Asynchronous replication timing of imprinted loci is independent of DNA methylation,but consistent with differential subnuclear localization

Genomic imprinting in mammals marks the two parental alleles resulting in differential gene expression. Imprinted loci are characterized by distinct epigenetic modifications such as differential DNA methylation and asynchronous replication timing. To determine the role of DNA methylation in replication timing of imprinted loci, we analyzed replication timing in Dnmt1- and Dnmt3L-deficient embryonic stem (ES) cells, which lack differential DNA methylation and imprinted gene expression. Asynchronous replication is maintained in these ES cells, indicating that asynchronous replication is parent-specific without the requirement for differential DNA methylation. Imprinting centers are required for regional control of imprinted gene expression. Analysis of replication fork movement and three-dimensional RNA and DNA fluorescent in situ hybridization (FISH) analysis of the Igf2-H19 locus in various cell types indicate that the Igf2-H19 imprinting center differentially regulates replication timing of nearby replicons and subnuclear localization. Based on these observations, we suggest a model in which cis elements containing nonmethylation imprints are responsible for the movement of parental imprinted loci to distinct nuclear compartments with different replication characteristics resulting in asynchronous replication timing.     

Epigenetic analysis of the Dlk1-Gtl2 imprinted domain on mouse chromosome 12: implications for imprinting control from comparison with Igf2-H19.

Dlk1 and Gtl2 are reciprocally imprinted genes located 80 kb apart on mouse chromosome 12. Similarities between this domain and that of the well characterized Igf2-H19 locus have been previously noted. Comparative genomic and epigenetic analysis of these two domains might help identify allele-specific epigenetic regulatory elements and common features involved in aspects of imprinting control. Here we describe a detailed methylation analysis of the Dlk1-Gtl2 domain on both parental alleles in the mouse. Like the Igf2-H19 domain, areas of differential methylation are hypermethylated on the paternal allele and hypomethylated on the maternal allele. Three differentially methylated regions (DMRs), each with different epigenetic characteristics, have been identified. One DMR is intergenic, contains tandem repeats and is the only region that inherits a paternal methylation mark from the germline. An intronic DMR contains a conserved putative CTCF-binding domain. All three DMRs have both unique and common features compared to those identified in the Igf2-H19 domain.     

A human H19 transgene exhibits impaired paternal-specific imprint acquisition and maintenance in mice

Genomic imprinting, the differential expression of autosomal genes based on their parent of origin, is observed in all eutherian mammals that have been examined. In most instances the genes that are imprinted in one species are imprinted in others as well, suggesting that imprinting predated eutherian radiation. For example, the RNA-coding H19 gene is repressed upon paternal inheritance in all species examined to date. Thus, it is surprising that there is remarkably little sequence conservation among the cis-acting DNA regulatory elements that are required for imprinting of H19 and the tightly linked Igf2 gene. The most conserved characteristic in the imprinting control region (ICR) is the presence of multiple binding sites for the zinc finger protein CTCF, raising the possibility that CTCF binding might be sufficient for the reciprocal imprinting of H19 and Igf2. To investigate whether a human H19 transgene, harboring seven CTCF sites, is correctly recognized and imprinted in the mouse, a 100 kb transgene containing the human H19 gene was introduced into the mouse germline. The human transgene was specifically methylated after passage through the male germline in a copy number-dependent manner, but the methylation was unstable, undergoing progressive loss during development. Consequently, the transgene was highly expressed upon both maternal and paternal inheritance. These results argue that the signals for both the acquisition and maintenance of methylation imprinting are diverging rapidly.     

The H19 methylation imprint is erased and re-established differentially on the parental alleles during male germ cell development

Differences in DNA methylation distinguish the maternal and paternal alleles of many imprinted genes. Allele-specific methylation that is inherited from the gametes and maintained throughout development has been proposed as a candidate imprinting mark. To determine how methylation is established in the germline, we have analyzed the allelic methylation patterns of the maternally expressed, paternally methylated H19 gene during gametogenesis in the mouse embryo. We show here that both parental alleles are devoid of methylation in male and female mid-gestation embryonic germ cells, suggesting that methylation imprints are erased in the germ cells prior to this time. In addition, we demonstrate that the subsequent hypermethylation of the paternal and maternal alleles in the male germline occurs at different times. Although the paternal allele becomes hypermethylated during fetal stages, methylation of the maternal allele begins during perinatal stages and continues postnatally through the onset of meiosis. The differential acquisition of methylation on the parental H19 alleles during gametogenesis implies that the two unmethylated alleles can still be distinguished from each other. Thus, in the absence of DNA methylation, other epigenetic mechanism(s) appear to maintain parental identity at the H19 locus during male germ cell development.     

