Differential maintenance and de novo methylating activity by three DNA methyltransferases in aging and immortalized fibroblasts.

Genomic methylation, which influences many cellular processes such as gene expression and chromatin organization, generally declines with cellular senescence although some genes undergo paradoxical hypermethylation during cellular aging and immortalization. To explore potential mechanisms for this process, we analyzed the methylating activity of three DNA methyltransferases (Dnmts) in aging and immortalized WI-38 fibroblasts. Overall maintenance methylating activity by the Dnmts greatly decreased during cellular senescence. In immortalized WI-38 cells, maintenance methylating activity was similar to that of normal young cells. Combined de novo methylation activity of the Dnmts initially decreased but later increased as WI-38 cells aged and was strikingly elevated in immortalized cells. To further elucidate the mechanisms for changes in DNA methylation in aging and immortalized cells, the individual Dnmts were separated and individually assessed for maintenance and de novo methylating activity. We resolved three Dnmt fractions, one of which was the major maintenance methyltransferase, Dnmt1, which declined steadily in activity with cellular senescence and immortalization. However, a more basic Dnmt, which has significant de novo methylating activity, increased markedly in activity in aging and immortalized cells. We have identified this methyltransferase as Dnmt3b which has an important role in neoplastic transformation but its role in cellular senescence and immortalization has not previously been reported. An acidic Dnmt we isolated also had increased de novo methylating activity in senescent and immortalized WI-38 cells. These studies indicate that reduced genome-wide methylation in aging cells may be attributed to attenuated Dnmt1 activity but that regional or gene-localized hypermethylation in aging and immortalized cells may be linked to increased de novo methylation by Dnmts other than the maintenance methyltransferase.     

Autonomous silencing of the imprinted Cdkn1c gene in stem cells

Parent-of-origin specific expression of imprinted genes relies on the differential DNA methylation of specific genomic regions. Differentially methylated regions (DMRs) acquire DNA methylation either during gametogenesis (primary DMR) or after fertilisation when allele-specific expression is established (secondary DMR). Little is known about the function of these secondary DMRs. We investigated the DMR spanning Cdkn1c in mouse embryonic stem cells, androgenetic stem cells and embryonic germ stem cells. In all cases, expression of Cdkn1c was appropriately repressed in in vitro differentiated cells. However, stem cells failed to de novo methylate the silenced gene even after sustained differentiation. In the absence of maintained DNA methylation (Dnmt1^-/-), Cdkn1c escapes silencing demonstrating the requirement for DNA methylation in long term silencing in vivo. We propose that postfertilisation differential methylation reflects the importance of retaining single gene dosage of a subset of imprinted loci in the adult.     

Methylation status of differentially methylated regions at Igf2/H19 locus in porcine gametes and preimplantation embryos

The aim of this study was to demonstrate how differential methylation imprints are established during porcine preimplantation embryo development. For the methylation analysis, the primers for the three Igf2/H19 DMRs were designed and based upon previously published sequences. The methylation marks of Igf2/H19 DMRs were analysed in sperm and MII oocytes with our results showing that these regions are fully methylated in sperm but remain unmethylated in MII oocytes. In order to identify the methylation pattern at the pronuclear stage, we indirectly compared the methylation profile of Igf2/H19 DMR3 in each zygote derived by in vitro fertilization, parthenogenesis, and androgenesis. Interestingly, this region was found to be differently methylated according to parental origins; DMR3 was hemimethylated in in vitro fertilized zygotes, fully methylated in parthenogenetic zygotes, and demethylated in androgenetic zygotes. These results indicate that the methylation mark of the paternal allele is erased by active demethylation, and that of the maternal one is de novo methylated. We further examined the methylation imprints of Igf2/H19 DMR3 during early embryonic development. The hemimethylated pattern as seen in zygotes fertilized in vitro was observed up to the 4-cell embryo stage. However, this mark was exclusively demethylated at the 8-cell stage and then restored at the morula stage. These results suggest that methylation imprints are established via dynamic changes during early embryonic development in porcine embryos.     

