Conservation of a maternal-specific methylation signal at the human IGF2R locus

The human 1GF2R gene has been reported to be either blallelicallyor very rarely monoallelically expressed, in contrast to thematernally expressed mouse counterpart. We describe here ananalysis of the 5' portion of the human IGF2R gene and showthat it contains a maternally methylated CpG island in the secondintron. A similar maternally methylated intronic element hasbeen proposed to be the imprinting box for the mouse gene andalthough the relevance of this element has yet to be directlydemonstrated, methylation has been reported to be essentialto maintain allele-specific expression of imprinted genes. Allelicexpression analysis of human IGF2R in 70 lymphoblastoid celllines identified only one iine showing monoallelic expression.Thus, in this tissue monoparental methylatlon of the IGF2R genedoes not correlate with allele-specific expression. We alsoconfirm here that the human IGF2R gene is located in an asynchronouslyreplicating chromosomal region, as are all other imprinted genesso far analyzed. The mouse and human IGF2R intronic CpG islandsboth contain numerous large direct repeats that are methylatedfollowing maternal, but not paternal, transmittance. Thus featuresthat attract maternal-specific methylatlon are conserved betweenthe mouse and human genes. Since these intronic CpG islandsshare organizational rather than sequence homology, this suggeststhat secondary DNA structure may play a role in attracting amaternal methylation imprint.     

Oocyte growth-dependent progression of maternal imprinting in mice

In mammals, some genes categorized as imprinted genes are exclusively expressed either from maternal or paternal allele. This parental-origin-specific gene expression is regulated by epigenetic modification of DNA methylation in differentially methylated region (DMR), which is independently imposed during oogenesis and spermatogenesis. It is known that methylation of DMR in the female germ line is established during oocyte growth phase. However, the cause of the progression of methylation on DMR, due to either aging of mice or growth-size of oocyte was unclear up to now. Here, we analyzed the methylation of DMR for each eight imprinted genes (Igf2r, Lit1, Zac1, Snrpn, Peg1/Mest, Impact, Meg1/Grb10, and H19) by bisulfite sequencing methylation assay, using oocytes from 10 dpp (days post partum), 15 dpp, 20 dpp, and adult mice. To find whether the size of oocytes is the cause of methylation, above oocytes were classified into seven groups (each oocyte diameter ranging from 40 to 75 microm with intervals of 5 microm). The results from juvenile mice oocytes showed that DMR methylation progressed according to oocyte growth each imprinted gene. More than 85% of DMR methylation was achieved for both Igf2r, Zac1 & Lit1 with oocyte size of reaching 55 microm and Snrpn, Peg1/Mest, Impact, and Meg1/Grb10 with oocyte size of reaching 60 microm. Preferential methylation of maternal allele was observed in Zac1 and Peg1/Mest of juvenile oocytes and in Snrpn of juvenile and adult oocytes. The oocyte size-dependent-methylation progressed equally for all three different-age juvenile mice. The size-dependent-methylation was also recognized in the growing oocytes collected from adult mice, although the progress is slightly slower than that of juvenile mice. From these results, we concluded that DNA methylation is established with oocyte size dependent manner, not with aging of mice.     

Maternal repression of the human GRB10 gene in the developing central nervous system; evaluation of the role for GRB10 in Silver-Russell syndrome

