methylation acquisition during spermatogenesis and absence of methylation imprints in Dnmt3a-difecient spermatogonia. (A) Methylation status of three Gpr1-Zdbf2 DMRs in E14.5, E16.5 and E18.5 prospermatogonia. As a control, the paternally methylated H19 DMR and the maternally methylated Lit1 DMR were included. The regions we analyzed and the CpG sites in this study are shown in Figure 2A. (B) Methylation profile of DMRs in normal and Dnmt3a-deficient prospermatogonia. Male germ cells at P7 were isolated from testes of normal and the conditional Dnmt3a knockout mice by FACS. The bisulfite-PCR-based assays for the three paternally methylated Gpr1-Zdbf2 and the H19 DMRs and maternally methylated Lit1 DMR.
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Figure 3: Methylation acquisition during spermatogenesis and absence of methylation imprints in Dnmt3a-difecient spermatogonia. (A) Methylation status of three Gpr1-Zdbf2 DMRs in E14.5, E16.5 and E18.5 prospermatogonia. As a control, the paternally methylated H19 DMR and the maternally methylated Lit1 DMR were included. The regions we analyzed and the CpG sites in this study are shown in Figure 2A. (B) Methylation profile of DMRs in normal and Dnmt3a-deficient prospermatogonia. Male germ cells at P7 were isolated from testes of normal and the conditional Dnmt3a knockout mice by FACS. The bisulfite-PCR-based assays for the three paternally methylated Gpr1-Zdbf2 and the H19 DMRs and maternally methylated Lit1 DMR.

Mentions: In E14.5 prospermatogonia, DMR2 was ∼15% methylated while DMR1 and DMR3 were unmethylated (Figure 3A). In contrast, the paternally methylated H19 DMR was unmethylated in E14.5 prospermatogonia. This was different to the Kato’s et al. (16) paper that reported that the H19 DMR was hypomethylated (5–15%) in E14.5 prospermatogonia. The maternally methylated Lit1 DMR was almost unmethylated. In E16.5 prospermatogonia, methylation at DMR2 had increased, methylation was observed at DMR1 but methylation at DMR3 was mosaic. In E18.5, methylation of DMR2 and DMR3 further increased but DMR1 methylation was still mosaic. These data suggested that the DMR2 region was the first to acquire DNA methylation followed by DMR3 and then DMR1.Figure 3.


A tripartite paternally methylated region within the Gpr1-Zdbf2 imprinted domain on mouse chromosome 1 identified by meDIP-on-chip.

Hiura H, Sugawara A, Ogawa H, John RM, Miyauchi N, Miyanari Y, Horiike T, Li Y, Yaegashi N, Sasaki H, Kono T, Arima T - Nucleic Acids Res. (2010)

Methylation acquisition during spermatogenesis and absence of methylation imprints in Dnmt3a-difecient spermatogonia. (A) Methylation status of three Gpr1-Zdbf2 DMRs in E14.5, E16.5 and E18.5 prospermatogonia. As a control, the paternally methylated H19 DMR and the maternally methylated Lit1 DMR were included. The regions we analyzed and the CpG sites in this study are shown in Figure 2A. (B) Methylation profile of DMRs in normal and Dnmt3a-deficient prospermatogonia. Male germ cells at P7 were isolated from testes of normal and the conditional Dnmt3a knockout mice by FACS. The bisulfite-PCR-based assays for the three paternally methylated Gpr1-Zdbf2 and the H19 DMRs and maternally methylated Lit1 DMR.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2926594&req=5

Figure 3: Methylation acquisition during spermatogenesis and absence of methylation imprints in Dnmt3a-difecient spermatogonia. (A) Methylation status of three Gpr1-Zdbf2 DMRs in E14.5, E16.5 and E18.5 prospermatogonia. As a control, the paternally methylated H19 DMR and the maternally methylated Lit1 DMR were included. The regions we analyzed and the CpG sites in this study are shown in Figure 2A. (B) Methylation profile of DMRs in normal and Dnmt3a-deficient prospermatogonia. Male germ cells at P7 were isolated from testes of normal and the conditional Dnmt3a knockout mice by FACS. The bisulfite-PCR-based assays for the three paternally methylated Gpr1-Zdbf2 and the H19 DMRs and maternally methylated Lit1 DMR.
Mentions: In E14.5 prospermatogonia, DMR2 was ∼15% methylated while DMR1 and DMR3 were unmethylated (Figure 3A). In contrast, the paternally methylated H19 DMR was unmethylated in E14.5 prospermatogonia. This was different to the Kato’s et al. (16) paper that reported that the H19 DMR was hypomethylated (5–15%) in E14.5 prospermatogonia. The maternally methylated Lit1 DMR was almost unmethylated. In E16.5 prospermatogonia, methylation at DMR2 had increased, methylation was observed at DMR1 but methylation at DMR3 was mosaic. In E18.5, methylation of DMR2 and DMR3 further increased but DMR1 methylation was still mosaic. These data suggested that the DMR2 region was the first to acquire DNA methylation followed by DMR3 and then DMR1.Figure 3.

Bottom Line: In both humans and mice, the adjacent gene, Gpr1/GPR1, which encodes a G-protein-coupled receptor 1 protein with transmembrane domain, was also imprinted and paternally expressed.The Gpr1-Zdbf2 domain was most similar to the Rasgrf1 domain as both DNA methylation and the actively expressed allele were in cis on the paternal chromosome.This work demonstrates the effectiveness of meDIP-on-chip as a technique for identifying DMRs.

View Article: PubMed Central - PubMed

Affiliation: Innovation of New Biomedical Engineering Center, University of Tohoku, Aoba-ku, Sendai, Japan.

ABSTRACT
The parent-of-origin specific expression of imprinted genes relies on DNA methylation of CpG-dinucleotides at differentially methylated regions (DMRs) during gametogenesis. To date, four paternally methylated DMRs have been identified in screens based on conventional approaches. These DMRs are linked to the imprinted genes H19, Gtl2 (IG-DMR), Rasgrf1 and, most recently, Zdbf2 which encodes zinc finger, DBF-type containing 2. In this study, we applied a novel methylated-DNA immunoprecipitation-on-chip (meDIP-on-chip) method to genomic DNA from mouse parthenogenetic- and androgenetic-derived stem cells and sperm and identified 458 putative DMRs. This included the majority of known DMRs. We further characterized the paternally methylated Zdbf2/ZDBF2 DMR. In mice, this extensive germ line DMR spanned 16 kb and possessed an unusual tripartite structure. Methylation was dependent on DNA methyltransferase 3a (Dnmt3a), similar to H19 DMR and IG-DMR. In both humans and mice, the adjacent gene, Gpr1/GPR1, which encodes a G-protein-coupled receptor 1 protein with transmembrane domain, was also imprinted and paternally expressed. The Gpr1-Zdbf2 domain was most similar to the Rasgrf1 domain as both DNA methylation and the actively expressed allele were in cis on the paternal chromosome. This work demonstrates the effectiveness of meDIP-on-chip as a technique for identifying DMRs.

Show MeSH
Related in: MedlinePlus