The paternal methylation imprint of the mouse H19 locus is acquired in the gonocyte stage during foetal testis development

BACKGROUND: Germline-specific differential DNA methylation that persists through fertilization and embryonic development is thought to be the 'imprint' distinguishing the parental alleles of imprinted genes. If such methylation is to work as the imprinting mechanism, however, it has to be reprogrammed following each passage through the germline. Previous studies on maternally methylated genes have shown that their methylation imprints are first erased in primordial germ cells (PGCs) and then re-established during oocyte growth. RESULTS: We have examined the timing of the reprogramming of the paternal methylation imprint of the mouse H19 gene during germ cell development. In both male and female PGCs, the paternal allele is partially methylated whereas the maternal allele is unmethylated. This partial methylation is completely erased in the female germline by entry into meiosis, establishing the oocyte methylation pattern. In the male germline, both alleles become methylated, mainly during the gonocyte stage, establishing the sperm methylation pattern. CONCLUSION: The paternal methylation imprint of H19 is established in the male germline and erased in the female germline at specific developmental stages. The identification of the timings of the methylation and demethylation should help to identify and characterize the biochemical basis of the reprogramming of imprinting.     

IVF results in de novo DNA methylation and histone methylation at an Igf2-H19 imprinting epigenetic switch

Recent studies suggest that IVF and assisted reproduction technologies (ART) may result in abnormal genomic imprinting, leading to an increased frequency of Angelman syndrome (AS) and Beckwith-Weidemann syndrome (BWS) in IVF children. To learn how ART might alter the epigenome, we examined morulas and blastocysts derived from C57BL/6J X M. spretus F1 mice conceived in vivo and in vitro and determined the allelic expression of four imprinted genes: Igf2, H19, Cdkn1c and Slc221L. IVF-derived mouse embryos that were cultured in human tubal fluid (HTF) (Quinn's advantage) media displayed a high frequency of aberrant H19 imprinting, whereas in vivo and IVF embryos showed normal maternal expression of Cdkn1c and normal biallelic expression of Igf2 and Slc221L. Embryonic stem (ES) cells derived from IVF blastocysts also showed abnormal Igf2/H19 imprinting. Allele-specific bisulphite PCR reveals abnormal DNA methylation at a CCCTC-binding factor (CTCF) site in the imprinting control region (ICR), as the normally unmethylated maternal allele acquired a paternal methylation pattern. Chromatin immunoprecipitation (ChIP) assays indicate an increase of lysine 4 methylation (dimethyl Lys4-H3) on the paternal chromatin and a gain in lysine 9 methylation (trimethyl Lys9-H3) on the maternal chromatin at the same CTCF-binding site. Our results indicate that de novo DNA methylation on the maternal allele and allele-specific acquisition of histone methylation lead to aberrant Igf2/H19 imprinting in IVF-derived ES cells. We suggest that ART, which includes IVF and various culture media, might cause imprinting errors that involve both aberrant DNA methylation and histone methylation at an epigenetic switch of the Igf2-H19 gene region.     

Chromatin conformation of the H19 epigenetic mark

Genomic imprinting in mammals is an epigenetic process that results in differential expression of the two parental alleles. The tightly linked murine H19 and Igf2 genes are reciprocally imprinted: H19 is expressed from the maternal chromosome while Igf2 is expressed from the paternal chromosome. A single regulatory region in the 5' flank of the H19 gene has been implicated in silencing both genes. On the paternal chromosome, this region is heavily methylated at CpG residues, leading to repression of the H19 gene. The mechanism by which the same region in an unmethylated state on the maternal chromosome silences Igf2 is less well understood. We have probed the chromatin structure of the region by assessing its sensitivity to nuclease digestion. Two regions of nuclease hypersensitivity that are specific to the maternal chromosome were identified. These coincide with the region that is most heavily methylated on the paternal chromosome. As is the case with paternal methylation, hypersensitivity is present in all tissues surveyed, irrespective of H19 expression. We suggest that the chromatin structure of the maternal 5' flank of the H19 gene may represent an epigenetic mark involved in the silencing of Igf2.      

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.     