Timing of establishment of paternal methylation imprints in the mouse

Imprinted genes are characterized by predominant expression from one parental allele and differential DNA methylation. Few imprinted genes have been found to acquire a methylation mark in the male germ line, however, and only one of these, H19, has been studied in detail. We examined methylation of the Rasgrf1 and Gtl2 differentially methylated regions (DMR) to determine whether methylation is erased in male germ cells at e12.5 and when the paternal allele acquires methylation. We also compared their methylation dynamics with those of H19 and the maternally methylated gene Snrpn. Our results show that methylation is erased on Rasgrf1, H19, and Snrpn at e12.5, but that Gtl2 retains substantial methylation at this stage. Erasure of methylation marks on Gtl2 appears to occur later in female germ cells to give the unmethylated profile seen in mature MII oocytes. In the male germ line, de novo methylation of Rasgrf1, Gtl2, and H19 occurs in parallel between e12.5 and e17.5, but the DMR are not completely methylated until the mature sperm stage, suggesting a methylation dynamic different from that of IAP, L1, and minor satellite sequences, which have been shown to become fully methylated by e17.5 in male germ cells. This study also indicates important differences between different imprinted DMR in timing and extent of methylation in the germ cells.     

Erasure of methylation imprinting of Igf2r during mouse primordial germ-cell development

During germ cell differentiation in mice, the genome undergoes specific epigenetic modifications. These include demethylation of imprinted genes and subsequent establishment of parental allele-specific methylation. The mouse Igf2r gene is an imprinted gene that shows maternal-specific expression. Maternal-specific methylation of differentially methylated region 2 (DMR2) of this gene may be necessary for its maternal-specific expression. Before the allele-specific methylation is established, DMR2 is demethylated in both male and female primordial germ cells (PGCs) by 13.5 days post coitum (dpc), indicating that the demethylation of this region occurs earlier in PGC development. The timing of the demethylation has been, however, unknown. In this study, we attempted to determine the timing of methylation erasure of Igf2r DMR2 in developing PGCs, using transgenic mice expressing green fluorescent protein specifically in the germ line. We purified migrating PGCs from the transgenic mice and examined the methylation status of DMR2. The results show that some CpG sites within DMR2 start demethylation at 9.5 dpc in some migrating PGCs, before the cells colonize genital ridges, and the progression of demethylation is rapid after colonization of the genital ridges. To examine whether the gonadal environment is involved in demethylation, we analyzed the methylation of DMR2 after culturing migrating PGCs in the absence of a gonadal environment. These culture experiments support the idea that a gonadal environment is not required for demethylation of the region in at least a fraction of PGCs.     

DNA methylation and demethylation events during meiotic prophase in the mouse testis.

The genes encoding three different mammalian testis-specific nuclear chromatin proteins, mouse transition protein 1, mouse protamine 1, and mouse protamine 2, all of which are expressed postmeiotically, are marked by methylation early during spermatogenesis in the mouse. Analysis of DNA from the testes of prepubertal mice and isolated testicular cells revealed that transition protein 1 became progressively less methylated during spermatogenesis, while the two protamines became progressively more methylated; in contrast, the methylation of beta-actin, a gene expressed throughout spermatogenesis, did not change. These findings provide evidence that both de novo methylation and demethylation events are occurring after the completion of DNA replication, during meiotic prophase in the mouse testis.     

Distinct methylation changes at the IGF2-H19 locus in congenital growth disorders and cancer

Background

Differentially methylated regions (DMRs) are associated with many imprinted genes. In mice methylation at a DMR upstream of the H19 gene known as the Imprint Control region (IC1) is acquired in the male germline and influences the methylation status of DMRs 100 kb away in the adjacent Insulin-like growth factor 2 (Igf2) gene through long-range interactions. In humans, germline-derived or post-zygotically acquired imprinting defects at IC1 are associated with aberrant activation or repression of IGF2, resulting in the congenital growth disorders Beckwith-Wiedemann (BWS) and Silver-Russell (SRS) syndromes, respectively. In Wilms tumour and colorectal cancer, biallelic expression of IGF2 has been observed in association with loss of methylation at a DMR in IGF2. This DMR, known as DMR0, has been shown to be methylated on the silent maternal IGF2 allele presumably with a role in repression. The effect of IGF2 DMR0 methylation changes in the aetiology of BWS or SRS is unknown.

Methodology/Principal Findings

We analysed the methylation status of the DMR0 in BWS, SRS and Wilms tumour patients by conventional bisulphite sequencing and pyrosequencing. We show here that, contrary to previous reports, the IGF2 DMR0 is actually methylated on the active paternal allele in peripheral blood and kidney. This is similar to the IC1 methylation status and is inconsistent with the proposed silencing function of the maternal IGF2 allele. Beckwith-Wiedemann and Silver-Russell patients with IC1 methylation defects have similar methylation defects at the IGF2 DMR0, consistent with IC1 regulating methylation at IGF2 in cis. In Wilms tumour, however, methylation profiles of IC1 and IGF2 DMR0 are indicative of methylation changes occurring on both parental alleles rather than in cis.

