Loss of inherited genomic imprints in mice leads to severe disruption in placental lipid metabolism.

INTRODUCTION: Monoallelic expression of imprinted genes is necessary for placental development and normal fetal growth. Differentially methylated domains (DMDs) largely determine the parental-specific monoallelic expression of imprinted genes. Maternally derived DNA (cytosine-5-) -methyltransferase 1o (DNMT1o) maintains DMDs during the eight-cell stage of development. DNMT1o-deficient mouse placentas have a generalized disruption of genomic imprints. Previous studies have demonstrated that DNMT1o deficiency alters placental morphology and broadens the embryonic weight distribution in late gestation. Lipids are critical for fetal growth. Thus, we assessed the impact of disrupted imprinting on placental lipids. METHODS: Lipids were quantified from DNMT1o-deficient mouse placentas and embryos at E17.5 using a modified Folch method. Expression of select genes critical for lipid metabolism was quantified with RT-qPCR. Mitochondrial morphology was assessed by TEM and mitochondrial aconitase and cytoplasmic citrate concentrations quantified. DMD methylation was determined by EpiTYPER. RESULTS: We found that DNMT1o deficiency is associated with increased placental triacylglycerol levels. Neither fetal triacylglycerol concentrations nor expression of select genes that mediate placental lipid transport were different from wild type. Placental triacylglycerol accumulation was associated with impaired beta-oxidation and abnormal citrate metabolism with decreased mitochondrial aconitase activity and increased cytoplasmic citrate concentrations. Loss of methylation at the MEST DMD was strongly associated with placental triacylglycerol accumulation. DISCUSSION: A generalized disruption of genomic imprints leads to triacylglycerol accumulation and abnormal mitochondrial function. This could stem directly from a loss of methylation at a given DMD, such as MEST, or represent a consequence of abnormal placental development.

IFPA Meeting 2011 workshop report III: Placental immunology; epigenetic and microRNA-dependent gene regulation; comparative placentation; trophoblast differentiation; stem cells.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2011 there were twelve themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology: 1) immunology; 2) epigenetics; 3) comparative placentation; 4) trophoblast differentiation; 5) stem cells.

Methyltransferases mediate cell memory of a genotoxic insult.

Characterization of the direct effects of DNA-damaging agents shows how DNA lesions lead to specific mutations. Yet, serum from Hiroshima survivors, Chernobyl liquidators and radiotherapy patients can induce a clastogenic effect on naive cells, showing indirect induction of genomic instability that persists years after exposure. Such indirect effects are not restricted to ionizing radiation, as chemical genotoxins also induce heritable and transmissible genomic instability phenotypes. Although such indirect induction of genomic instability is well described, the underlying mechanism has remained enigmatic. Here, we show that mouse embryonic stem cells exposed to γ-radiation bear the effects of the insult for weeks. Specifically, conditioned media from the progeny of exposed cells can induce DNA damage and homologous recombination in naive cells. Notably, cells exposed to conditioned media also elicit a genome-destabilizing effect on their neighbouring cells, thus demonstrating transmission of genomic instability. Moreover, we show that the underlying basis for the memory of an insult is completely dependent on two of the major DNA cytosine methyltransferases, Dnmt1 and Dnmt3a. Targeted disruption of these genes in exposed cells completely eliminates transmission of genomic instability. Furthermore, transient inactivation of Dnmt1, using a tet-suppressible allele, clears the memory of the insult, thus protecting neighbouring cells from indirect induction of genomic instability. We have thus demonstrated that a single exposure can lead to long-term, genome-destabilizing effects that spread from cell to cell, and we provide a specific molecular mechanism for these persistent bystander effects. Collectively, our results impact the current understanding of risks from toxin exposures and suggest modes of intervention for suppressing genomic instability in people exposed to carcinogenic genotoxins.

The role of DMDs in the maintenance of epigenetic states.

An important aspect of genome reprogramming is the establishment and maintenance of gamete-specific DNA methylation patterns that distinguish the parental alleles of imprinted genes. Disrupting the accurate transmission of genomic imprints by interfering with these methylation patterns causes severe defects in fetal growth and development. The inheritance of sex-specific DNA methylation patterns from both parents is thus a fundamental molecular definition of genomic imprinting. The other cardinal aspect is the regulation of imprinted gene expression over a long genomic distance, spanning a few clustered imprinted genes. There is converging experimental evidence that differentially methylated domains (DMDs), located in non-coding regions of imprinted genes, are involved in both processes. As such, DMDs are the imprinting backbone upon which the fundamental processes of sex-specific methylation and imprinted gene expression are built.