Deletion of the H19 differentially methylated domain results in loss of imprinted expression of H19 and Igf2

Differentially methylated sequences associated with imprinted genes are proposed to control genomic imprinting. A 2-kb region located 5′ to the imprinted mouse H19 gene is hypermethylated on the inactive paternal allele throughout development. To determine whether this differentially methylated domain (DMD) is required for imprinted expression at the endogenous locus, we have generated mice harboring a 1.6-kb targeted deletion of the DMD and assayed for allelic expression of H19 and the linked, oppositely imprinted Igf2 gene. H19 is activated and Igf2 expression is reduced when the DMD deletion is paternally inherited; conversely, upon maternal transmission of the mutation, H19 expression is reduced and Igf2 is activated. Consistent with the DMD’s hypothesized role of setting up the methylation imprint, the mutation also perturbs allele-specific methylation of the remaining H19 sequences. In conclusion, these experiments show that the H19 hypermethylated 5′ flanking sequences are required to silence paternally derived H19. Additionally, these experiments demonstrate a novel role for the DMD on the maternal chromosome where it is required for the maximal expression of H19 and the silencing of Igf2. Thus, the H19 differentially methylated sequences are required for both H19 and Igf2 imprinting.     

DNA methylation patterns in human tissues of uniparental origin using a zinc-finger gene (ZNF127) from the Angelman/Prader-Willi region

In order to further our understanding of the epigenetic modifications of DNA and its role in imprinting, we examined DNA methylation patterns of human tissues of uniparental origin. We used complete hydatidiform moles (CHM), which are totally androgenetic conceptions, to examine the paternal methylation pattern in the absence of a maternal contribution and we used ovarian teratomas to represent the maternal counterpart. We carried out an analysis of DNA methylation of a gene which has been shown to contain sites which are differentially methylated in a parent-specific fashion. The gene, ZNF127, is located on chromosome 15q11-q13 in the region associated with Prader-Willi and Angelman syndromes. The parent-of-origin DNA methylation has been postulated to reflect the presence of an imprint and recent studies have confirmed that ZNF127 is differentially expressed only from the paternal chromosome. We identified a unique pattern of hyper- and hypomethylated sites in androgenetic conceptions which was nearly identical to the paternal pattern found in sperm. This may represent the paternal germ-line methylation imprint. We also studied partial hydatidiform moles, non-molar triploid conceptions, normal chorionic villi, and somatic tissue. These all demonstrated a modified DNA methylation pattern characteristic of normal chorionic villi with only limited findings of the imprint. Our results suggest that human androgenetic conceptions may provide an excellent model to analyze epigenetic DNA modifications, such as methylation, in imprinted genes. The paternal allele-specific methylation imprint will also be useful clinically to confirm the androgenetic nature of suspected molar conceptions in which parental blood samples may not be available. © 1996 Wiley-Liss, Inc.     

Genomic imprinting and its relevance to congenital disease, infertility, molar pregnancy and induced pluripotent stem cell

Genomic imprinting is an epigenetic gene-marking phenomenon that occurs in the germline, whereby genes are expressed from only one of the two parental copies in embryos and adults. Imprinting is essential for normal mammalian development and its disruption can cause various developmental defects and diseases. The process of imprinting in the germline involves DNA methylation of the imprint control regions (ICRs), and resulting parental-specific methylation imprints are maintained in the zygote and act as the marks controlling imprinted gene expression. Recent studies in mice have revealed new factors involved in imprint establishment during gametogenesis and maintenance during early development. Clinical studies have identified cases of imprinting disorders where involvement of factors shared by multiple ICRs for establishment or maintenance is suspected. These include Beckwith-Wiedemann syndrome, transient neonatal diabetes, Silver-Russell syndrome and others. More severe disruptions can lead to recurrent molar pregnancy, miscarriage or infertility. Imprinting defects may also occur during assisted reproductive technology or cell reprogramming. In this review, we summarize our current knowledge on the mechanisms of imprint establishment and maintenance, and discuss the relationship with various human disorders.     