The GRB10 gene encodes a growth suppressor and maps to human chromosome 7p11.2-p13. Maternal duplication (matdup) of this region has recently been associated with Silver-Russell syndrome (SRS), which is characterised by pre- and postnatal growth restriction, craniofacial dysmorphism and lateral asymmetry. Maternal uniparental disomy for chromosome 7 (mUPD7) occurs in approximately 7% of SRS patients. Exposure of a recessive allele due to isodisomy has been ruled out in five mUPD7 cases, suggesting genomic imprinting as the basis for disease. Assuming SRS patients with matdup of 7p11.2-p13 and mUPD7 share a common aetiology, this would implicate a maternally expressed gene from this interval, which is involved in growth inhibition. Murine Grb10 was identified as a maternally expressed gene by subtractive hybridisation using normal and androgenetic mouse embryos. Grb10 maps to the homologous region of proximal mouse chromosome 11, for which mUPD incurs reduced birthweight. A role for GRB10 in SRS was evaluated by determining its imprinting status in multiple human foetal tissues using expressed polymorphisms, and by screening the coding region for mutations in 18 classic non-mUPD7 SRS patients. Maternal repression of GRB10 was observed specifically in the developing central nervous system including brain and spinal cord, with biallelic expression in peripheral tissues. This is in contrast to mouse Grb10, and represents the first example of opposite imprinting in human and mouse homologues. While a role for GRB10 in mUPD7 SRS cases can not be ruled out on the basis of imprinting status, no mutations were identified in the patients screened.     

Comparative genome analysis of the mouse imprinted gene impact and its nonimprinted human homolog IMPACT: toward the structural basis for species-specific imprinting

Mouse Impact is a paternally expressed gene encoding an evolutionarily conserved protein of unknown function. Here we identified IMPACT, the human homolog of Impact, on chromosome 18q11. 2-12.1, a region syntenic to the mouse Impact locus. IMPACT was expressed biallelically in brain and in various tissues from two informative fetuses and in peripheral blood from an informative adult. To reveal the structural basis for the difference in allelic expression between the two species, we elucidated complete genome sequences for both mouse Impact ( approximately 38 kb) and human IMPACT ( approximately 30 kb). Sequence comparison revealed that the two genes share a well-conserved exon-intron organization but bear significantly different CpG islands. The mouse island lies in the first intron and contains characteristic tandem repeats. Furthermore, this island serves as a differentially methylated region (DMR) consisting of a hypermethylated maternal allele and an unmethylated paternal allele. Intriguingly, this intronic island is missing from the nonimprinted human IMPACT, whose sole CpG island spans the first exon, lacks any apparent repeats, and escapes methylation on both chromosomes. These results suggest that the intronic DMR plays a role in the imprinting of Impact.     

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.     

Sequence-based bioinformatic prediction and QUASEP identify genomic imprinting of the KCNK9 potassium channel gene in mouse and human

Genomic imprinting is the epigenetic marking of gene subsets resulting in monoallelic or predominant expression of one of the two parental alleles according to their parental origin. We describe the systematic experimental verification of a prioritized 16 candidate imprinted gene set predicted by sequence-based bioinformatic analyses. We used Quantification of Allele-Specific Expression by Pyrosequencing (QUASEP) and discovered maternal-specific imprinted expression of the Kcnk9 gene as well as strain-dependent preferential expression of the Rarres1 gene in E11.5 (C57BL/6 x Cast/Ei)F1 and informative (C57BL/6 x Cast/Ei) x C57BL/6 backcross mouse embryos. For the remaining 14 candidate imprinted genes, we observed biallelic expression. In adult mouse tissues, we found that Kcnk9 expression was restricted to the brain and also was maternal-specific. QUASEP analysis of informative human fetal brain samples further demonstrated maternal-specific imprinted expression of the human KCNK9 orthologue. The CpG islands associated with the mouse and human Kcnk9/KCNK9 genes were not differentially methylated, but strongly hypomethylated. Thus, we speculate that mouse Kcnk9 imprinting may be regulated by the maternal germline differentially methylated region in Peg13, an imprinted non-coding RNA gene in close proximity to Kcnk9 on distal mouse chromosome 15. Our data have major implications for the proposed role of Kcnk9 in neurodevelopment, apoptosis and tumourigenesis, as well as for the efficiency of sequence-based bioinformatic predictions of novel imprinted genes.     

No evidence of PEG1/MEST gene mutations in Silver-Russell syndrome patients.

