title: De novo DNA methylation through the 5'-segment of the H19 ICR maintains its imprint during early embryogenesis.
abstract: Genomic imprinting is a major monoallelic gene expression regulatory mechanism in mammals, and depends on gamete-specific DNA methylation of specialized cis-regulatory elements called imprinting control regions (ICRs). Allele-specific DNA methylation of the ICRs is faithfully maintained at the imprinted loci throughout development, even in early embryos where genomes undergo extensive epigenetic reprogramming, including DNA demethylation, to acquire totipotency. We previously found that an ectopically introduced H19 ICR fragment in transgenic mice acquired paternal allele-specific methylation in the somatic cells of offspring, whereas it was not methylated in sperm, suggesting that its gametic and postfertilization modifications were separable events. We hypothesized that this latter activity might contribute to maintenance of the methylation imprint in early embryos. Here, we demonstrate that methylation of the paternally inherited transgenic H19 ICR commences soon after fertilization in a maternal DNMT3A- and DNMT3L-dependent manner. When its germline methylation was partially obstructed by insertion of insulator sequences, the endogenous paternal H19 ICR also exhibited postfertilization methylation. Finally, we refined the responsible sequences for this activity in transgenic mice and found that deletion of the 5' segment of the endogenous paternal H19 ICR decreased its methylation after fertilization and attenuated Igf2 gene expression. These results demonstrate that this segment of the H19 ICR is essential for its de novo postfertilization DNA methylation, and that this activity contributes to the maintenance of imprinted methylation at the endogenous H19 ICR during early embryogenesis.
authors.affiliation: Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan Life Science Center of Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan.
authors.forename: Hitomi
authors.initials: H
authors.lastname: Matsuzaki
authors.affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan.
authors.forename: Eiichi
authors.initials: E
authors.lastname: Okamura
authors.affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Takahashi
authors.affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan.
authors.forename: Aki
authors.initials: A
authors.lastname: Ushiki
authors.affiliation: Department of Animal Bio-Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan.
authors.forename: Toshinobu
authors.initials: T
authors.lastname: Nakamura
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Nakano
authors.affiliation: Department of Maternal-Fetal Biology, National Research Institute for Child Health and Development, Setagaya, Tokyo 157-8535, Japan.
authors.forename: Kenichiro
authors.initials: K
authors.lastname: Hata
authors.affiliation: Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan Life Science Center of Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan.
authors.forename: Akiyoshi
authors.initials: A
authors.lastname: Fukamizu
authors.affiliation: Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan Life Science Center of Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan keiji@tara.tsukuba.ac.jp.
authors.forename: Keiji
authors.initials: K
authors.lastname: Tanimoto
mesh_terms: D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D019175:DNA Methylation / Q000502:physiology*; D000090205:DNA Methyltransferase 3A; D017931:DNA Primers / Q000235:genetics; D047108:Embryonic Development / Q000502:physiology*; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D018392:Genomic Imprinting / Q000502:physiology*; D007335:Insulin-Like Growth Factor II / Q000378:metabolism; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D062085:RNA, Long Noncoding / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; C000715535:Dnmt3a protein, mouse; C094335:H19 long non-coding RNA; C513121:IGF2 protein, mouse; D062085:RNA, Long Noncoding; D007335:Insulin-Like Growth Factor II; C498796:Dnmt3l protein, mouse; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A
keywords: DNA methylation; Early embryogenesis; Genomic imprinting; Igf2/H19 locus