IGF2 Differentially Methylated Regions Research Articles

DNA methylation modulates H19 and IGF2 expression in porcine female eye

The sexually dimorphic expression of H19/IGF2 is evolutionarily conserved. To investigate whether the expression of H19/IGF2 in the female porcine eye is sex-dependent, gene expression and methylation status were evaluated using quantitative real-time PCR (qPCR) and bisulfite sequencing PCR (BSP). We hypothesized that H19/IGF2 might exhibit a different DNA methylation status in the female eye. In order to evaluate our hypothesis, parthenogenetic (PA) cells were used for analysis by qPCR and BSP. Our results showed that H19 and IGF2 were over-expressed in the female eye compared with the male eye (3-fold and 2-fold, respectively). We observed a normal monoallelic methylation pattern for H19 differentially methylated regions (DMRs). Compared with H19 DMRs, IGF2 DMRs showed a different methylation pattern in the eye. Taken together, these results suggest that elevated expression of H19/IGF2 is caused by a specific chromatin structure that is regulated by the DNA methylation status of IGF2 DMRs in the female eye.

In vitro lead exposure changes DNA methylation and expression of IGF2 and PEG1/MEST

Epigenetic processes, such as changes in DNA methylation, likely mediate the link between environmental exposures in utero and altered gene expression. Differentially methylated regions (DMRs) that regulate imprinted genes may be especially vulnerable to environmental exposures since imprinting is established and maintained largely through DNA methylation, resulting in expression from only one parental chromosome. We used the human embryonic kidney cell line, HEK-293, to investigate the effects of exposure to physiologically relevant doses of lead acetate (Pb) on the methylation status of nine imprinted gene DMRs. We assessed mean methylation after seventy-two hours of Pb exposure (0–25μg/dL) using bisulfite pyrosequencing. The PEG1/MEST and IGF2 DMRs had maximum methylation decreases of 9.6% (20μg/dL; p<0.005) and 3.8% (25μg/dL; p<0.005), respectively. Changes at the MEG3 DMRs had a maximum decrease in methylation of 2.9% (MEG3) and 1.8% (MEG3-IG) at 5μg/dL Pb, but were not statistically significant. The H19, NNAT, PEG3, PLAGL1, and SGCE/PEG10 DMRs showed a less than 0.5% change in methylation, across the dose range used, and were deemed non-responsive to Pb in our model. Pb exposure below reportable/actionable levels increased expression of PEG1/MEST concomitant with decreased methylation. These results suggest that Pb exposure can stably alter the regulatory capacity of multiple imprinted DMRs.

Methylation and expression changes in imprinted genes H19 and Igf2 during serial somatic cell nuclear transfer using piglet fibroblasts

Cloned pigs produced by somatic cell nuclear transfer (SCNT) are important as a potential alternative source of organs. Although SCNT has created new possibilities for targeted gene modification, the successful cloning of pigs is rare. Here, we successfully conducted serial SCNT for three generations. We determined that the piglet genome was inherited from donor cell nuclei using microsatellite analysis of each generation. The methylation of differentially methylated regions (DMRs) in H19 was gradually reduced over the three generations of serial SCNT. By contrast, methylation of the insulin-like growth factor 2 (Igf2) DMR increased in the F1 generation, compared to the F0, and remained at the higher level in the F2 and F3 generations. The methylation patterns of housekeeping genes such as GAPDH and β-actin were unchanged in the serially cloned pigs. In addition, expression levels of H19 and Igf2 were variable for each generation of serial SCNT piglets, but there was no clear relationship between the methylation and gene expression patterns. Our study conclusively demonstrates that the methylation patterns of DMRs in H19 and Igf2 were altered, compare to the F0 donor, during serial SCNT, but housekeeping genes were unaffected.

Increased methylation at differentially methylated region of GNAS in infants born to gestational diabetes.