Genomic imprinting disrupted by a maternal effect mutation in the Dnmt1 gene.

Maintenance of genomic methylation patterns in mammalian somatic cells depends on DNA methyltransferase-1 (Dnmt1). Mouse oocytes and preimplantation embryos lack Dnmt1 but express a variant of this protein called Dnmt1o. We eliminated Dnmt1o by deletion of the oocyte-specific promoter and first exon from the Dnmt1 locus. Homozygous animals were normal, but most heterozygous fetuses of homozygous females died during the last third of gestation. Although genomic methylation patterns were established normally in Dnmt1o-deficient oocytes, embryos derived from such oocytes showed a loss of allele-specific expression and methylation at certain imprinted loci. Transient nuclear localization of Dnmt1o in 8-cell embryos suggests that this variant of Dnmt1 provides maintenance methyltransferase activity specifically at imprinted loci during the fourth embryonic S phase.

Forced expression of the homeobox-containing gene Pem blocks differentiation of embryonic stem cells.

Similarities in the differentiation of mouse embryos and ES cell embryoid bodies suggest that aspects of early mammalian embryogenesis can be studied in ES cell embryoid bodies. In an effort to understand the regulation of cellular differentiation during early mouse embryogenesis, we altered the expression of the Pem homeobox-containing gene in ES cells. Pem is normally expressed in the preimplantation embryo and expressed in a lineage-restricted fashion following implantation, suggesting a role for Pem in regulating cellular differentiation in the early embryo. Here, we show that the forced expression of Pem from the mouse Pgk-1 promoter in ES cells blocks the in vitro and in vivo differentiation of the cells. In particular, embryoid bodies produced from these Pgk-Pem ES cells do not differentiate into primitive endoderm or embryonic ectoderm, which are prominent features of early embryoid bodies from normal ES cells. This Pgk-Pem phenotype is also different from the null phenotype, as embryoid bodies derived from ES cells in which endogenous Pem gene expression has been blocked show a pattern of differentiation similar to that of normal ES cells. When the Pgk-Pem ES cells were introduced into subcutaneous sites of nude mice, only undifferentiated EC-like cells were found in the teratomas derived from the injected cells. The Pem-dependent block of ES cell differentiation appears to be cell autonomous; Pgk-Pem ES cells did not differentiate when mixed with normal, differentiating ES cells. A block to ES cell differentiation, resulting from the forced expression of Pem, can also be produced by the forced expression of the nonhomeodomain region of Pem. These studies are consistent with a role for Pem in regulating the transition between undifferentiated and differentiated cells of the early mouse embryo.

cis-Acting signal for inheritance of imprinted DNA methylation patterns in the preimplantation mouse embryo.

The inheritance of gametic methylation patterns is a critical event in the imprinting of genes. In the case of the imprinted RSVIgmyc transgene, the methylation pattern in the unfertilized egg is maintained by the early mouse embryo, whereas the sperm's methylation pattern is lost in the early embryo. To investigate the cis-acting requirements for this preimplantation stage of genomic imprinting, we examined the fate of different RSVIgmyc methylation patterns, preimposed on RSVIgmyc and introduced into the mouse zygote by pronuclear injection. RSVIgmyc methylation patterns with a low percentage of methylated CpG dinucleotides, generated by using bacterial cytosine methylases with four-base recognition sequences, were lost in the early embryo. In contrast, methylation was maintained when all CpG dinucleotides were methylated with the bacterial SssI (CpG) methylase. This singular maintenance of RSVIgmyc methylation preimposed with SssI methylase appears to be specific to the early, undifferentiated embryo; differentiated NIH 3T3 fibroblasts transfected with methylated versions of RSVIgmyc maintained all methylation patterns, independent of the level of preimposed methylation. The methylation pattern of the RSVIgmyc allele in adult founder transgenic mice that was produced by pronuclear injection of an SssI-methylated construct could not be distinguished from the maternal RSVIgmyc methylation pattern. Thus, a highly methylated allele in adult mice, normally generated by transmission of RSVIgmyc through the female germ line, was also produced in founder transgenic mice by bypassing gametogenesis and introducing a highly methylated RSVIgmyc into the mouse zygote. These results suggest that RSVIgmyc methylation itself is a cis-acting signal for the preimplantation maintenance of the oocyte's methylation pattern and, therefore, a cis-acting signal for RSVIgmyc imprinting. Furthermore, our inability to identify a sequence element within RSVIgmyc that was absolutely required for its imprinting suggests that the extent of RSVIgmyc methylation, rather than a particular pattern of methylation, is the principal feature of this imprinting signal.

