A Randomly Integrated Transgenic <i>H19</i> Imprinting Control Region Acquires Methylation Imprinting Independently of Its Establishment in Germ Cells

Matsuzaki, Hitomi; Okamura, Eiichi; Shimotsuma, Motoshi; Fukamizu, Akiyoshi; Tanimoto, Koichi

doi:10.1128/mcb.00275-09

Cited by 33 publications

(43 citation statements)

References 39 publications

(68 reference statements)

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“…Because the endogenous H19 ICR is fully methylated in sperm, the postfertilization methylation activity at the endogenous locus, if present, is normally difficult to reveal. Our previous results (Matsuzaki et al, 2009) and those of others (Gebert et al, 2010;Park et al, 2004;Puget et al, 2015) suggested that the gametic methylation of the H19 ICR was governed by signals from surrounding sequences, i.e. those located outside the 2.9-kb H19 ICR region.…”

Section: Methylation Acquisition At the Transgenic H19 Icr In Early Ementioning

confidence: 99%

“…Preimplantation embryos were embedded in agarose beads and treated with sodium bisulfite as described previously (Matsuzaki et al, 2009). Genomic DNA extracted from postimplantation embryos (E8.5), adult male sperm, or the tail tips of ∼1-week-old animals was treated with sodium bisulfite using the EZ DNA Methylation Kit (Zymo Research).…”

Section: Dna Methylation Analysis By Bisulfite Sequencingmentioning

confidence: 99%

“…Surprisingly, however, the transgenic H19 ICR was not methylated in the testes. In addition, randomly integrated H19 ICR fragments in the mouse genome were hypermethylated in the paternal allele after fertilization, irrespective of their variable methylation levels in the testes of multiple TgM lines (Matsuzaki et al, 2009). It was therefore presumed that the H19 ICR was marked by an epigenetic modification other than DNA methylation in the germ line, and that paternal allele-specific methylation was acquired after fertilization by referring to this hypothetical mark.…”

Section: Introductionmentioning

confidence: 99%

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De novoDNA methylation through 5'-segment of theH19ICR maintains its imprint during early embryogenesis

et al. 2015

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Genomic imprinting is a major monoallelic gene expression regulatory mechanism in mammals, and depends on gametespecific 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.

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Section: Methylation Acquisition At the Transgenic H19 Icr In Early Ementioning

confidence: 99%

Section: Dna Methylation Analysis By Bisulfite Sequencingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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De novoDNA methylation through 5'-segment of theH19ICR maintains its imprint during early embryogenesis

et al. 2015

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“…Transgene-specific nested PCR, cloning, and sequencing analysis were performed as described previously (23,24). Subregions of the H19 ICR (region I, nucleotides 1245 to 1995; region II, nucleotides 2845 to 3432; region II=, nucleotides 3050 to 3432; GenBank accession no.…”

Section: Methodsmentioning

confidence: 99%

“…The cumulus cells were removed by treatment with hyaluronidase and then washed repeatedly with M2 medium and PBS. Four to 14 blastocysts or 21 to 38 oocytes were embedded in agarose beads and treated with sodium bisulfite as described previously (24).…”

Section: Methodsmentioning

confidence: 99%

The H19 Imprinting Control Region Mediates Preimplantation Imprinted Methylation of Nearby Sequences in Yeast Artificial Chromosome Transgenic Mice

Okamura

Matsuzaki

Sakaguchi

et al. 2013

Molecular and Cellular Biology

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cIn the mouse Igf2/H19 imprinted locus, differential methylation of the imprinting control region (H19 ICR) is established during spermatogenesis and is maintained in offspring throughout development. Previously, however, we observed that the paternal H19 ICR, when analyzed in yeast artificial chromosome transgenic mice (YAC-TgM), was preferentially methylated only after fertilization. To identify the DNA sequences that confer methylation imprinting, we divided the H19 ICR into two fragments (1.7 and 1.2 kb), ligated them to both ends of a DNA fragment into which CTCF binding sites had been inserted, and analyzed this in YAC-TgM. The maternally inherited sequence, normally methylated after implantation in the absence of H19 ICR sequences, became hypomethylated, demonstrating protective activity against methylation within the ICR. Meanwhile, the paternally inherited sequence was hypermethylated before implantation only when a 1.7-kb fragment was ligated. Consistently, when two subfragments of the H19 ICR were individually investigated for their activities in YAC-TgM, only the 1.7-kb fragment was capable of introducing paternal allele-specific DNA methylation. These results show that postfertilization methylation imprinting is conferred by a paternal allele-specific methylation activity present in a 1.7-kb DNA fragment of the H19 ICR, while maternal allele-specific activities protect the allele from de novo DNA methylation.

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H19ICR mediated transcriptional silencing does not require target promoter methylation

Gebert

Jeong

et al. 2016

Biochemical and Biophysical Research Communications

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Transcription of the reciprocally imprinted genes Insulin-like growth factor 2 (Igf2) and H19 is orchestrated by the 2.4-kb H19 Imprinting Control Region (H19ICR) located upstream of H19. Three known functions are associated with the H19ICR: (1) it is a germline differentially methylated region, (2) it is a transcriptional insulator, and (3) it is a transcriptional silencer. The molecular mechanisms of the DMR and insulator functions have been well characterized but the basis for the ICR’s silencer function is less well understood. In order to study the role the H19ICR intrinsically plays in gene silencing, we transferred the 2.4-kb H19ICR to a heterologous non-imprinted location on chromosome 5, upstream of the alpha fetoprotein (Afp) promoter. Independent of its orientation, the 2.4-kb H19ICR silences transcription from the paternal Afp promoter. Thus silencing is a function intrinsic to this DNA element. Further, ICR mediated silencing is a developmental process that, unexpectedly, does not occur through DNA methylation at the target promoter.

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A Randomly Integrated Transgenic H19 Imprinting Control Region Acquires Methylation Imprinting Independently of Its Establishment in Germ Cells

Cited by 33 publications

References 39 publications

De novoDNA methylation through 5'-segment of theH19ICR maintains its imprint during early embryogenesis

De novoDNA methylation through 5'-segment of theH19ICR maintains its imprint during early embryogenesis

The H19 Imprinting Control Region Mediates Preimplantation Imprinted Methylation of Nearby Sequences in Yeast Artificial Chromosome Transgenic Mice

H19ICR mediated transcriptional silencing does not require target promoter methylation

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