BackgroundOffspring of pregnancy complicated with gestational diabetes (GDM) are at high risk for metabolic diseases. The mechanisms behind the association of intrauterine exposure to GDM and high risk of health problems in later life remain largely unknown. The aim of this study was to clarify the alteration in methylation levels at differentially methylated regions (DMRs) of GNAS and IGF2 in fetuses of GDM women and to explore the possible mechanisms linking maternal GDM with high risk of metabolic diseases in later life of GDM offspring.MethodsLymphocytes were isolated from umbilical cord blood of infants born to 87 women with GDM and 81 women with normal pregnancy. Genomic DNA was extracted and DNA methylation levels of GNAS and IGF2 DMRs were determined by Massarray quantitative methylation analysis.ResultsThe methylation levels were detected in 7 CpG sites of GNAS DMRs and 6 sites of IGF2 DMRs. Methylation levels were significantly higher at sites 4, 5 and 7 of GNAS DMR in GDM compared to normal pregnancy (P = 0.007, 0.008 and 0.008, respectively). The methylation level at site 4 of GNAS was significantly correlated with the presence of GDM (P = 0.003), the methylation levels at site 5 and 7 were significantly correlated with the presence of GDM (P = 0.002 for both) and gestational age (P = 0.027 for both). There was no significant difference in any sites of IGF2 DMR (P > 0.05 for all).ConclusionsWe concluded maternal GDM-induced hypermethylation at GNAS DMR and this condition may be among the mechanisms associating maternal GDM with increased risk of metabolic diseases in later life of offspring.

Differential methylation status of IGF2‐H19 locus does not affect the fertility of crossbred bulls but some of the CTCF binding sites could be potentially important

Associations between abnormal methylation of spermatozoan DNA with male infertility have been sought in recent years to identify a molecular explanation of differential spermatozoan function. The present work was undertaken to investigate the methylation profile of differentially methylated regions (DMRs) in the IGF2-H19 locus of Bos taurus X Bos indicus crossbred bull spermatozoa. Bulls having more than at least 100 insemination records over a period of 12 years were classified into two groups of five bulls each belonging to low- and high-fertility groups. The IGF2 and H19 DMR sequences in B. indicus cattle were observed to be in absolute homology with B. taurus cattle. The DNA of crossbred bull spermatozoa was isolated, bisulfite treated, and amplified for specific DMR regions using methylation-change-specific primers. The overall degree of methylation at IGF2-H19 DMRs was not found to be significantly different among two groups of bulls. The sixth CTCF binding site (CCCTC) identified in H19 DMR, however, had a significant methylation difference between the high- and low-fertility bulls. It was concluded that alteration of the methylation levels at IGF2-H19 DMRs might not be responsible for the fertility difference of crossbred bulls, although the role played by the specific CTCF binding sites at this locus, which could influence IGF2 expression during spermatogenesis and early embryonic development, deserves further attention.

Depression in pregnancy, infant birth weight and DNA methylation of imprint regulatory elements

Depressed mood in pregnancy has been linked to low birth weight (LBW, < 2,500 g), a risk factor for adult-onset chronic diseases in offspring. We examined maternal depressed mood in relation to birth weight and evaluated the role of DNA methylation at regulatory sequences of imprinted genes in this association. We measured depressed mood among 922 pregnant women using the CES-D scale and obtained birth weight data from hospital records. Using bisulfite pyrosequencing of cord blood DNA from 508 infants, we measured methylation at differentially methylated regions (DMRs) regulating imprinted genes IGF2/H19, DLK1/MEG3, MEST, PEG3, PEG10/SGCE, NNAT and PLAGL1. Multiple regression models were used to examine the relationship between depressed mood, birth weight and DMR methylation levels. Depressed mood was associated with a more that 3-fold higher risk of LBW, after adjusting for delivery mode, parity, education, cigarette smoking, folic acid use and preterm birth. The association may be more pronounced in offspring of black women and female infants. Compared with infants of women without depressed mood, infants born to women with severe depressed mood had a 2.4% higher methylation at the MEG3 DMR. Whereas LBW infants had 1.6% lower methylation at the IGF2 DMR, high birth weight (> 4,500 g) infants had 5.9% higher methylation at the PLAGL1 DMR compared with normal birth weight infants. Our findings confirm that severe maternal depressed mood in pregnancy is associated with LBW, and that MEG3 and IGF2 plasticity may play important roles.