Conclusions/Significance

These results support a model in which DMR0 and IC1 have opposite susceptibilities to global hyper and hypomethylation during tumorigenesis independent of the parent of origin imprint. In contrast, during embryogenesis DMR0 is methylated or demethylated according to the germline methylation imprint at the IC1, indicating different mechanisms of imprinting loss in neoplastic and non-neoplastic cells.     

CTCF elements direct allele-specific undermethylation at the imprinted H19 locus

The H19 imprinted gene locus is regulated by an upstream 2 kb imprinting control region (ICR) that influences allele-specific expression, DNA methylation, and replication timing. This ICR becomes de novo methylated during late spermatogenesis in the male but emerges from oogenesis in an unmethylated form, and this allele-specific pattern is then maintained throughout early development and in all tissues of the mouse. We have used a genetic approach involving transfection into embryonic stem (ES) cells in order to decipher how the maternal allele is protected from de novo methylation at the time of implantation. Our studies show that CCCTC binding factor (CTCF) boundary elements within the ICR have the ability to prevent de novo methylation on the maternal allele. Since CTCF does not recognize its binding sequence when methylated, this reaction does not occur on the paternal allele, thus preserving the gamete-derived, allele-specific pattern. These results suggest that CTCF may play a general role in the maintenance of differential methylation patterns in vivo.     

The Testis-Specific Factor CTCFL Cooperates with the Protein Methyltransferase PRMT7 in H19 Imprinting Control Region Methylation

Expression of imprinted genes is restricted to a single parental allele as a result of epigenetic regulation—DNA methylation and histone modifications. Igf2/H19 is a reciprocally imprinted locus exhibiting paternal Igf2 and maternal H19 expression. Their expression is regulated by a paternally methylated imprinting control region (ICR) located between the two genes. Although the de novo DNA methyltransferases have been shown to be necessary for the establishment of ICR methylation, the mechanism by which they are targeted to the region remains unknown. We demonstrate that CTCFL/BORIS, a paralog of CTCF, is an ICR-binding protein expressed during embryonic male germ cell development, coinciding with the timing of ICR methylation. PRMT7, a protein arginine methyltransferase with which CTCFL interacts, is also expressed during embryonic testis development. Symmetrical dimethyl arginine 3 of histone H4, a modification catalyzed by PRMT7, accumulates in germ cells during this developmental period. This modified histone is also found enriched in both H19 ICR and Gtl2 differentially methylated region (DMR) chromatin of testis by chromatin immunoprecipitation (ChIP) analysis. In vitro studies demonstrate that CTCFL stimulates the histone-methyltransferase activity of PRMT7 via interactions with both histones and PRMT7. Finally, H19 ICR methylation is demonstrated by nuclear co-injection of expression vectors encoding CTCFL, PRMT7, and the de novo DNA methyltransferases, Dnmt3a, -b and -L, in Xenopus oocytes. These results suggest that CTCFL and PRMT7 may play a role in male germline imprinted gene methylation.     

The testis-specific factor CTCFL cooperates with the protein methyltransferase PRMT7 in H19 imprinting control region methylation

Expression of imprinted genes is restricted to a single parental allele as a result of epigenetic regulation—DNA methylation and histone modifications. Igf2/H19 is a reciprocally imprinted locus exhibiting paternal Igf2 and maternal H19 expression. Their expression is regulated by a paternally methylated imprinting control region (ICR) located between the two genes. Although the de novo DNA methyltransferases have been shown to be necessary for the establishment of ICR methylation, the mechanism by which they are targeted to the region remains unknown. We demonstrate that CTCFL/BORIS, a paralog of CTCF, is an ICR-binding protein expressed during embryonic male germ cell development, coinciding with the timing of ICR methylation. PRMT7, a protein arginine methyltransferase with which CTCFL interacts, is also expressed during embryonic testis development. Symmetrical dimethyl arginine 3 of histone H4, a modification catalyzed by PRMT7, accumulates in germ cells during this developmental period. This modified histone is also found enriched in both H19 ICR and Gtl2 differentially methylated region (DMR) chromatin of testis by chromatin immunoprecipitation (ChIP) analysis. In vitro studies demonstrate that CTCFL stimulates the histone-methyltransferase activity of PRMT7 via interactions with both histones and PRMT7. Finally, H19 ICR methylation is demonstrated by nuclear co-injection of expression vectors encoding CTCFL, PRMT7, and the de novo DNA methyltransferases, Dnmt3a, -b and -L, in Xenopus oocytes. These results suggest that CTCFL and PRMT7 may play a role in male germline imprinted gene methylation.     

