Methylation Imprinting of H19 and SNRPN Genes in Human Benign Ovarian Teratomas

In humans, studies of female germ cells are very limited by ethics. The current study investigated the usefulness of benign ovarian teratomas as a substitute for ova in analyses of imprinted genes. Twenty-five human benign ovarian teratomas were typed with 45 microsatellite DNA markers and classified according to their genotypic features. Two oppositely imprinted genes, H19 and SNRPN, were then chosen for analysis of their methylation states in these tumors. These analyses revealed that benign ovarian teratomas consist of a mixture of genetically and epigenetically heterogeneous cell populations. In contrast to previous reports, we could document only one case rising from germ cells by meiosis-II nondisjunction. H19 and SNRPN were methylated in individual teratomas to various degrees, ranging from normal somatic cell to expected ovum levels. The allele with residual methylation of H19 was consistent with that methylated in the patient's blood DNA, thus being of paternal origin. Degrees of H19 hypomethylation and SNRPN hypermethylation increased as the cellular origin of the tumors advanced in oogenesis and were closely correlated in individual teratomas. These results could be best explained by the assumption that the primary imprinting is a progressively organized process and suggest that the establishment of primary imprints on different genes might be mechanistically linked, even when those genes are oppositely imprinted.     

Analysis of the methylation status of imprinted genes based on methylation-specific polymerase chain reaction combined with denaturing high-performance liquid chromatography

A procedure for the analysis of the methylation status of imprinted genes is described. The method offers a rapid and reliable alternative to conventional methods such as Southern blots and methylation-specific polymerase chain reaction (PCR) (i.e., allele-specific methylation-specific PCR). The efficient resolution of the differentially methylated alleles is demonstrated for three human imprinted genes: SNRPN, LIT1 (alias KCNQ1OT1), and H19. Abnormal imprinting of SNRPN is associated with the Angelman/Prader-Willi syndromes, and that of LIT1 and H19 with the Beckwith-Wiedemann syndrome. The method is based on methylation-specific PCR followed by denaturing high-performance liquid chromatography (MSP/DHPLC). Briefly, genomic DNA is initially subjected to an in vitro bisulfite treatment, whereby unmethylated cytosines are deaminated. Subsequent PCR amplifications, using primers specific for modified DNA, are aimed at DNA segments that show parent-of-origin-specific methylation. PCR conditions are chosen that allow an efficient amplification of both alleles. The PCR products representing the two alleles are identical in size; they differ, however, at a number of positions within the amplified DNA segment. The DHPLC analysis allows very efficient resolution of the two populations of PCR products, providing qualitative and quantitative results.     

Expression of H19 does not influence the timing of replication of the Igf2/H19 imprinted region

Allele specific timing of replication is believed to be a hallmark of imprinted genes, however recent evidence suggests that this might not be the case for the insulin-like growth factor 2 (Igf2) and H19 locus. In this report, we assayed the timing of replication of Igf2 and H19 in two mouse embryonic cell lines expressing both H19 and Igf2, and one cell line maternally disomic for the Igf2/H19 mouse locus which expresses H19 but not Igf2. In all cell lines, Igf2 and H19 were replicated early in the S phase of the cell cycle, and both alleles replicated at the same time. This indicates that any differences in the timing of replication at the Igf2/H19 locus are of a lesser magnitude than those found in other imprinted regions. Dev Genet 20:29–35, 1997. © 1997 Wiley-Liss, Inc.     