Silver-Russell syndrome (SRS) is characterized by prenatal and postnatal growth retardation with morphologic anomalies. Maternal uniparental disomy 7 has been reported in some SRS patients. PEG1/MEST is an imprinted gene on chromosome 7q32 that is expressed only from the paternal allele and is a candidate gene for SRS. To clarify its biological function and role in SRS, we screened PEG1/MEST abnormalities in 15 SRS patients from various standpoints. In the lymphocytes of SRS patients, no aberrant expression patterns of two splice variants (alpha and beta) of PEG1/MEST were detected when they were compared with normal samples. Direct sequence analysis failed to detect any mutations in the PEG1/MEST alpha coding region, and there were no significant mutations in the 5'-flanking upstream region containing the predicted promoter and the highly conserved human/mouse genomic region. Differential methylation patterns of the CpG island for PEG1/MEST alpha were normally maintained and resulted in the same pattern as in the normal control, suggesting that there was no loss of imprinting. These findings suggest that PEG1/MEST can be excluded as a major determinant of SRS.     

Establishing the epigenetic status of the Prader-Willi/Angelman imprinting center in the gametes and embryo

The Prader-Willi/Angelman imprinted domain on human chromosome 15q11-q13 is regulated by an imprinting control center (IC) composed of a sequence around the SNRPN promoter (PWS-SRO) and a sequence located 35 kb upstream (AS-SRO). We have previously hypothesized that the primary imprint is established on AS-SRO, which then confers imprinting on PWS-SRO. Here we examine this hypothesis using a transgene that includes both AS-SRO and PWS-SRO sequences and carries out the entire imprinting process. The epigenetic features of this transgene resemble those previously observed on the endogenous locus, thus allowing analyses in the gametes and early embryo. We demonstrate that the primary imprint is in fact established in the gametes, creating a differentially methylated CpG cluster (DMR) on AS-SRO, presumably by an adjacent de novo signal (DNS). The DMR and DNS bind specific proteins: an allele-discrimination protein (ADP) and a de novo methylation protein, respectively. ADP, being a maternal protein, is involved in both the establishment of DMR in the gametes and in its maintenance through implantation when methylation of PWS-SRO on the maternal allele takes place. Importantly, while the AS-SRO is required in the gametes to confer methylation on PWS-SRO, it is dispensable later in development.     

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.     

Detailed methylation analysis of CpG islands on human chromosome region 9p21

Deletion of 9p21 is the most commonly reported chromosomal abnormality in pediatric acute lymphoblastic leukemia, and published data suggest that the maternal chromosome is preferentially deleted. Preferential maternal deletion of 9p21 and reports of a differentially methylated region (DMR) and of parental effects in mice with lymphoma suggest there may be an unrecognized imprinted locus in this region. To screen for DMRs, we used the mcrBC/HpaII screening method and peripheral-blood DNA. Of 36 CpG islands within an 8.5-Mb region of 9p21, seven were identified as putative DMRs and were further analyzed by bisulfite sequencing. Neither any of the CpG islands nor a previously published putative DMR nearby showed evidence of differential parental methylation; however, the published DMR did demonstrate sequence-dependent differential methylation. Our data, which showed heterogeneous and low-level methylation of CpG islands, have obvious implications for methylation studies.     

Post-weaning diet affects genomic imprinting at the insulin-like growth factor 2 (Igf2) locus

