Maintenance Of Imprinting Research Articles

Genetic imprinting and embryonic development

Erasure, establishment and maintenance of genetic imprinting are indispensable for normal embryonic development. All these processes depend on accurate expression and intimate cooperation of kinds of DNA methyltransferases. Many genetic syndromes and embryo developmental anomalies are caused by abnormality of genetic imprinting. Genetic imprinting is important for the nucleus totipotential of primordial germ cell, maturation of gamete,growth and development of embryo, structure and function of placenta as well as postnatal growth and development of individuals.

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.

A mechanism for the establishment and maintenance of methylation imprinting by the H19 ICR in human beta-globin YAC TgM

A mechanism for the establishment and maintenance of methylation imprinting by the H19 ICR in human beta-globin YAC TgM

Bovine SNRPN Methylation Imprint in Oocytes and Day 17 In Vitro-Produced and Somatic Cell Nuclear Transfer Embryos1

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.

Maintenance of DNA methylation during the Arabidopsis life cycle is essential for parental imprinting.

Imprinted genes are expressed predominantly from either their paternal or their maternal allele. To date, all imprinted genes identified in plants are expressed in the endosperm. In Arabidopsis thaliana, maternal imprinting has been clearly demonstrated for the Polycomb group gene MEDEA (MEA) and for FWA. Direct repeats upstream of FWA are subject to DNA methylation. However, it is still not clear to what extent similar cis-acting elements may be part of a conserved molecular mechanism controlling maternally imprinted genes. In this work, we show that the Polycomb group gene FERTILIZATION-INDEPENDENT SEED2 (FIS2) is imprinted. Maintenance of FIS2 imprinting depends on DNA methylation, whereas loss of DNA methylation does not affect MEA imprinting. DNA methylation targets a small region upstream of FIS2 distinct from the target of DNA methylation associated with FWA. We show that FWA and FIS2 imprinting requires the maintenance of DNA methylation throughout the plant life cycle, including male gametogenesis and endosperm development. Our data thus demonstrate that parental genomic imprinting in plants depends on diverse cis-elements and mechanisms dependent or independent of DNA methylation. We propose that imprinting has evolved under constraints linked to the evolution of plant reproduction and not by the selection of a specific molecular mechanism.

Limited evolutionary conservation of imprinting in the human placenta.

The epigenetic phenomenon of genomic imprinting provides an additional level of gene regulation that is confined to a limited number of genes, frequently, but not exclusively, important for embryonic development. The evolution and maintenance of imprinting has been linked to the balance between the allocation of maternal resources to the developing fetus and the mother's well being. Genes that are imprinted in both the embryo and extraembryonic tissues show extensive conservation between a mouse and a human. Here we examine the human orthologues of mouse genes imprinted only in the placenta, assaying allele-specific expression and epigenetic modifications. The genes from the KCNQ1 domain and the isolated human orthologues of the imprinted genes Gatm and Dcn all are expressed biallelically in the human, from first-trimester trophoblast through to term. This lack of imprinting is independent of promoter CpG methylation and correlates with the absence of the allelic histone modifications dimethylation of lysine-9 residue of H3 (H3K9me2) and trimethylation of lysine-27 residue of H3 (H3K27me3). These specific histone modifications are thought to contribute toward regulation of imprinting in the mouse. Genes from the IGF2R domain show polymorphic concordant expression in the placenta, with imprinting demonstrated in only a minority of samples. Together these findings have important implications for understanding the evolution of mammalian genomic imprinting. Because most human pregnancies are singletons, this absence of competition might explain the comparatively relaxed need in the human for placental-specific imprinting.

Establishment and maintenance of H19 imprinting in the germline and preimplantation embryo

The mouse H19 and Igf2 genes are oppositely imprinted and share enhancers that reside 3′ to the genes. The imprinted expression of these genes is coordinated by a 2-kb regulatory element, the differentially methylated domain (DMD), positioned between the two genes. The methylation status of this region determines the ability of the insulator factor CTCF to bind to its sites in the DMD. Deletions and mutations of the DMD that affect imprinting in the soma have little effect on the methylation pattern of H19 in the germline, suggesting that additional sequences and factors contribute to the earliest stages of imprinting regulation at this locus. Less is known about these initial steps, which include the marking of the parental alleles, the onset of allele-specific expression patterns and maintenance of the imprints in the preimplantation embryo. Here, we will focus on these early steps, summarizing what is known and what questions remain to be addressed.

Genomic imprinting in Dicer1-hypomorphic mice

To address the function of RNA interference (RNAi) in transcriptional silencing in mammals, we analyzed genomic imprinting in Dicer1-hypomorphic mice, in which Dicer1 expression was significantly reduced. We did not observe any abnormality in the allelic expression of imprinted genes in these mice or their offspring, suggesting that reduced expression of Dicer1 did not significantly affect the maintenance and reprogramming of imprinting.