Disruption of imprinting and aberrant embryo development in completely inbred embryonic stem cell-derived mice

The completely embryonic stem (ES) cell-derived mice (ES mice) produced by tetraploid embryo complementation provide us with a rapid and powerful approach for functional genome analysis. However, inbred ES cell lines often fail to generate ES mice. The genome of mouse ES cells is extremely unstable during in vitro culture and passage, and the expression of the imprinted genes is most likely to be affected. Whether the ES mice retain or repair the abnormalities of the donor ES cells has still to be determined. Here we report that the inbred ES mice were efficiently produced with the inbred ES cell line (SCR012). The ES fetuses grew more slowly before day 17.5 after mating, but had an excessive growth from day 17.5 to birth. Five imprinted genes examined (H19, Igf2, Igf2r, Peg1, Peg3) were expressed abnormally in ES fetuses. Most remarkably, the expression of H19 was dramatically repressed in the ES fetuses through the embryo developmental stage, and this repression was associated with abnormal biallelic methylation of the H19 upstream region. The altered methylation pattern of H19 was further demonstrated to have arisen in the donor ES cells and persisted on in vivo differentiation to the fetal stage. These results indicate that the ES fetuses did retain the epigenetic alterations in imprinted genes from the donor ES cells.     

Bovine SNRPN methylation imprint in oocytes and day 17 in vitro-produced and somatic cell nuclear transfer embryos

Findings from recent studies have suggested that the low survival rate of animals derived via somatic cell nuclear transfer (SCNT) may be in part due to epigenetic abnormalities brought about by this procedure. DNA methylation is an epigenetic modification of DNA that is implicated in the regulation of imprinted genes. Genes subject to genomic imprinting are expressed monoallelically in a parent of origin-dependent manner and are important for embryo growth, placental function, and neurobehavioral processes. The vast majority of imprinted genes have been studied in mice and humans. Herein, our objectives were to characterize the bovine SNRPN gene in gametes and to compare its methylation profile in in vivo-produced, in vitro-produced, and SCNT-derived Day 17 elongating embryos. A CpG island within the 5' region of SNRPN was identified and examined using bisulfite sequencing. SNRPN alleles were unmethylated in sperm, methylated in oocytes, and approximately 50% methylated in somatic samples. The examined SNRPN region appeared for the most part to be normally methylated in three in vivo-produced Day 17 embryos and in eight in vitro-produced Day 17 embryos examined, while alleles from Day 17 SCNT embryos were severely hypomethylated in seven of eight embryos. In this study, we showed that the SNRPN methylation profiles previously observed in mouse and human studies are also conserved in cattle. Moreover, SCNT-derived Day 17 elongating embryos were abnormally hypomethylated compared with in vivo-produced and in vitro-produced embryos, which in turn suggests that SCNT may lead to faulty reprogramming or maintenance of methylation imprints at this locus.     

Oppositely imprinted genes H19 and insulin-like growth factor 2 are coexpressed in human androgenetic trophoblast.

Human uniparental gestations such as gynogenetic ovarian teratomas and androgenetic complete hydatidiform moles provide a model to evaluate the integrity of parent-specific gene expression--i.e., imprinting--in the absence of a complementary parental genetic contribution. We studied expression, in these tissues, of the oppositely imprinted genes H19, which is an embryonic nontranslated RNA, and insulin-like growth factor type 2 (IGF2). Normal gestations only express H19 from the maternal allele and express IGF2 from the paternal allele, whereas neither is expressed from the maternal genome of gynogenetic gestations, and both are expressed from the paternal genome of androgenetic gestations. Coexpression of H19 and IGF2 in the androgenetic tissues was in a single population of cells, mononuclear trophoblast--the same cell type expressing these genes in biparental placentas. These results demonstrate that a biparental genome may be required for expression of the reciprocal IGF2/H19 imprint. Alternatively, biparental expression may be a normal feature of some imprinted genes in specific cell types. Additional experiments with other imprinted genes will clarify whether this reflects global failure of the imprinting process or a change specific to the IGF2/H19 locus.     

