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Tandem repeat hypothesis in imprinting: deletion of a conserved direct repeat element upstream of H19 has no effect on imprinting in the Igf2-H19 region.

ABSTRACT: Igf2 and H19 are reciprocally imprinted genes on mouse distal chromosome 7. They share several regulatory elements, including a differentially methylated region (DMR) upstream of H19 that is paternally methylated throughout development. The cis-acting sequence requirements for targeting DNA methylation to the DMR remain unknown; however, it has been suggested that direct tandem repeats near DMRs could be involved. Previous studies of the imprinted Rasgrf1 locus demonstrate indeed that a direct repeat element adjacent to a DMR is responsible for establishing paternal allele-specific methylation at the DMR and therefore allelic expression of the Rasgrf1 transcript. We identified a prominent and conserved direct tandem repeat 1 kb upstream of the H19 DMR and proposed that it played a similar role in imprinted regulation of H19. To test our hypothesis, we generated mice harboring a 1.7-kb targeted deletion of the direct repeat element and analyzed fetal growth, allelic expression, and methylation within the Igf2-H19 region. Surprisingly the deletion had no effect on imprinting. These results together with deletions of other repeats close to imprinted genes suggest that direct repeats may not be important for the targeting of methylation at the majority of imprinted loci and that the Rasgrf1 locus may be an exception to this rule.

SUBMITTER: Lewis A

PROVIDER: S-EPMC480907 | biostudies-literature | 2004 Jul

REPOSITORIES: biostudies-literature

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Tandem repeat hypothesis in imprinting: deletion of a conserved direct repeat element upstream of H19 has no effect on imprinting in the Igf2-H19 region.

Lewis Annabelle A Mitsuya Kohzoh K Constancia Miguel M Reik Wolf W

Molecular and cellular biology 20040701 13

Igf2 and H19 are reciprocally imprinted genes on mouse distal chromosome 7. They share several regulatory elements, including a differentially methylated region (DMR) upstream of H19 that is paternally methylated throughout development. The cis-acting sequence requirements for targeting DNA methylation to the DMR remain unknown; however, it has been suggested that direct tandem repeats near DMRs could be involved. Previous studies of the imprinted Rasgrf1 locus demonstrate indeed that a direct r ...[more]

PMID: 15199123

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Project description:BackgroundBeckwith-Wiedemann syndrome (BWS) is a loss-of-imprinting pediatric overgrowth syndrome. The primary features of BWS include macrosomia, macroglossia, and abdominal wall defects. Secondary features that are frequently observed in BWS patients are hypoglycemia, nevus flammeus, polyhydramnios, visceromegaly, hemihyperplasia, cardiac malformations, and difficulty breathing. BWS is speculated to occur primarily as the result of the misregulation of imprinted genes associated with two clusters on chromosome 11p15.5, namely the KvDMR1 and H19/IGF2. A similar overgrowth phenotype is observed in bovine and ovine as a result of embryo culture. In ruminants this syndrome is known as large offspring syndrome (LOS). The phenotypes associated with LOS are increased birth weight, visceromegaly, skeletal defects, hypoglycemia, polyhydramnios, and breathing difficulties. Even though phenotypic similarities exist between the two syndromes, whether the two syndromes are epigenetically similar is unknown. In this study we use control Bos taurus indicus X Bos taurus taurus F1 hybrid bovine concepti to characterize baseline imprinted gene expression and DNA methylation status of imprinted domains known to be misregulated in BWS. This work is intended to be the first step in a series of experiments aimed at determining if LOS will serve as an appropriate animal model to study BWS.ResultsThe use of F1 B. t. indicus x B. t. taurus tissues provided us with a tool to unequivocally determine imprinted status of the regions of interest in our study. We found that imprinting is conserved between the bovine and human in imprinted genes known to be associated with BWS. KCNQ1OT1 and PLAGL1 were paternally-expressed while CDKN1C and H19 were maternally-expressed in B. t. indicus x B. t. taurus F1 concepti. We also show that in bovids, differential methylation exists at the KvDMR1 and H19/IGF2 ICRs.ConclusionsBased on these findings we conclude that the imprinted gene expression of KCNQ1OT1, CDKN1C, H19, and PLAGL1 and the methylation patterns at the KvDMR1 and H19/IGF2 ICRs are conserved between human and bovine. Future work will determine if LOS is associated with misregulation at these imprinted loci, similarly to what has been observed for BWS.