DEMETER DNA Glycosylase Establishes MEDEA Polycomb Gene Self-Imprinting by Allele-Specific Demethylation

MEDEA (MEA) is an Arabidopsis Polycomb group gene that is imprinted in the endosperm. The maternal allele is expressed and the paternal allele is silent. MEA is controlled by DEMETER (DME), a DNA glycosylase required to activate MEA expression, and METHYLTRANSFERASE I (MET1), which maintains CG methylation at the MEA locus. Here we show that DME is responsible for endosperm maternal-allele-specific hypomethylation at the MEA gene. DME can excise 5-methylcytosine in vitro and when expressed in E. coli. Abasic sites opposite 5-methylcytosine inhibit DME activity and might prevent DME from generating double-stranded DNA breaks. Unexpectedly, paternal-allele silencing is not controlled by DNA methylation. Rather, Polycomb group proteins that are expressed from the maternal genome, including MEA, control paternal MEA silencing. Thus, DME establishes MEA imprinting by removing 5-methylcytosine to activate the maternal allele. MEA imprinting is subsequently maintained in the endosperm by maternal MEA silencing the paternal allele.

X‐linked imprinting: effects on brain and behaviour

Imprinted genes are monoallelically expressed in a parent-of-origin-dependent manner and can affect brain and behavioural phenotypes. The X chromosome is enriched for genes affecting neurodevelopment and is donated asymmetrically to male and female progeny. Hence, X-linked imprinted genes could potentially influence sexually dimorphic neurobiology. Consequently, investigations into such loci may provide new insights into the biological basis of behavioural differences between the sexes and into why men and women show different vulnerabilities to certain mental disorders. In this review, we summarise recent advances in our knowledge of X-linked imprinted genes and the brain substrates that they may act upon. In addition, we suggest strategies for identifying novel X-linked imprinted genes and their downstream effects and discuss evolutionary theories regarding the origin and maintenance of X-linked imprinting.

A human imprinting centre demonstrates conserved acquisition but diverged maintenance of imprinting in a mouse model for Angelman syndrome imprinting defects

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are caused by the loss of imprinted gene expression from chromosome 15q11-q13. Imprinted gene expression in the region is regulated by a bipartite imprinting centre (IC), comprising the PWS-IC and the AS-IC. The PWS-IC is a positive regulatory element required for bidirectional activation of a number of paternally expressed genes. The function of the AS-IC appears to be to suppress PWS-IC function on the maternal chromosome through a methylation imprint acquired during female gametogenesis. Here we have placed the entire mouse locus under the control of a human PWS-IC by targeted replacement of the mouse PWS-IC with the equivalent human region. Paternal inheritance of the human PWS-IC demonstrates for the first time that a positive regulatory element in the PWS-IC has diverged. These mice show postnatal lethality and growth deficiency, phenotypes not previously attributed directly to the affected genes. Following maternal inheritance, the human PWS-IC is able to acquire a methylation imprint in mouse oocytes, suggesting that acquisition of the methylation imprint is conserved. However, the imprint is lost in somatic cells, showing that maintenance has diverged. This maternal imprinting defect results in expression of maternal Ube3a-as and repression of Ube3a in cis, providing evidence that Ube3a is regulated by its antisense and creating the first reported mouse model for AS imprinting defects.

Loss of imprinting and promoter usage of the IGF2 in laryngeal squamous cell carcinoma

The gene for insulin-like growth factor two, IGF2 is maternally imprinted. Fifteen heterozygous samples were analyzed for the IGF2 imprinting status and promoter usage. IGF2 LOI was detected in four non-tumorous tissues and in six laryngeal squamous cell carcinoma (LSCC) tumors. There was no clear pattern of specific promoter activity in LSCC tumors and the adjacent normal tissues. P1 promoter usage was active in eight LSCCs, among them four with LOI. As it was activated in four tumors with maintenance of imprinting (MOI) and four non-tumors, we concluded that P1 promoter is not exclusively connected with IGF2 LOI in LSCC.

Beckwith–Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is a clinically heterogeneous overgrowth syndrome associated with an increased risk for embryonal tumor development. BWS provides an ideal model system to study epigenetic mechanisms. This condition is caused by a variety of genetic or epigenetic alterations within two domains of imprinted growth regulatory genes on human chromosome 11p15. Molecular studies of BWS have provided important data with respect to epigenotype/genotype-phenotype correlations; for example, alterations of Domain 1 are associated with the highest risk for tumor development, specifically Wilms' tumor. Further, the elucidation of the molecular basis for monozygotic twinning in BWS defined a critical period for imprint maintenance during pre-implantation embryonic development. In the future, such molecular studies in BWS will permit enhanced medical management and targeted genetic counseling.