DHPLC-based method for DNA methylation analysis of differential methylated regions from imprinted genes

The bisulfite genomic sequencing method is one of the most widely used techniques for methylation analysis in heterogeneous unbiased PCR, amplifying for both methylated and unmethylated alleles simultaneously. However, it requires labor-intensive and time-consuming cloning and sequencing steps. In the current study, we used a denaturing high-performance liquid chromatography (DHPLC) procedure in a complementary way with the bisulfite genomic sequencing to analyze the methylation of differentially methylated regions (DMRs) of imprinted genes. We showed reliable and reproducible results in distinguishing overall methylation profiles of DMRs regions of human SNRPN, H19, MEST/PEG1, LIT1, IGF2, TSSC5, WT1 antisense, and mouse H19, Mest/Peg1, Igf2R imprinted genes. These DHPLC profiles were in accordance with bisulfite genomic sequencing data and may serve as a type of "fingerprint," revealing the overall methylation status of DMRs associated with sample heterogeneity. We conclude that DHPLC analysis could be used to increase the throughput efficiency of methylation pattern analysis of imprinted genes after the bisulfite conversion of genomic DNA and unbiased PCR amplification.     

Characterization of differentially methylated regions in 3 bovine imprinted genes: a model for studying human germ-cell and embryo development

Correct imprinting is crucial for normal fetal and placental development in mammals. Experimental evidence in animal models and epidemiological studies in humans suggest that assisted reproductive technologies (ARTs) can interfere with imprinted gene regulation in gametogenesis and early embryogenesis. Bos taurus is an agriculturally important species in which ARTs are commonly employed. Because this species exhibits a similar preimplantation development and gestation length as humans, it is increasingly being used as a model for human germ-cell and embryo development. However, in contrast to humans and mice, there is relatively little information on bovine imprinted genes. Here, we characterized the bovine intergenic IGF2-H19 imprinting control region (ICR) spanning approximately 3 kb. We identified a 300-bp differentially methylated region (DMR) approximately 6 kb upstream of the H19 promoter, containing a CpG island with CTCF-binding site and high sequence similarity with the human intergenic ICR. Additional differentially methylated CpG islands lie -6 kb to -3 kb upstream of the promoter, however these are less conserved. Both classical bisulfite sequencing and bisulfite pyrosequencing demonstrated complete methylation of the IGF2-H19 ICR in sperm, complete demethylation in parthenogenetic embryos having only the female genome, and differential methylation in placental and somatic tissues. In addition, we established pyrosequencing assays for the previously reported bovine SNRPN and PEG3 DMRs. The observed methylation patterns were consistent with genomic imprinting in all analyzed tissues/cell types. The identified IGF2-H19 ICR and the developed quantitative methylation assays may prove useful for further studies on the relationship between ARTs and imprinting defects in the bovine model.     

Analysis of murine Snrpn and human SNRPN gene imprinting in transgenic mice

The SNRPN gene is known to be expressed exclusively from the paternal allele and to map to the critical region for the neurobehavioral disorder, Prader-Willi syndrome (PWS). As a means to investigate the mechanism of imprinting for the SNRPN gene, we have sought to recapitulate the imprinted expression of the endogenous gene. Using an 85-kb murine Snrpn clone, containing 33 kb of 5′ and 30 kb of 3′ flanking DNA, we obtained two intact transgenic lines. One line, containing two copies of the Snrpn transgene, recapitulated the imprinted expression pattern of the endogenous locus, whereas the other transgenic line, containing a single copy, was expressed upon both maternal and paternal inheritance. This suggests that a 6.6-kb region of maternal-specific DNA methylation that we have identified may be sufficient to confer imprinted expression, but not in a copy-number independent manner. Finally, we produced five lines of transgenic mice using a 76-kb human SNRPN clone containing 45 kb and 7 kb of 5′ and 3′ flanking DNA, respectively. We found all the lines were expressed upon both maternal and paternal inheritance, regardless of copy number, suggesting that the imprinting machinery in mouse and human may have diverged. Received: 11 November 1998 / Accepted: 29 January 1999     

In vitro culture conditions favoring selection of chromosomal abnormalities in human ES cells