Project description:BackgroundPorcine IGF2 and the H19 genes are imprinted. The IGF2 is paternally expressed, while the H19 gene is maternally expressed. Extensive studies in mice established a boundary model indicating that the H19 differentially methylated domain (DMD) controls, upon binding with the CTCF protein, reciprocal imprinting of the IGF2 and the H19 genes. IGF2 transcription is tissue and development specific involving the use of 4 promoters. In the liver of adult Large White boars IGF2 is expressed from both parental alleles, whereas in skeletal muscle and kidney tissues we observed variable relaxation of IGF2 imprinting. We hypothesized that IGF2 expression from both paternal alleles and relaxation of IGF2 imprinting is reflected in differences in DNA methylation patterns at the H19 DMD and IGF2 differentially methylated regions 1 and 2 (DMR1 and DMR2).ResultsBisulfite sequencing analysis did not show any differences in DNA methylation at the three porcine CTCF binding sites in the H19 DMD between liver, muscle and kidney tissues of adult pigs. A DNA methylation analysis using methyl-sensitive restriction endonuclease SacII and 'hot-stop' PCR gave consistent results with those from the bisulfite sequencing analysis. We found that porcine H19 DMD is distinctly differentially methylated, at least for the region formally confirmed by two SNPs, in liver, skeletal muscle and kidney of foetal, newborn and adult pigs, independent of the combined imprinting status of all IGF2 expressed transcripts. DNA methylation at CpG sites in DMR1 of foetal liver was significantly lower than in the adult liver due to the presence of hypomethylated molecules. An allele specific analysis was performed for IGF2 DMR2 using a SNP in the IGF2 3'-UTR. The maternal IGF2 DMR2 of foetal and newborn liver revealed a higher DNA methylation content compared to the respective paternal allele.ConclusionsOur results indicate that the IGF2 imprinting status is transcript-specific. Biallelic IGF2 expression in adult porcine liver and relaxation of IGF2 imprinting in porcine muscle were a common feature. These results were consistent with the IGF2 promoter P1 usage in adult liver and IGF2 promoter P2, P3 and P4 usages in muscle. The results showed further that bialellic IGF2 expression in liver and relaxation of imprinting in muscle and kidney were not associated with DNA methylation variation at and around at least one CTCF binding site in H19 DMD. The imprinting status in adult liver, muscle and kidney tissues were also not reflected in the methylation patterns of IGF2 DMRs 1 and 2.

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Project description:Genomic imprinting leads to parent-of-origin specific gene expression and is determined by epigenetic modification of genes. The paternally expressed gene insulin-like growth-factor 2 (IGF2) is located about ~100kb from the maternally expressed non-coding gene H19 on human chromosome 11, and both genes play major roles in embryonic and placental growth. Given adverse gestational environments can influence DNA methylation patterns in extra-embryonic tissues, we hypothesized that prenatal exposure to endocrine disrupting chemicals (EDCs) alters H19 and IGF2 methylation in placenta. Our study was restricted to a total of 196 women co-enrolled in the Predictors of Preeclampsia Study and the Harvard Epigenetic Birth Cohort. First trimester urine concentrations of 8 phenols and 11 phthalate metabolites were measured and used to characterize EDC exposure profiles. We assessed methylation of differentially methylated regions (DMRs) by pyrosequencing of H19, IGF2DMR0, and IGF2DMR2 and correlated values with phenol and phthalate metabolites. We also assessed overall expression and allele-specific expression of H19 and IGF2. We found several significant associations between DNA methylation and additive biomarker measurements. A significant decrease in H19 methylation was associated with high levels of the sum (?) of phthalate metabolites and metabolites of low molecular weight (LMW) phthalates. ?phthalate and LMW phthalate concentrations were inversely associated with IGF2DMR0 methylation values. Variation in methylation was not associated with changes in allele-specific expression. However increased deviation of allele-specific expression of H19 was associated with ?di(2-ethylhexyl) phthalate metabolites and high molecular weight phthalates. Neither methylation nor expression of these imprinted regions had a significant impact on birth length or birth weight. Overall, our study provides new insight into an epigenetic mechanism that occurs following EDC exposure.

Dataset Information

Tandem repeat hypothesis in imprinting: deletion of a conserved direct repeat element upstream of H19 has no effect on imprinting in the Igf2-H19 region.

Publications

Tandem repeat hypothesis in imprinting: deletion of a conserved direct repeat element upstream of H19 has no effect on imprinting in the Igf2-H19 region.

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