Developmentally regulated functions of the H19 differentially methylated domain

Igf2 and H19 are physically linked imprinted genes. In embryonic liver, their reciprocal expression (paternal for Igf2 and maternal for H19) is controlled by a paternally methylated region (H19 DMD) located 5' of H19. This region contains a methylation-sensitive insulator that prevents the Igf2 promoters being activated by downstream enhancers on the maternal chromosome. In adult liver, Igf2 is normally not expressed but is reactivated upon tumour formation. By analysing three deletions of the H19 locus, we investigated the mechanism regulating the imprinted expression of the Igf2 gene in the course of liver tumourigenesis. We observed that the role of the H19 DMD in the control of Igf2 expression changes during tumourigenesis. The H19 DMD is required on the paternal chromosome for Igf2 activation in the early stages while its maternal allele is necessary for maintaining Igf2 imprinting only in the late stages. A positive regulatory function of the paternal H19 DMD is also evident in normal neonatal liver, but its relevance for Igf2 expression becomes higher in the second post-natal week. Our results support a model in which both methylated and non-methylated parental copies of the H19 DMD have active roles in the regulation of Igf2 expression in the liver and these activities are under developmental control.     

Aberrant DNA methylation of imprinted loci in sperm from oligospermic patients

Recent studies suggest that assisted reproductive technologies (ART), which involve the isolation, handling and culture of gametes and early embryos, are associated with an increased incidence of rare imprinting disorders. Major epigenetic events take place during this time and the process of ART may expose the epigenome to external influences, preventing the proper establishment and maintenance of genomic imprints. However, the risks of ART cannot be simply evaluated because the patients who receive ART may differ both demographically and genetically from the general population at reproductive age. In this study, we examined the DNA methylation status of seven imprinted genes using a combined bisulphite-PCR restriction analysis and sequencing technique on sperm DNA obtained from 97 infertile men. We found an abnormal paternal methylation imprint in 14 patients (14.4%) and abnormal maternal imprint in 20 patients (20.6%). The majority of these doubly defective samples were in men with moderate or severe oligospermia. These abnormalities were specific to imprinted loci as we found that global DNA methylation was normal in these samples. The outcome of ART with sperm shown to have an abnormal DNA methylation pattern was generally poor. However, one sample of sperm with both paternal and maternal methylation errors used in ICSI produced a child of normal appearance without any abnormalities in their imprinted methylation pattern. Our data suggest that sperm from infertile patients, especially those with oligospermia, may carry a higher risk of transmitting incorrect primary imprints to their offspring, highlighting the need for more research into ART.     

Imprinting mutations in the Beckwith--Wiedemann syndrome suggested by an altered imprinting pattern in the IGF2-H19 domain

Regional regulation of parental imprinting in the IGF2–H19domain of imprinted genes was studied in the Beckwith—Wiedemannsyndrome (BWS). We identified BWS patients who had inheriteda normal biparental chromosome complement of the chromosome11p15.5 region (where IGF2 and H19 reside), but had an alteredpattern of allelic methylation of both genes, with the maternalchromosome carrying a paternal imprinting pattern. In fibroblasts,IGF2 was expressed from both parental alleles and H19 was notexpressed, precisely as predicted from the altered pattern ofallelic methylation. Interestingly, DNA replication patternsin the 11p15.5 region remained asynchronous as in controls.Our results therefore provide the first example of a dissociationof regional control of DNA replication from regional controlof allelic methylation and expression in imprinting. We suggestthat the altered pattern of allelic methylation and expressionarises in the germline or in the early embryo from defects inresetting or setting of imprinting in the maternal germline.Potential candidate regions for mutations include the previouslyidentified translocation breakpoint clusters and the H19 geneitself. The finding of possible ‘imprinting mutations’in BWS raises the prospect of identifying genetic factors thatcontrol imprinting in this region.     

Loss of imprinting and cancer

Imprinting is defined as the parental allele-specific expression of a very limited set of genes (about 50-80). This regulation depends upon an epigenetic marking of parental alleles during gametogenesis. Monoallelic expression ensures that the levels of the proteins encoded by imprinted genes, important factors of embryonic growth, placental growth or adult metabolism, are assured. Without precise control of their expression, developmental abnormalities result, as is shown by a number of hereditary over-growth syndromes, including Beckwith-Wiedemann syndrome. The regulation of imprinted genes is largely dependent on methylation marks, which are laid down during embryological development of germ cells. Once in place, the methylation status of precise chromosomal regions, Imprinting Control Regions (ICRs), is read by either of two mechanisms, chromatin barrier formation or untranslated RNAs, thereby ensuring that only the maternal or paternal allele is expressed. Each imprinted gene is classified as maternal or paternal according to the expressed allele. The stability of the marked regions in somatic cells is maintained through each cellular replication by a methylation enzyme complex containing Dnmt1. Although the major reading mechanisms of imprinted status are known, chromatin boundary formation by CTCF and untranslated RNAs, the molecules elaborating the initial ICR methylation, are just being uncovered. Mis-regulation of imprinted gene expression (loss of imprinting [LOI]) is seen frequently and precociously in a large variety of human tumours, making LOI a potentially valuable tool for both diagnosis and treatment. In fact, LOI is presently considered the most abundant and most precocious alteration in cancer. The present review proposes a mechanism responsible for LOI, as well as its eventual value in tumour diagnosis and prognosis.     