Parental alleles of an imprinted mouse transgene replicate synchronously.

Molecular features of imprinted genes include differences in expression, methylation, and the timing of DNA replication between parental alleles. Whereas methylation differences always seem to be associated with differences in expression, differences in the timing of replication between parental homologs are not always seen at imprinted loci. These observations raise the possibility that differences in replication timing may not be an essential feature underlying genomic imprinting. In this study, we examined the timing of replication of the two alleles of the imprinted RSVIgmyc transgene in individual embryonic cells using fluorescence in situ hybridization (FISH). The cis-acting signals for RSVIgmyc imprinting are within RSVIgmyc itself. Thus, allele-specific differences in replication, if they indeed govern RSVIgmyc imprinting, should be found in RSVIgmyc sequences. We found that the parental alleles of RSVIgmyc, which exhibit differences in methylation, replicated at the same time. Synchronous replication was also seen in embryonic cells containing a modified version of RSVIgmyc that exhibited parental allele differences in both methylation and expression. These findings indicate that maintenance of expression and methylation differences between alleles does not require a difference in replication timing. The differences in replication timing of endogenous imprinted alleles detected by FISH might therefore reflect structural differences between the two alleles that could be a consequence of imprinting or, alternatively, could be unrelated to imprinting.

Phenotypic variation in a genetically identical population of mice.

The parental alleles of an imprinted gene acquire their distinctive methylation patterns at different times in development. For the imprinted RSVIgmyc transgene, methylation of the maternal allele is established in the oocyte and invariably transmitted to the embryo. In contrast, the methylation of the paternal allele originates during embryogenesis. Here, we show that the paternal methylation pattern among mice with identical genetic backgrounds is subject to extensive variation. In addition to this nongenetic variation, the process underlying RSVIgmyc methylation in the embryo is also subject to considerable genetic regulation. The paternal transgene allele is highly methylated in an inbred C57BL/6J strain, whereas it is relatively undermethylated in an inbred FVB/N strain. Individual methylation patterns of paternal alleles, and therefore all of the variation (nongenetic and genetic) in methylation patterns within an RSVIgmyc transgenic line, are established in early embryogenesis. For each mouse, the paternal RSVIgmyc allele is unmethylated at the day-3.5 blastocyst stage, and the final, adult methylation pattern is found no later than day 8.5 of embryogenesis. Because of the strong relationship between RSVIgmyc methylation and expression, the variation in methylation is also manifest as variation in transgene expression. These results identify embryonic de novo methylation as an important source of both genetic and nongenetic contributions to phenotypic variation and, as such, further our understanding of the developmental origin of imprinted genes.

Ovarian teratomas associated with the insertion of an imprinted transgene.

Ovarian teratomas are tumors that arise from female germ cells and are often a mixture of immature embryonal carcinoma cells and mature embryonic cells. Tissues derived from all three primary embryonic lineages (ectoderm, mesoderm, and endoderm) are typically found in the mature elements of a teratoma. In the case of the transgenic mouse line TG.KD, created with an imprinted transgene construct, malignant ovarian teratomas of a mixed germ cell tumor morphology occur in 15-20% of hemizygous female carriers of the transgene. The tumors frequently metastasize and can result in death of the mouse. Genetic analysis indicates that the tumors are associated with the transgenes integration site. Inbred FVB/N and female mice of other transgenic lines, also created in the inbred FVB/N strain with the same DNA construct as TG.KD, do not develop teratomas. In addition to teratomas, the integration of the transgene on Chromosome (Chr) 8 is associated with a perinatal lethality in homozygous transgenic carriers. The hemizygous genotypes of the teratomas suggest that they arise from early germ cells, prior to the completion of meiosis I.

Regulation of genomic imprinting by gametic and embryonic processes.