Narrowed abrogation of the Angelman syndrome critical interval on human chromosome 15 does not interfere with epigenotype maintenance in somatic cells

Human chromosome 15q11-q13 involves a striking imprinted gene cluster of more than 2 Mb that is concomitant with multiple neurological disorders manifested by Prader-Willi syndrome (PWS) and Angelman syndrome (AS). PWS and AS patients with imprinting mutation have microdeletions, which share a 4.3 kb short region of overlap (SRO) at the 5' end of the paternal SNURF-SNRPN gene in PWS, or on the maternal allele, which shares a 880 bp SRO located at the 35 kb upstream of the SNURF-SNRPN promoter in AS. Recent studies have revealed an essential role of PWS-SRO in the postzygotic maintenance of the appropriate epigenotype on the paternal chromosome. For AS-SRO, however, there is insufficient experimental evidence exists to determine the direct functions. Here we show that the complete deletion of AS-SRO does not cause any anomalies of imprinted gene expression or DNA methylation on the mutated human chromosome 15, further supporting the idea that AS-SRO is dispensable for post implantation imprint maintenance. This implies that AS-SRO is not essential for the robust epigenotype preservation in somatic cells.

Lsh controls silencing of the imprinted Cdkn1c gene

Epigenetic regulation, such as DNA methylation plays an important role in the control of imprinting. Lsh, a member of the SNF2 family of chromatin remodeling proteins, controls DNA methylation in mice. To investigate whether Lsh affects imprinting, we examined CpG methylation and allelic expression of individual genes in Lsh-deficient embryos. We report here that loss of Lsh specifically alters expression of the Cdkn1c gene (also known as p57(Kip2)) but does not interfere with maintenance of imprints at the H19, Igf2, Igf2r, Zac1 and Meg9 genes. The reactivation of the silenced paternal Cdkn1c allele correlates closely with a loss of CpG methylation at the 5' DMR at the Cdkn1c promoter, whereas KvDMR1 and DMRs of other imprinted genes were not significantly changed. Chromatin immunoprecipitations demonstrate a direct association of Lsh with the 5' DMR at the Cdkn1c promoter, but not with Kv DMR1 or other imprinted loci. These data suggest that methylation of the 5' DMR plays an important role in the imprinting of the Cdkn1c gene. Furthermore, it suggests that Lsh is not required for maintenance of imprinting marks in general, but is only crucial for imprinting at distinct genomic sites.

Paternal imprints can be established on the maternal Igf2-H19 locus without altering replication timing of DNA.

Genomic imprinting in mammals marks the parental alleles in gametes, resulting in differential gene expression in offspring. A number of epigenetic features are associated with imprinted genes. These include differential DNA methylation, histone acetylation and methylation, subnuclear localization and DNA replication timing. While DNA methylation has been shown to be necessary both for establishment and maintenance of imprinting, the connections with the other types of epigenetic marking systems are not clear. Specifically, it is not known whether the other marking systems, either on their own or in conjunction with DNA methylation, are required for imprinting. Here we show that in the mouse mutant Minute (Mnt) the Igf2-H19 locus acquires a paternal methylation imprint in the maternal germline. DNA methylation of the H19 DMR is established in oogenesis, maintained during postzygotic development on the maternal allele, and erased in primordial germ cells. The fact that a paternal type methylation imprint can also be established in the maternal germline indicates that trans-acting factors that target methylation to this imprinted region are likely to be the same in both germlines. Surprisingly, however, asynchrony of DNA replication of the locus is maintained despite the altered expression and methylation imprint of Igf2 and H19. These results show clearly that replication asynchrony of this region is neither the determinant factor for, nor a consequence of, epigenetic modifications that are critical for genomic imprinting. Replication asynchrony may thus be regulated differently from methylation imprints and have a separate function.

Alterations in Promoter Usage and Expression Levels of Insulin-like Growth Factor-II and H19 Genes in Cervical and Endometrial Cancer.