Tandem B1 elements located in a mouse methylation center provide a target for de novo DNA methylation

A cis-acting methylation center that signals de novo DNA methylation is located upstream of the mouse Aprt gene. In the current study, two approaches were taken to determine if tandem B1 repetitive elements found at the 3' end of the methylation center contribute to the methylation signal. First, bisulfite genomic sequencing demonstrated that CpG sites within the B1 elements were methylated at relative levels of 43% in embryonal stem cells deficient for the maintenance DNA methyltransferase when compared with wild type embryonal stem cells. Second, the ability of the B1 elements to signal de novo methylation upon stable transfection into mouse embryonal carcinoma cells was examined. This approach demonstrated that the B1 elements were methylated de novo to a high level in the embryonal carcinoma cells and that the B1 elements acted synergistically. The results from these experiments provide strong evidence that the tandem B1 repetitive elements provide a significant fraction of the methylation center signal. By extension, they also support the hypothesis that one role for DNA methylation in mammals is to protect the genome from expression and transposition of parasitic elements.     

Extensive tissue-specific variation of allelic methylation in the Igf2 gene during mouse fetal development: relation to expression and imprinting

The imprinted Igf2 gene is active only on the paternal allele in most tissues. Its imprinting involves a cis-acting imprinting-control region (ICR) located upstream of the neighboring and maternally expressed H19 gene. It is thought that differential methylation of the parental alleles at the ICR is crucial for parental imprinting of both genes. Differentially methylated regions (DMRs) have also been identified within the Igf2 gene and their differential methylation is thought to be established during early development. To gain further insight into the function of these DMRs, we performed a quantitative analysis of their allelic methylation levels in different tissues during fetal development and the postnatal period in the mouse. Surprisingly, we found that the methylation levels of Igf2 DMRs vary extensively during fetal development, mostly on the expressed paternal allele. In particular, in skeletal muscle, differential allelic methylation in both DMR 1 and DMR 2 occurs only after birth, whereas correct paternal monoallelic expression is always observed, including in the embryonic stages. This suggests that differential methylation in the DMR 1 and DMR 2 of the Igf2 gene is dispensable for its imprinting in skeletal muscle. Furthermore, progressive methylation of the Igf2 paternal allele appears to be correlated with concomitant postnatal down-regulation and silencing of the gene. We discuss possible relations between Igf2 allelic methylation and expression during fetal development.     

Establishment of paternal allele-specific DNA methylation at the imprinted mouse Gtl2 locus

The monoallelic expression of imprinted genes is controlled by epigenetic factors including DNA methylation and histone modifications. In mouse, the imprinted gene Gtl2 is associated with two differentially methylated regions: the IG-DMR, which serves as a gametic imprinting mark at which paternal allele-specific DNA methylation is inherited from sperm, and the Gtl2-DMR, which acquires DNA methylation on the paternal allele after fertilization. The timeframe during which DNA methylation is acquired at secondary DMRs during post-fertilization development and the relationship between secondary DMRs and imprinted expression have not been well established. In order to better understand the role of secondary DMRs in imprinting, we examined the methylation status of the Gtl2-DMR in pre- and post-implantation embryos. Paternal allele-specific DNA methylation of this region correlates with imprinted expression of Gtl2 during post-implantation development but is not required to implement imprinted expression during pre-implantation development, suggesting that this secondary DMR may play a role in maintaining imprinted expression. Furthermore, our developmental profile of DNA methylation patterns at the Cdkn1c- and Gtl2-DMRs illustrates that the temporal acquisition of DNA methylation at imprinted genes during post-fertilization development is not universally controlled.Key words: genomic imprinting, DNA methylation, Gtl2, secondary DMR, epigenetics     

The bovine IGF2 gene is differentially methylated in oocyte and sperm DNA

The insulin-like growth factor 2 gene (IGF2) encodes an essential growth factor and is imprinted in various mammalian species. Differentially methylated regions (DMRs) are often located within CpG islands and are critically involved in the regulation of monoallelic Igf2 expression in the mouse. Only partial sequence information is available for the bovine IGF2 gene and no DMR has currently been identified. The goal of this study was to identify a DMR within the bovine IGF2 gene as a prerequisite for further studies on gene-specific methylation patterns during preimplantation development. Here we describe the sequence analysis of a CpG-rich DNA fragment from the 5' untranslated region spanning exons and introns 4 and 5 and the identification of a previously unknown DMR in exon 10 of the bovine IGF2 gene. Bisulfite analysis revealed that this DMR is differentially methylated in mature oocytes and sperm. The identification of an intragenic DMR within a developmentally important gene such as the bovine IGF2 gene provides a useful tool to evaluate the methylation patterns of embryos derived in vivo and in vitro. Our study is the first report of a differentially methylated region in a bovine imprinted gene discovered by the analysis of female and male gametes.     

