Towards unravelling the Igf2/H19 imprinted domain

Genomic imprinting is an epigenetic marking process that confers parent-of-origin-dependent expression on certain genes. These imprinted genes are sometimes found in clusters, suggesting a possible involvement of higher order regulatory elements controlling expression and imprinting of genes organised in such clusters. In the distal chromosome 7 there are at least four imprinted genes: Mash2, Ins2, Igf2 and H19. Recent evidence⁽¹⁾ suggests that imprinting and expression of at least Igf2 and H19 may be mechanistically linked.     

The mouse Chromosome 7 distal imprinting domain maps to G-bands F4/F5

Mouse Chromosome (Chr) 7 distal to band F3 on the physical map is known to be subject to imprinting, maternal duplication (MatDp) of the region leading to a late embryonic lethality, while paternal duplication (PatDp) causes death in utero before 11.5 dpc. Using a new mouse reciprocal translocation T(7;11)65H to produce MatDp for distal Chr 7, we have mapped the region subject to imprinting more precisely to bands 7F4/F5 on the cytogenetic map. Fluorescence in situ hybridization (FISH) studies on mitotic and meiotic chromosomes of a T65H heterozygote show that the imprinted gene Igf2 is located in the same region. This was confirmed by the finding that embryos with MatDp of bands 7F4/F5 did not express Igf2. We suggest that other members of the imprinted domain containing Igf2, namely Mash2, H19, Ins2, and p57 ^K1P2 , are also located in 7F4/F5 and that some or all of these genes may be responsible for the two imprinting lethalities seen with MatDp and PatDp for this region. Received: 13 October 1996 / Accepted: 8 December 1996     

Replication Timing Properties within the Mouse Distal Chromosome 7 Imprinting Cluster

Genomic imprinting is characterized by allele-specific expression of genes within chromosomal domains. Here we show, using fluorescence in situ hybridization (FISH) analysis, that the large chromosomal domain of the mouse distal chromosome 7 imprinting cluster, approximately 1 Mb in length between p57^Kip2 and H19 genes, replicates asynchronously between the two alleles during S-phase. At the telomeric side of this domain, we found a transition from asynchronous replication at the imprinted p57^Kip2 gene to synchronous replication at the Nap2 gene. Two-color FISH suggested that the paternal allele of this whole domain replicates earlier than its maternal allele. Treatment of the cells with a histone deacetylase inhibitor abolished this allele-specific feature accompanied with accelerated replication of the later-replicating allele at a domain level. Allele-specific asynchronous replication was observed even in ES cells. These results suggest that this imprinting cluster consists of a large replication domain which is already found at the early stage in development.     

Loss of Igf2 imprinting in monoclonal mouse hepatic tumor cells is not associated with abnormal methylation patterns for the H19, Igf2, and Kvlqt1 differentially methylated regions

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

A differentially methylated imprinting control region within the Kcnq1 locus harbors a methylation-sensitive chromatin insulator

The mechanisms underlying the phenomenon of genomic imprinting remain poorly understood. In one instance, a differentially methylated imprinting control region (ICR) at the H19 locus has been shown to involve a methylation-sensitive chromatin insulator function that apparently partitions the neighboring Igf2 and H19 genes in different expression domains in a parent of origin-dependent manner. It is not known, however, if this mechanism is unique to the Igf2/H19 locus or if insulator function is a common feature in the regulation of imprinted genes. To address this question, we have studied an ICR in the Kcnq1 locus that regulates long range repression on the paternally derived p57Kip2 and Kcnq1 alleles in an imprinting domain that includes Igf2 and H19. We show that this ICR appears to possess a unidirectional chromatin insulator function in somatic cells of both mesodermal and endodermal origins. Moreover, we document that CpG methylation regulates this insulator function suggesting that a methylation-sensitive chromatin insulator is a common theme in the phenomenon of genomic imprinting.     

TGFα Reactivates Imprinted <Emphasis Type="Italic">Igf2</Emphasis> in the Parthenogenetic Mice Embryos and Placenta

Imprinted genes play important roles in the mammalian development. In the parthenogenetic embryos (PE), there is only expression of maternally expressed genes. Therefore, PEs are appropriate experimental models to study genomic imprinting controlling mechanisms. The maternally expressed H19 and paternally expressed Igf2 are reciprocally imprinted genes in normal embryos. Here, we studied effect of transforming growth factor alpha (TGFα) treatment in vitro (10 ng/ml at the morula stage) on the expression of Igf2/H19 locus in mice PE (9.5 days of gestation, 25 somites) and their placentas (PP). Using RT-PCR, we showed that TGFα reactivated maternally imprinted Igf2 gene in parthenogenetic embryos and placentas. In spite of similar Tgfα expression in the preimplantation stages, its expression in the 9.5-day parthenogenetic embryos is significantly less than in normal embryos (NE). In our experiments, it was shown that reactivation of Igf2 gene occurred independently of H19 gene. In vitro TGFα treatment of mouse PE reactivated paternally expressed Igf2 gene in the PE and PP. In the PE and PP, both Igf2 and H19 were expressed. It seems that TGFα can play an important role as modulator of the Igf2/H19 locus.     