Hematopoietic reconstitution with androgenetic and gynogenetic stem cells

Parthenogenetic embryonic stem (ES) cells with two oocyte-derived genomes (uniparental) have been proposed as a source of autologous tissue for transplantation. The therapeutic applicability of any uniparental cell type is uncertain due to the consequences of genomic imprinting that in mammalian uniparental tissues causes unbalanced expression of imprinted genes. We transplanted uniparental fetal liver cells into lethally irradiated adult mice to test their capacity to replace adult hematopoietic tissue. Both maternal (gynogenetic) and paternal (androgenetic) derived cells conveyed long-term, multilineage reconstitution of hematopoiesis in recipients, with no associated pathologies. We also establish that uniparental ES cells can differentiate into transplantable hematopoietic progenitors in vitro that contribute to long-term hematopoiesis in recipients. Hematopoietic tissue in recipients maintained fidelity of parent-of-origin methylation marks at the Igf2/H19 locus; however, variability occurred in the maintenance of parental-specific methylation marks at other loci. In summary, despite genomic imprinting and its consequences on development that are particularly evident in the androgenetic phenotype, uniparental cells of both parental origins can form adult-transplantable stem cells and can repopulate an adult organ.     

Genomic imprinting: potential function and mechanisms revealed by the Prader-Willi and Angelman syndromes

The Prader-Willi (PWS) and Angelman (AS) syndromes are two clinically distinct syndromes which result from lack of expression of imprinted genes within chromosome 15q11-q13. These two syndromes result from 15q11-q13 deletions, chromosome 15 uniparental disomy (UPD), imprinting centre mutations and, for AS, probable mutations in a single gene. The differential phenotype results from a paternal genetic deficiency in PWS patients and a maternal genetic deficiency in AS patients. Within 15q11-q13, four genes (SNRPN, IPW, ZNF127, FNZ127) and two expressed sequence tags (PAR1 and PAR5) have been found to be expressed only from the paternally inherited chromosome, and therefore all must be considered candidate genes involved in the pathogenesis of PWS. A candidate AS gene (UBE3A) has very recently been identified. The mechanisms of imprinted gene expression are not yet understood, but it is clear that DNA methylation is involved in both somatic cell expression and inheritance of the imprint. The presence of DNA methylation imprints that distinguish the paternally and maternally inherited alleles is a common characteristic of all known imprinted genes which have been studied extensively, including SNRPN and ZNF127. Recently, several PWS and AS patients have been found that have microdeletions in a region upstream of the SNRPN gene referred to as the imprinting centre, or IC. Paternal IC deletions in PWS patients and maternal IC deletions in AS patients result in uniparental DNA methylation and uniparental gene expression at biparentally inherited loci. The IC is a novel genetic element which controls initial resetting of the parental imprint in the germline for all imprinted gene expression over a 1.5-2.5 Mb region within chromosome 15q11-q13.      

Biparental hydatidiform moles: a maternal effect mutation affecting imprinting in the offspring

Highly recurrent hydatidiform moles (HMs) studied to date are not androgenetic but have biparental genomic contribution (BiHM). Affected women have an autosomal recessive mutation that causes their pregnancies to develop into HM. Although there is genetic heterogeneity, a major locus maps to chromosome 19q13.42, but a mutated gene has not yet been identified. Molecular studies have shown that maternal imprinting marks are deregulated in the BiHM trophoblast. The mutations that cause this condition are, therefore, hypothesized to occur in genes that encode transacting factors required for the establishment of imprinting marks in the maternal germline or for their maintenance in the embryo. Although only DNA methylation marks at imprinted loci have been studied in the BiHM, the mutation may affect genes that are essential for other forms of chromatin remodelling at imprinted loci and necessary for correct maternal allele-specific DNA methylation and imprinted gene expression. Normal pregnancies interspersed with BiHM have been reported in some of the pedigrees, but affected women repeatedly attempting pregnancy should be counselled about the risk for invasive trophoblastic disease with each subsequent BiHM.