Dnmt1 overexpression causes genomic hypermethylation, loss of imprinting, and embryonic lethality

Biallelic expression of Igf2 is frequently seen in cancers because Igf2 functions as a survival factor. In many tumors the activation of Igf2 expression has been correlated with de novo methylation of the imprinted region. We have compared the intrinsic susceptibilities of the imprinted region of Igf2 and H19, other imprinted genes, bulk genomic DNA, and repetitive retroviral sequences to Dnmt1 overexpression. At low Dnmt1 methyltransferase levels repetitive retroviral elements were methylated and silenced. The nonmethylated imprinted region of Igf2 and H19 was resistant to methylation at low Dnmt1 levels but became fully methylated when Dnmt1 was overexpressed from a bacterial artificial chromosome transgene. Methylation caused the activation of the silent Igf2 allele in wild-type and Dnmt1 knockout cells, leading to biallelic Igf2 expression. In contrast, the imprinted genes Igf2r, Peg3, Snrpn, and Grf1 were completely resistant to de novo methylation, even when Dnmt1 was overexpressed. Therefore, the intrinsic difference between the imprinted region of Igf2 and H19 and of other imprinted genes to postzygotic de novo methylation may be the molecular basis for the frequently observed de novo methylation and upregulation of Igf2 in neoplastic cells and tumors. Injection of Dnmt1-overexpressing embryonic stem cells in diploid or tetraploid blastocysts resulted in lethality of the embryo, which resembled embryonic lethality caused by Dnmt1 deficiency.     

A tripartite paternally methylated region within the Gpr1-Zdbf2 imprinted domain on mouse chromosome 1 identified by meDIP-on-chip

The parent-of-origin specific expression of imprinted genes relies on DNA methylation of CpG-dinucleotides at differentially methylated regions (DMRs) during gametogenesis. To date, four paternally methylated DMRs have been identified in screens based on conventional approaches. These DMRs are linked to the imprinted genes H19, Gtl2 (IG-DMR), Rasgrf1 and, most recently, Zdbf2 which encodes zinc finger, DBF-type containing 2. In this study, we applied a novel methylated-DNA immunoprecipitation-on-chip (meDIP-on-chip) method to genomic DNA from mouse parthenogenetic- and androgenetic-derived stem cells and sperm and identified 458 putative DMRs. This included the majority of known DMRs. We further characterized the paternally methylated Zdbf2/ZDBF2 DMR. In mice, this extensive germ line DMR spanned 16 kb and possessed an unusual tripartite structure. Methylation was dependent on DNA methyltransferase 3a (Dnmt3a), similar to H19 DMR and IG-DMR. In both humans and mice, the adjacent gene, Gpr1/GPR1, which encodes a G-protein-coupled receptor 1 protein with transmembrane domain, was also imprinted and paternally expressed. The Gpr1-Zdbf2 domain was most similar to the Rasgrf1 domain as both DNA methylation and the actively expressed allele were in cis on the paternal chromosome. This work demonstrates the effectiveness of meDIP-on-chip as a technique for identifying DMRs.     

Allele-specific histone modifications regulate expression of the Dlk1-Gtl2 imprinted domain

Dlk1 and Gtl2 are reciprocally expressed imprinted genes located on mouse chromosome 12. The Dlk1-Gtl2 locus carries three differentially methylated regions (DMRs), which are methylated only on the paternal allele. Of these, the intergenic (IG) DMR, located 12 kb upstream of Gtl2, is required for proper imprinting of linked genes on the maternal chromosome, while the Gtl2 DMR, located across the promoter of the Gtl2 gene, is implicated in imprinting on both parental chromosomes. In addition to DNA methylation, modification of histone proteins is also an important regulator of imprinted gene expression. Chromatin immunoprecipitation was therefore used to examine the pattern of histone modifications across the IG and Gtl2 DMRs. The data show maternal-specific histone acetylation at the Gtl2 DMR, but not at the IG DMR. In contrast, only low levels of histone methylation were observed throughout the region, and there was no difference between the two parental alleles. An existing mouse line carrying a deletion/insertion upstream of Gtl2 is unable to imprint the Dlk1-Gtl2 locus properly and demonstrates loss of allele-specific methylation at the Gtl2 DMR. Further analysis of these animals now shows that the loss of allele-specific methylation is accompanied by increased paternal histone acetylation at the Gtl2 DMR, with the activated paternal allele adopting a maternal acetylation pattern. These data indicate that interactions between DNA methylation and histone acetylation are involved in regulating the imprinting of the Dlk1-Gtl2 locus.