A 210-kb segment of tandem repeats and retroelements located between imprinted subdomains of mouse distal chromosome 7.

Mammalian genes subject to genomic imprinting often form clusters and are regulated by long-range mechanisms. The distal imprinted domain of mouse chromosome 7 is orthologous to the Beckwith-Wiedemann syndrome domain in human chromosome 11p15.5 and contains at least 13 imprinted genes. This domain consists of two subdomains, which are respectively regulated by an imprinting center. We here report the finished-quality sequence of a 0.6-Mb region encompassing the more centromeric subdomain. The sequence contains four imprinted genes (Ascl2/Mash2, Ins2, Igf2 and H19) and reveals previously unidentified CpG islands and tandem repeats, which may be features of imprinted genes. Most interestingly, a unique 210-kb segment consisting almost exclusively of tandem repeats and retroelements is identified. This segment, located between Th and Ins2, has features of heterochromatin-forming DNA and is highly methylated at CpG sites. The segment exhibits asynchronous replication on the parental chromosomes, a feature of the imprinted domains. We propose that this repeat segment could serve either as a boundary between the two subdomains or as a target for epigenetic chromatin modifications that regulate imprinting.     

Replication timing properties within the mouse distal chromosome 7 imprinting cluster

Genomic imprinting is characterized by allele-specific expression of genes within chromosomal domains. Here we show, using fluorescence in situ hybridization (FISH) analysis, that the large chromosomal domain of the mouse distal chromosome 7 imprinting cluster, approximately 1 Mb in length between p57Kip2 and H19 genes, replicates asynchronously between the two alleles during S-phase. At the telomeric side of this domain, we found a transition from asynchronous replication at the imprinted p57Kip2 gene to synchronous replication at the Nap2 gene. Two-color FISH suggested that the paternal allele of this whole domain replicates earlier than its maternal allele. Treatment of the cells with a histone deacetylase inhibitor abolished this allele-specific feature accompanied with accelerated replication of the later-replicating allele at a domain level. Allele-specific asynchronous replication was observed even in ES cells. These results suggest that this imprinting cluster consists of a large replication domain which is already found at the early stage in development.     

Ubiquitous expression and imprinting of Snrpn in the mouse

Snrpn is known to be abundantly expressed in rodent brain and heart, and in two separate studies with neonatal mouse brain it has been shown to be maternally imprinted, that is, the maternal allele is normally repressed. We now provide evidence on the expression profile and imprinting status of Snrpn throughout development. Using RT-PCR, we have established that Snrpn is further expressed at low levels in lung, liver, spleen, kidney, skeletal muscle, and gonads. Moreover, using mice with only maternal copies of Snrpn (maternal duplication for the chromosome region involved and parthenogenotes), we have shown that the gene is imprinted in all of these tissues and, generally, from the time the gene is first expressed at 7.5 days gestation. In contrast to the findings made with the imprinted genes, Igf2, Ins1, and Ins2, there is no evidence of tissue-specific imprinting in the embryo with Snrpn. Nor, as found with Igf2 and Igf2r, is there evidence of a window of biallelic expression between the germ line imprint and the time of gene repression. The absence of Snrpn expression in early embryos contrasts with the findings in ES cells.     

TGFalpha reactivates imprinted Igf2 in the parthenogenetic mice embryos and placenta

Imprinted genes play important roles in the mammalian development. In the parthenogenetic embryos (PE) there is only expression of maternally expressed genes. Therefore, PEs are appropriate experimental models to study genomic imprinting controlling mechanisms. The maternally expressed H19 and paternally expressed Igf2 are reciprocally imprinted genes in normal embryos. Here we studied effect of transforming growth factor alpha (TGFalpha) treatment in vitro (10 ng/ml at the morula stage) on the expression of Igf2/H19 locus in mice PE (9.5-days of gestation, 25 somites) and their placentas (PP). Using RT-PCR we showed that TGFalpha reactivated maternally imprinted Igf2 gene in parthenogenetic embryos and placentas. In spite of similar Tgfalpha expression in the pre-implantation stages, its expression in the 9.5-day parthenogenetic embryos is significantly less than in normal embryos (NE). In our experiments it was shown that reactivation of Igf2 gene occurred independently of H19 gene. In vitro TGFalpha treatment of mouse PE reactivated paternally expressed Igf2 gene in the PE and PP. In the PE and PP both Igf2 and H19 were expressed. It seems that TGFalpha can play an important role as modulator of the Igf2/H19 locus.     