Prenatal famine and genetic variation are independently and additively associated with DNA methylation at regulatory loci within IGF2/H19.

Both the early environment and genetic variation may affect DNA methylation, which is one of the major molecular marks of the epigenome. The combined effect of these factors on a well-defined locus has not been studied to date. We evaluated the association of periconceptional exposure to the Dutch Famine of 1944–45, as an example of an early environmental exposure, and single nucleotide polymorphisms covering the genetic variation (tagging SNPs) with DNA methylation at the imprinted IGF2/H19 region, a model for an epigenetically regulated genomic region. DNA methylation was measured at five differentially methylated regions (DMRs) that regulate the imprinted status of the IGF2/H19 region. Small but consistent differences in DNA methylation were observed comparing 60 individuals with periconceptional famine exposure with unexposed same-sex siblings at all IGF2 DMRs (PBH<0.05 after adjustment for multiple testing), but not at the H19 DMR. IGF2 DMR0 methylation was associated with IGF2 SNP rs2239681 (PBH = 0.027) and INS promoter methylation with INS SNPs, including rs689, which tags the INS VNTR, suggesting a mechanism for the reported effect of the VNTR on INS expression (PBH = 3.4×10−3). Prenatal famine and genetic variation showed similar associations with IGF2/H19 methylation and their contributions were additive. They were small in absolute terms (<3%), but on average 0.5 standard deviations relative to the variation in the population. Our analyses suggest that environmental and genetic factors could have independent and additive similarly sized effects on DNA methylation at the same regulatory site.

Normal epigenetic inheritance in mice conceived by in vitro fertilization and embryo transfer

An association between assisted reproductive technology (ART) and neurobehavioral imprinting disorders has been reported in many studies, and it seems that ART may interfere with imprint reprogramming. However, it has never been explored whether epigenetic errors or imprinting disease susceptibility induced by ART can be inherited transgenerationally. Hence, the aim of this study was to determine the effect of in vitro fertilization and embryo transfer (IVF-ET) on transgenerational inheritance in an inbred mouse model. Mice derived from IVF-ET were outcrossed to wild-type C57BL/6J to obtain their female and male line F2 and F3 generations. Their behavior, morphology, histology, and DNA methylation status at several important differentially methylated regions (DMRs) were analyzed by Morris water maze, hematoxylin and eosin (H&E) staining, and bisulfite genomic sequencing. No significant differences in spatial learning or phenotypic abnormality were found in adults derived from IVF (F1) and female and male line F2 and F3 generations. A borderline trend of hypomethylation was found in H19 DMR CpG island 3 in the female line-derived F3 generation (0.40±0.118, P=0.086). Methylation status in H19/Igf2 DMR island 1, Igf2 DMR, KvDMR, and Snrpn DMR displayed normal patterns. Methylation percentage did not differ significantly from that of adults conceived naturally, and the expression of the genes they regulated was not disturbed. Transgenerational integrity, such as behavior, morphology, histology, and DNA methylation status, was maintained in these generations, which indicates that exposure of female germ cells to hormonal stimulation and gamete manipulation might not affect the individuals and their descendents.