IGF2 loss of imprinting (LOI) is fairly prevalent and implicated in the pathogenesis of human cancer and developmental disease; however, the causes of this phenomenon are largely unknown. We determined whether the post-weaning diet of mice affects allelic expression and CpG methylation of Igf2. C57BL/6JxCast/EiJ F1 hybrid mice were weaned onto (1) a standard natural ingredient control diet, (2) a synthetic control diet or (3) a synthetic methyl-donor-deficient diet lacking folic acid, vitamin B(12), methionine and choline. Maternal Igf2 expression in kidney was negligible at birth, but increased to approximately 10% of total expression after 60 days on the natural control diet. By 60 days post-weaning, both synthetic diets caused significant LOI of Igf2 relative to animals weaned onto the natural control diet. Total Igf2 expression was significantly reduced in these groups, however, indicating that the increase in relative maternal Igf2 expression was caused by specific down-regulation of the paternal allele. The LOI induced by the synthetic-deficient diet persisted during a subsequent 100-day 'recuperation' period on natural ingredient diet. There were no group differences in overall or allele-specific CpG methylation in the H19 differentially methylated region (DMR), Igf2 DMR0 or Igf2 DMR1. At 30 and 60 days post-weaning, however, the paternal allele of Igf2 DMR2 was hypermethylated in the kidneys of mice on the control synthetic diet. These results indicate that post-weaning diet can permanently affect expression of Igf2, suggesting that childhood diet could contribute to IGF2 LOI in humans.     

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.      

No evidence of PEG1/MEST gene mutations in Silver‐Russell syndrome patients

Silver‐Russell syndrome (SRS) is characterized by prenatal and postnatal growth retardation with morphologic anomalies. Maternal uniparental disomy 7 has been reported in some SRS patients. PEG1/MEST is an imprinted gene on chromosome 7q32 that is expressed only from the paternal allele and is a candidate gene for SRS. To clarify its biological function and role in SRS, we screened PEG1/MEST abnormalities in 15 SRS patients from various standpoints. In the lymphocytes of SRS patients, no aberrant expression patterns of two splice variants (α and β) of PEG1/MEST were detected when they were compared with normal samples. Direct sequence analysis failed to detect any mutations in the PEG1/MEST α coding region, and there were no significant mutations in the 5′‐flanking upstream region containing the predicted promoter and the highly conserved human/mouse genomic region. Differential methylation patterns of the CpG island for PEG1/MEST α were normally maintained and resulted in the same pattern as in the normal control, suggesting that there was no loss of imprinting. These findings suggest that PEG1/MEST can be excluded as a major determinant of SRS. © 2001 Wiley‐Liss, Inc.     

A genome-wide screen for normally methylated human CpG islands that can identify novel imprinted genes

DNA methylation is a covalent modification of the nucleotide cytosine that is stably inherited at the dinucleotide CpG by somatic cells, and 70% of CpG dinucleotides in the genome are methylated. The exception to this pattern of methylation are CpG islands, CpG-rich sequences that are protected from methylation, and generally are thought to be methylated only on the inactive X-chromosome and in tumors, as well as differentially methylated regions (DMRs) in the vicinity of imprinted genes. To identify chromosomal regions that might harbor imprinted genes, we devised a strategy for isolating a library of normally methylated CpG islands. Most of the methylated CpG islands represented high copy number dispersed repeats. However, 62 unique clones in the library were characterized, all of which were methylated and GC-rich, with a GC content >50%. Of these, 43 clones also showed a CpG(obs)/CpG(exp) >0.6, of which 30 were studied in detail. These unique methylated CpG islands mapped to 23 chromosomal regions, and 12 were differentially methylated regions in uniparental tissues of germline origin, i.e., hydatidiform moles (paternal origin) and complete ovarian teratomas (maternal origin), even though many apparently were methylated in somatic tissues. We term these sequences gDMRs, for germline differentially methylated regions. At least two gDMRs mapped near imprinted genes, HYMA1 and a novel homolog of Elongin A and Elongin A2, which we term Elongin A3. Surprisingly, 18 of the methylated CpG islands were methylated in germline tissues of both parental origins, representing a previously uncharacterized class of normally methylated CpG islands in the genome, and which we term similarly methylated regions (SMRs). These SMRs, in contrast to the gDMRs, were significantly associated with telomeric band locations (P =.0008), suggesting a potential role for SMRs in chromosome organization. At least 10 of the methylated CpG islands were on average 85% conserved between mouse and human. These sequences will provide a valuable resource in the search for novel imprinted genes, for defining the molecular substrates of the normal methylome, and for identifying novel targets for mammalian chromatin formation.