CTCF regulates allelic expression of Igf2 by orchestrating a promoter-polycomb repressive complex 2 intrachromosomal loop

CTCF is a zinc finger DNA-binding protein that regulates the epigenetic states of numerous target genes. Using allelic regulation of mouse insulin-like growth factor II (Igf2) as a model, we demonstrate that CTCF binds to the unmethylated maternal allele of the imprinting control region (ICR) in the Igf2/H19 imprinting domain and forms a long-range intrachromosomal loop to interact with the three clustered Igf2 promoters. Polycomb repressive complex 2 is recruited through the interaction of CTCF with Suz12, leading to allele-specific methylation at lysine 27 of histone H3 (H3-K27) and to suppression of the maternal Igf2 promoters. Targeted mutation or deletion of the maternal ICR abolishes this chromatin loop, decreases allelic H3-K27 methylation, and causes loss of Igf2 imprinting. RNA interference knockdown of Suz12 also leads to reactivation of the maternal Igf2 allele and biallelic Igf2 expression. CTCF and Suz12 are coprecipitated from nuclear extracts with antibodies specific for either protein, and they interact with each other in a two-hybrid system. These findings offer insight into general epigenetic mechanisms by which CTCF governs gene expression by orchestrating chromatin loop structures and by serving as a DNA-binding protein scaffold to recruit and bind polycomb repressive complexes.     

Nucleotide Sequence of a 28-kb Mouse Genomic Region Comprising the Imprinted Igf2 Gene

The mouse insulin-like growth factor II gene (Igf2) is physicallylinked to the insulin II gene (Ins2) and both are subject totissue-specific genomic imprinting. The paternal-specific expressionof Igf2 has been associated with hypermethylation of some CpGsites in the 5' flanking region and in the body of the gene.As a first step in analyzing the structural features of thisimprinted locus, we here report the complete nucleotide sequenceof Igf2, including all introns and the intergenic region adjacentto Ins2. This 28-kb segment of mouse chromosome 7 exhibits 80%overall identity with the corresponding rat sequence and hasa high GC content of 52%. In addition to the known CpG islandwithin the second Igf2 promoter, another island was identifiedapproximately 2 kb 5' to the first exon. Other features of thislocus include a 35-fold tandem repeat of an 11-bp sequence thatoverlaps Igf2 pseudo-exon 2, and a B2 repeat element in theintergenic region between Ins2 and Igf2. The GC-richness andthe presence of CpG islands associated with tandem repeats arecommon features of imprinted genes and thus may play a rolein the imprinting mechanism.     

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.     

Pluripotential stem cells derived from migrating primordial germ cells

Pluripotent stem cells termed embryonic germ cells (EGCs) have earlier been derived from pre- and post-migrating mouse primordial germ cells (PGCs). We have recently obtained four EGC lines from migrating PGCs of 9.5 days post coitum (dpc) embryos. All lines were male with normal karyotype and showed properties that are similar to previously established EGC lines, including colony morphology, expression of alkaline phosphatase (AP), and expression of SSEA-1 antigen. The developmental potency of two of these lines was tested in vivo. They contributed to a range of tissues in fetal chimeras including heart, lung, kidney, intestine, muscle, brain and skin. We also examined the methylation status of the imprinted genes: Igf2r, p57Kip2, Lit1, H19 and Igf2. Igf2r, p57Kip2 and Lit1 were unmethylated in all analysed EGC lines, whereas H19 and Igf2 showed significant hypo-methylation in the 9.5 dpc EGC-1 line when compared to previously derived 11.5 dpc male EGC lines. This suggests that imprint erasure in the male germ line occurs prior to 9.5 dpc for all imprinted genes examined.     

Tissue-specific relationship of S-adenosylhomocysteine with allele-specific H19/Igf2 methylation and imprinting in mice with hyperhomocysteinemia

DNA methylation is linked to homocysteine metabolism through the generation of S-adenosylmethionine (AdoMet) and S-Adenosylhomocysteine (AdoHcy). The ratio of AdoMet/AdoHcy is often considered an indicator of tissue methylation capacity. The goal of this study is to determine the relationship of tissue AdoMet and AdoHcy concentrations to allele-specific methylation and expression of genomically imprinted H19/Igf2. Expression of H19/Igf2 is regulated by a differentially methylated domain (DMD), with H19 paternally imprinted and Igf2 maternally imprinted. F1 hybrid C57BL/6J x Castaneous/EiJ (Cast) mice with (+/−), and without (+/+), heterozygous disruption of cystathionine-β-synthase (Cbs) were fed a control diet or a diet (called HH) to induce hyperhomocysteinemia and changes in tissue AdoMet and AdoHcy. F1 Cast x Cbs+/− mice fed the HH diet had significantly higher plasma total homocysteine concentrations, higher liver AdoHcy, and lower AdoMet/AdoHcy ratios and this was accompanied by lower liver maternal H19 DMD allele methylation, lower liver Igf2 mRNA levels, and loss of Igf2 maternal imprinting. In contrast, we found no significant differences in AdoMet and AdoHcy in brain between the diet groups but F1 Cast x Cbs+/− mice fed the HH diet had higher maternal H19 DMD methylation and lower H19 mRNA levels in brain. A significant negative relationship between AdoHcy and maternal H19 DMD allele methylation was found in liver but not in brain. These findings suggest the relationship of AdoMet and AdoHcy to gene-specific DNA methylation is tissue-specific and that changes in DNA methylation can occur without changes in AdoMet and AdoHcy.