Modulation of imprinted gene network in placenta results in normal development of in vitro manipulated mouse embryos

Genomic imprinting regulates the expression of a group of genes monoallelically expressed in a parent-of-origin specific manner. Allele-specific DNA methylation occurs at differentially methylated regions (DMRs) of these genes. We have previously shown that in vitro fertilization and embryo culture result in methylation defects at the imprinted H19-Igf2 locus at the blastocyst stage. The current study was designed to evaluate the consequences of these manipulations on genomic imprinting after implantation in the mouse. Blastocysts were produced following three experimental conditions: (i) embryos maintained in culture medium after in vivo fertilization or (ii) in vitro fertilization and (iii) a control group with embryos obtained after in vivo fertilization and timed mating. Blastocysts were all transplanted into pseudopregnant females. Embryos and placentas were collected on day 10.5 of development. DNA methylation patterns of the H19, Igf2, Igf2r and Dlk1-Dio3 DMRs were analyzed by quantitative pyrosequencing. In contrast to blastocyst stage, methylation profiles were normal both in embryonic and placental tissues after in vitro fertilization and culture. Expression of a selected set of imprinting genes from the recently described imprinted gene network (IGN) (including Igf2 and H19) was analyzed in placental tissues by quantitative RT-PCR. Placentas obtained after in vitro fertilization and embryo culture displayed significantly disturbed levels of H19 and Igf2 mRNA, as well as of most other genes from the IGN. As embryos were phenotypically normal, we hypothesize that the modulation of a coordinated network of imprinted genes results in a compensatory process capable of correcting potential dysfunction of placenta.

Analysis of IGF2 mRNA expression and its methylation status between cattle yaks and their parents

Cattle yaks are an F1 hybrid between cattle and yaks and exhibit significant hybrid vigor. However, cattle yaks are sterile males. To study the epigenetic aspects of this reproductive isolation, IGF2 mRNA expression in cattle yaks and their parents and the patterns of the IGF2 differentially methylated regions (DMR) were examined in blood and testes. The results showed that IGF2 expression in cattle yaks’ testes was lower than in their parents ( P < 0.01). The IGF2 DMR was highly methylated (above 90%) in blood and testes of cattle yaks, yaks and cattle, with the highest level in cattle yaks ( P > 0.05). Our study showed that IGF2 plays an important role in bovine spermatogenesis and might be involved in cattle-yak male sterility. The methylation level of the IGF2 DMR was irrelevant to the lower expression of IGF2 in cattle yaks; other factors perhaps play roles in its expression.

Heritable rather than age-related environmental and stochastic factors dominate variation in DNA methylation of the human IGF2/H19 locus

Epigenetic variation may significantly contribute to the risk of common disease. Currently, little is known about the extent and causes of epigenetic variation. Here, we investigated the contribution of heritable influences and the combined effect of environmental and stochastic factors to variation in DNA methylation of the IGF2/H19 locus. Moreover, we tested whether this locus was subject to age-related degeneration of epigenetic patterns as was previously suggested for global methylation. We measured methylation of the H19 and IGF2 differentially methylated regions (DMRs) in 196 adolescent and 176 middle-aged twins using a recently developed mass spectrometry-based method. We observed substantial variation in DNA methylation across individuals, underscoring that DNA methylation is a quantitative trait. Analysis of data in monozygotic and dizygotic twins revealed that a significant part of this variation could be attributed to heritable factors. The heritability of methylation of individual CpG sites varied between 20 and 74% for the H19 DMR and was even higher, between 57 and 97%, for the IGF2 DMR. Remarkably, the combined influence of environmental and stochastic factors on DNA methylation was not greater in middle-age than in adolescence, suggesting a limited role for age-related degeneration of methylation patterns at this locus. Single nucleotide polymorphisms in the IGF2/H19 locus were significantly associated with DNA methylation of the IGF2 DMR (P = 0.004). A preliminary analysis suggested an association between H19 DMR methylation and body size (P < 0.05). Our study shows that variation in DNA methylation of the IGF2/H19 locus is mainly determined by heritable factors and single nucleotide polymorphisms (SNPs) in cis, rather than the cumulative effect of environmental and stochastic factors occurring with age.