The biallelic expression of insulin-like growth factor-II (IGF2) and H19 has been reported to be associated with the progression of several tumors. Here, the promoter usage and expression levels of IGF2 and H19 are reported to be altered in cervical and endometrial cancers showing loss of imprinting (LOI). The imprinting status of IGF2 and H19 was examined in 32 cervical carcinomas, their matched normal tissues, 13 endometrial cancer and 33 normal endometrial tissues. The LOI of IGF2 was observed in 7 of 18 (39%) and 1 of 13 (8.3%) informative cervical carcinomas and informative endometrial cancers, respectively. The LOI of the H19 gene was detected in 5 of 14 (36%) and in all 11 (100%) informative cervical carcinoma cases and informative endometrial cancer cases, respectively. The use of promoter P1 was observed in the LOI tumors of IGF2, but not in the tumors showing maintenance of IGF2 imprinting (MOI), or in cervical and endometrial cancers. Unlike MOI tumors, some LOI tumors revealed a lack of IGF2 transcription from the promoter P3. The LOI tumors of IGF2 showed increased expression of the IGF2 level, but a down-regulation of the H19, relative to normal tissues, whereas the MOI tumors revealed no significant alterations. These results suggest that the promoter P1 could be involved in the biallelic expression of IGF2, and that the altered expression of the IGF2 and H19 levels might be associated with the progression of cervical and endometrial cancers that exhibit biallelic IGF2 expression.

Loss of imprinting of IGF2 and H19 in osteosarcoma is accompanied by reciprocal methylation changes of a CTCF-binding site.

The adjacent insulin-like growth factor 2 (IGF2) and H19 genes are imprinted in most normal human tissues, but imprinting is often lost in tumors. The mechanisms involved in maintenance of imprinting (MOI) and loss of imprinting (LOI) are unresolved. We show here that osteosarcoma (OS) tumors with IGF2/H19 MOI exhibit allele-specific differential methylation of a CTCF-binding site upstream of H19. LOI of IGF2 or H19 in OS occurs in a mutually exclusive manner, and occurs with monoallelic expression of the other gene. Bisulfite sequencing reveals IGF2 LOI occurs with biallelic CpG methylation of the CTCF-binding site, while H19 LOI occurs with biallelic hypomethylation of this site. Our data demonstrate that IGF2 LOI and H19 LOI are accompanied by reciprocal methylation changes at a critical CTCF-binding site. We propose a model in which incomplete gain or loss of methylation at this CTCF-binding site during tumorigenesis explains the complex and often conflicting expression patterns of IGF2 and H19 in tumors.

Discordant KCNQ1OT1 imprinting in sets of monozygotic twins discordant for Beckwith-Wiedemann syndrome.

Beckwith-Wiedemann syndrome (BWS) presents with visceromegaly, macroglossia, tumor predisposition and other congenital abnormalities, and is usually associated with abnormalities of chromosome 11p15. A number of identical twin pairs, mostly female, have been reported to be discordant for BWS. We show here that the incidence of female monozygotic twins among patients with BWS is dramatically increased over that of the general population. A cluster of imprinted genes within 11p15 is thought to be coordinately regulated via the imprinted expression of KCNQ1OT1, which encodes an untranslated RNA. In skin fibroblasts from five monozygotic twin pairs discordant for BWS, each affected twin had an imprinting defect at KCNQ1OT1 on 11p15, whereas the unaffected twin did not. Five additional monozygotic twin pairs, for whom only blood was available, also displayed an imprinting defect at KCNQ1OT1. It is possible that discordance for BWS in MZ twins is due to unequal splitting of the inner cell mass during twinning, thereby causing differential maintenance of imprinting at KCNQ1OT1. Alternatively, we propose that KCNQ1OT1 is especially vulnerable to a loss of imprinting event, caused by a lack of maintenance DNA methylation at a critical stage of preimplantation development, and that this loss of imprinting predisposes to twinning as well as to discordance for BWS. These data underscore the importance of continued surveillance of children born following assisted reproductive technologies that impact the preimplantation embryo.

Alterations in promoter usage and expression levels of insulin-like growth factor-II and H19 genes in cervical carcinoma exhibiting biallelic expression of IGF-II

Biallelic expression of insulin-like growth factor-II (IGF2) has been reported to be associated with progression of several tumors. Here, we report that the promoter usage and expression levels of IGF2 and H19 were altered in cervical carcinoma showing loss of imprinting. The imprinting status was examined in 32 cervical carcinomas and their matched normal tissues. Loss of imprinting (LOI) of IGF2 was observed in seven of 18 (39%) informative cases. LOI of H19 gene was detected in five of 14 informative cases. The usage of promoter P1 was observed in LOI tumors of IGF2, but not in the tumors showing maintenance of IGF2 imprinting (MOI) and in the normal cervical tissues. Unlike MOI tumors, some LOI tumors revealed the lack of IGF2 transcription from promoter P3. LOI tumors of IGF2 showed an increased expression level of IGF2 but a down-regulation of H19 relative to the normal tissues whereas MOI tumors did not reveal significant alterations. These results suggest that promoter P1 could be involved in the biallelic expression of IGF2 and that the altered expression levels of IGF2 and H19 gene might be associated with the progression of cervical carcinoma showing biallelic expression of IGF2.