Previous studies in several laboratories have demonstrated inadvertent chromosomal abnormalities in long-term cultured human embryonic stem cells (HESC). Here, using a two-step selection process we report a functional adaptation of a HESC line, HS181, towards a decreased dependence of extra cellular matrix (ECM) for in vitro survival, that is for growth directly onto a plastic surface. Successful adaptation was paralleled with a karyotype change in 100% of the cells to 47,XX,del(7)(q11.2),+i(12)(p10). The resulting adapted population showed increased survival and growth on plastic and also maintained expression of HESC markers, but showed a decreased pluripotency, as demonstrated by results from embryoid body (EB) formation in vitro. The finding of reduced pluripotency may not be totally unexpected since the variant cells were selected for self-renewal and proliferation, not differentiation during the adaptation to growth on plastic. In the light of recent models of a germ cell origin of HESC it is of particular interest that similar to many of the reported spontaneous HESC mutants, one of the identified specific chromosome abnormalities, i(12p), has also been strongly implicated for human germ cell cancer. However, the mutated HESC variant carrying this mutation failed to grow as a xeno-graft in a mouse model in vivo. This is surprising and needs a further mechanistic analysis for its explanation. Increased knowledge of genetic integrity of HESC may have significance on the understanding of mechanisms for tumor progression and thus strategy for treatments, particularly for tumors occurring in early life.     

DNA methylation variability at growth-related imprints does not contribute to overweight in monozygotic twins discordant for BMI

Defective genomic imprinting is often associated with syndromes that include abnormal growth as a clinical phenotype. However, whether differential methylation at imprinted loci also contributes to nonsyndromic abnormal body weight regulation is yet unknown. In this study, we investigated a potential contribution of aberrant DNA methylation at nine differentially methylated regions (DMRs) to the development of nonsyndromic overweight. Sixteen monozygotic (MZ) twins discordant for BMI (BMI difference ranging from 2.9-9.5 kg/m(2)) were recruited from the East Flanders Prospective Twin Survey. DNA extracted from saliva samples was bisulfite-treated followed by PCR amplification of target regions in DMRs most representative for abnormal growth syndromes: KvDMR1, H19 CTCF4, H19 CTCF6, IGF2 DMR0, IGF2 DMR2, GRB10, MEST, SNRPN, GNAS XL-α-s and GNAS Exon1A. At the DMRs analyzed, methylation-dependent primer extension experiments revealed only small intrapair differences in methylation indexes (MI) between the heavy and lean co-twins. In addition, no significant correlations between intrapair BMI differences and intrapair differences in MI were observed. In conclusion, DNA methylation variability at the nine DMRs analyzed does not seem to contribute to the discordancy in BMI observed in these MZ twins.     

An enhancer element at the Igf2/H19 locus drives gene expression in both imprinted and non-imprinted tissues

The insulin-like growth factor 2 (Igf2) gene encodes a potent growth factor that is expressed in multiple tissues during embryonic development. Expression at this locus is mediated by genomic imprinting. In the developing endodermal tissues, imprinting of Igf2 is mediated by the interaction of a set of enhancers downstream of the linked H19 gene with a differentially methylated domain (DMD) that lies approximately 2-4 kb upstream of H19 that has a boundary or insulator function in the hypomethylated state. In the remainder of tissues that express Igf2 and H19, the cis elements that drive their correct expression and imprinting are not well understood. In addition, enhancers driving expression of Igf2 in the choroid plexus and leptomeninges, tissues where the gene is thought not to be imprinted, have not been isolated. Here we show that biallelic (non-imprinted) expression within the choroid plexus is restricted to the epithelium, and we provide evidence that a conserved intergenic region functions as an enhancer for Igf2 both in tissues where the gene is imprinted, and where Igf2 is biallelically expressed. The presence of an enhancer for imprinted tissues in the intergenic region argues for the existence of imprinting controls distinct from the DMD, which may be provided by differential methylation at sites proximal to Igf2.