Loss of Igf2 Imprinting in Monoclonal Mouse Hepatic Tumor Cells Is Not Associated with Abnormal Methylation Patterns for the H19, Igf2, andKvlqt1 Differentially Methylated Regions

IGFII, the peptide encoded by the Igf2 gene, is a broad spectrum mitogen with important roles in prenatal growth as well as cancer progression. Igf2 is transcribed from the paternally inherited allele, whereas the linked H19 is transcribed from the maternal allele. Igf2 imprinting is thought to be maintained by differentially methylated regions (DMRs) located at multiple sites such as upstream of H19 and Igf2 and within Kvlqt1 loci. Biallelic expression (loss of imprinting (LOI)) of Igf2 is frequently observed in cancers, and a subset of Wilms' and intestinal tumors have been shown to exhibit abnormal methylation at H19DMR associated with loss of maternal H19 expression, but it is not known whether such changes are common in other neoplasms. Because cancers consist of diverse cell populations with and without Igf2 LOI, we established four independent monoclonal cell lines with Igf2 LOI from mouse hepatic tumors. We here demonstrate retention of normal differential methylation at H19, Igf2, or Kvlqt1 DMR by all of the cell lines. Furthermore, H19 was found to be expressed exclusively from the maternal allele, and levels of CTCF, a multifunctional nuclear factor that has an important role in the Igf2 imprinting, were comparable with those in normal hepatic tissues with no mutational changes detected. These data indicate that Igf2 LOI in tumor cells is not necessarily linked to abnormal methylation at H19, Igf2, or Kvlqt1 loci.

Epigenetic modifications in an imprinting cluster are controlled by a hierarchy of DMRs suggesting long-range chromatin interactions.

Imprinted genes and their control elements occur in clusters in the mammalian genome and carry epigenetic modifications. Observations from imprinting disorders suggest that epigenetic modifications throughout the clusters could be under regional control. However, neither the elements that are responsible for regional control, nor its developmental timing, particularly whether it occurs in the germline or postzygotically, are known. Here we examine regional control of DNA methylation in the imprinted Igf2-H19 region in the mouse. Paternal germline specific methylation was reprogrammed after fertilization in two differentially methylated regions (DMRs) in Igf2, and was reestablished after implantation. Using a number of knockout strains in the region, we found that the DMRs themselves are involved in regional coordination in a hierarchical fashion. Thus the H19 DMR was needed on the maternal allele to protect the Igf2 DMRs 1 and 2 from methylation, and Igf2 DMR1 was needed to protect DMR2 from methylation. This regional coordination occurred exclusively after fertilization during somatic development, and did not involve linear spreading of DNA methylation, suggesting a model in which long-range chromatin interactions are involved in regional epigenetic coordination. These observations are likely to be relevant to other gene clusters in which epigenetic regulation plays a role, and in pathological situations in which epigenetic regulation is disrupted.

Extensive tissue-specific variation of allelic methylation in the Igf2 gene during mouse fetal development: relation to expression and imprinting.

The imprinted Igf2 gene is active only on the paternal allele in most tissues. Its imprinting involves a cis-acting imprinting-control region (ICR) located upstream of the neighboring and maternally expressed H19 gene. It is thought that differential methylation of the parental alleles at the ICR is crucial for parental imprinting of both genes. Differentially methylated regions (DMRs) have also been identified within the Igf2 gene and their differential methylation is thought to be established during early development. To gain further insight into the function of these DMRs, we performed a quantitative analysis of their allelic methylation levels in different tissues during fetal development and the postnatal period in the mouse. Surprisingly, we found that the methylation levels of Igf2 DMRs vary extensively during fetal development, mostly on the expressed paternal allele. In particular, in skeletal muscle, differential allelic methylation in both DMR 1 and DMR 2 occurs only after birth, whereas correct paternal monoallelic expression is always observed, including in the embryonic stages. This suggests that differential methylation in the DMR 1 and DMR 2 of the Igf2 gene is dispensable for its imprinting in skeletal muscle. Furthermore, progressive methylation of the Igf2 paternal allele appears to be correlated with concomitant postnatal down-regulation and silencing of the gene. We discuss possible relations between Igf2 allelic methylation and expression during fetal development.