Functional Characterization of a Testis-Specific DNA Binding Activity at the <i>H19</i>/<i>Igf2</i> Imprinting Control Region

Bowman, Aaron B.; Levorse, John; Ingram, Robert S.; Tilghman, Shirley M.

doi:10.1128/mcb.23.22.8345-8351.2003

Cited by 16 publications

(13 citation statements)

References 30 publications

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“…Studies at the imprint control region of H19/Igf2 in mice as well as in the human showed around 5 and 6% intermediate methylation patterns (40 -50%) respectively. 28,29 These percentages are lower than the relaxation of allele-specific methylation observed at the IG-DMR.…”

Section: Discussionmentioning

confidence: 51%

Methylation analysis of the intergenic differentially methylated region of DLK1-GTL2 in human

et al. 2007

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Imprinting is a non-Mendelian form of inheritance where epigenetic modifications control mono-allelic expression depending on the parental origin. Methylation of CpG-dinucleotides at differentially methylated regions (DMRs) is one of the best-studied mechanisms directing expression to one specific parental allele. We studied the methylation patterns of the intergenic (IG)-DMR of DLK1 and GTL2. The methylation marks of the IG-DMR were analysed in human gametes, preimplantation embryos, amniocytes and blood of babies born after intracytoplasmic sperm injection (ICSI) and blood from adults using a bisulphite sequencing technique. In oocytes, the IG-DMR was mainly unmethylated while in sperm cells a generally methylated pattern was detected. This germ-line specific methylation mark was maintained in the preimplantation embryos until the second cleavage stage. Afterwards in the preimplantation embryos, intermediate methylation patterns (26 -74% methylation) occurred, which may point to relaxation of the imprints. Intermediate patterns were also present in amniocytes, blood from ICSI babies and adults. We hypothesise that in the early cleavage stage embryo a strict differential methylation pattern is needed for the correct imprint establishment of surrounding imprinted genes. Once correct imprinting of the involved gene(s) is acquired, a more relaxed state of the IG-region is allowed.

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Section: Discussionmentioning

confidence: 51%

Methylation analysis of the intergenic differentially methylated region of DLK1-GTL2 in human

et al. 2007

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“…Since no cis elements responsible for methylation establishment in germ cells have been identified so far (5,16,28,29), it is possible that allele-restricted histone modifications or the deposition of histone variants are used as epigenetic marks. According to our results, however, the methylation of the transgenic ICR was acquired after fertilization independently of methylation in the germ line.…”

Section: Discussionmentioning

confidence: 99%

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

Matsuzaki

Okamura

Shimotsuma

et al. 2009

Molecular and Cellular Biology

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The imprinted expression of the mouse Igf2/H19 locus is governed by the differential methylation of the imprinting control region (ICR), which is established initially in germ cells and subsequently maintained in somatic cells, depending on its parental origin. By grafting a 2.9-kbp H19 ICR fragment into a human ␤-globin yeast artificial chromosome in transgenic mice, we previously showed that the ICR could recapitulate imprinted methylation and expression at a heterologous locus, suggesting that the H19 ICR in the ␤-globin locus contained sufficient information to maintain the methylation mark (K. Tanimoto Curiously, however, the transgenic H19 ICR was not methylated in sperm, which was distinct from that seen in the endogenous locus. Here, we reevaluated the ability of the H19 ICR to mark the parental origin using more rigid criteria. In the testis, the methylation levels of the solitary 2.9-kbp transgenic ICR fragment varied significantly between six transgenic mouse lines. However, in somatic cells, the paternally inherited ICR fragment exhibited consistently higher methylation levels at five out of six randomly integrated sites in the mouse genome. These results clearly demonstrated that the H19 ICR could acquire parent-of-origin-dependent methylation after fertilization independently of the chromosomal integration site or the prerequisite methylation acquisition in male germ cells.

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“…Taken together, these experiments suggest that there may be one or more proteins other than CTCF that play a role in the establishment and maintenance of parental-specific epigenetic differences at the ICR, and in vivo footprinting of the ICR has reinforced this conclusion (44,45). Recently, two groups identified a series of sites within the ICR that bind specifically and exclusively to fetal and neonatal testes extracts derived from the stages of development when paternal methylation is being assembled on the ICR (5,45). These sites contain a core that is similar to the consensus sequence for nuclear receptor extended half sites, and mutations in the half site eliminate binding.…”

mentioning

confidence: 96%

“…These sites contain a core that is similar to the consensus sequence for nuclear receptor extended half sites, and mutations in the half site eliminate binding. However, when Bowman et al (5) mutated three of these sites in vivo, no effect on imprint establishment or maintenance was observed.…”

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confidence: 99%

Functional Characterization of a Novel Ku70/80 Pause Site at the H19/Igf2 Imprinting Control Region

Katz

Beer

Levorse

et al. 2005

Molecular and Cellular Biology

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The imprinted expression of the H19 and Igf2 genes in the mouse is controlled by an imprinting control center (ICR) whose activity is regulated by parent-of-origin differences in methylation. The only protein that has been implicated in ICR function is the zinc-finger protein CTCF, which binds at multiple sites within the maternally inherited ICR and is required to form a chromatin boundary that inhibits Igf2 expression. To identify other proteins that play a role in imprinting, we employed electrophoresis mobility shift assays to identify two novel binding sites within the ICR. The DNA binding activity was identified as the heterodimer Ku70/80, which binds nonspecifically to free DNA ends. The sites within the ICR bind Ku70/80 in a sequencespecific manner and with higher affinity than previously reported binding sites. The binding required the presence of Mg 2؉ , implying that the sequence is a pause site for Ku70/80 translocation from a free end. Chromatin immunoprecipitation assays were unable to confirm that Ku70/80 binds to the ICR in vivo. In addition, mutation of these binding sites in the mouse did not result in any imprinting defects. A genome scan revealed that the binding site is found in LINE-1 retrotransposons, suggesting a possible role for Ku70/80 in transposition.

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Functional Characterization of a Testis-Specific DNA Binding Activity at the H19/Igf2 Imprinting Control Region

Cited by 16 publications

References 30 publications

Methylation analysis of the intergenic differentially methylated region of DLK1-GTL2 in human

Methylation analysis of the intergenic differentially methylated region of DLK1-GTL2 in human

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

Functional Characterization of a Novel Ku70/80 Pause Site at the H19/Igf2 Imprinting Control Region

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