Parental genomic imprinting refers to the phenomenon by which alleles behave differently depending on the sex of the parent from which they are inherited. In the case of the murine transgene RSVIgmyc, imprinting is manifest in two ways: differential DNA methylation and differential expression. In inbred FVB/N mice, a transgene inherited from a male parent is undermethylated and expressed; a transgene inherited from the female parent is overmethylated and silent. Using a series of RSVIgmyc constructs and transgenic mice, we show that the imprinting of this transgene requires a cis-acting signal that is principally derived from the repeat sequences that make up the 3' portion of the murine immunoglobulin alpha heavy-chain switch region. Such imprinting is relatively independent of the site of transgene insertion but is influenced by the structure of the transgene itself. Imprinting is also modulated by genetic background. Detailed studies indicate that the paternal allele is undermethylated and expressed in inbred FVB/N mice and in heterozygous F1 FVB/N/C57Bl/6J mice but is overmethylated and silent in inbred C57Bl/6J mice. Consequently, the FVB/N genome appears to carry alleles of modulating genes that dominantly block methylation and permit expression of the paternally imprinted transgene. Furthermore, our results suggest that overmethylation is the default status of both parental alleles and that the paternal allele can be marked in trans by polymorphic factors that act in postblastocyst embryos.

Genomic imprinting: lessons from mouse transgenes.

Genomic imprinting is a non-Mendelian form of inheritance that results in an expression difference between the two parental alleles of an autosomal locus. The study of mouse transgenes has provided us with descriptions of a variety of imprinting or parent-of-origin effects, thereby anticipating similar inheritance phenomena in non-transgenic mice. Many mouse transgenes exhibit parent-of-origin behavior only on mixed strain backgrounds, whereas others are imprinted on inbred strain backgrounds. In the former cases, the parent-of-origin effects are due to strain-specific modifiers of DNA methylation and expression. These are inherited in a parent-specific fashion and exert their effects after fertilization. In the latter cases, true germline transgene imprinting, the creation of an imprinted locus occurs in a series of sequential steps. First, there is an erasure of the imprint from the previous generation in both male and female fetal germ cells. Second, upon completion of gametogenesis, distinctive methylation patterns have been placed on the transgene sequences of the two mature gametes. Third, only one of these inherited patterns is maintained in the early, pre-implantation embryo. The pattern of the other parental allele is erased. Finally, the methylation pattern of the alleles evolve in the later stages of development, but nonetheless the methylation difference (imprint) of the locus persists. Transgene imprinting behaviors, either on mixed strain backgrounds and on inbred genetic backgrounds, have counterparts in endogenous genetic phenomena.

The ontogeny of allele-specific methylation associated with imprinted genes in the mouse.

We have investigated the DNA methylation patterns in genomically imprinted genes of the mouse. Both Igf2 and H19 are associated with clear-cut regions of allele-specific paternal modification in late embryonic and adult tissues. By using a sensitive PCR assay, it was possible to follow the methylation state of individual HpaII sites in these genes through gametogenesis and embryogenesis. Most of these CpG moieties are not differentially modified in the mature gametes and also become totally demethylated in the early embryo in a manner similar to non-imprinted endogenous genes. Thus, the overall allele-specific methylation pattern at these sites must be established later during embryogenesis after the blastula stage. In contrast, sites in an Igf2r gene intron and one CpG residue in the Igf2 upstream region have allele-specific modification patterns which are established either in the gametes or shortly after fertilization and are preserved throughout pre-implantation embryogenesis. These studies suggest that only a few DNA modifications at selective positions in imprinted genes may be candidates for playing a role in the maintenance of parental identity during development.

Parental imprinting: potentially active chromatin of the repressed maternal allele of the mouse insulin-like growth factor II (Igf2) gene.

The mouse insulin-like growth factor II (Igf2) gene, which is located on distal chromosome 7 (Chr7), has been shown previously to undergo tissue-specific parental imprinting. This imprinting results in expression of Igf2 from the paternally inherited chromosome and repression of the maternally inherited allele in most tissues of the developing embryo. We are using embryos with the maternal duplication and paternal deficiency of distal Chr7 to characterize the mechanism that underlies repression of the maternal allele. We show that the chromatin of the 5' region of the repressed Igf2 allele is potentially active for transcription rather than heterochromatic. In particular, a CpG island that comprises one of the two strong promoters is unmethylated at both parental alleles, and DNase I hypersensitive sites in and around the strong promoters are consistently present on both parental chromosomes. In agreement with the chromatin state, primary transcripts from the maternal Igf2 allele have been detected at low but significant levels. These findings differ from observations in other instances of imprinting, namely, X-chromosome inactivation and transgene imprinting in mice. Although no parent-specific differences were detected in either DNA methylation or sensitivity to nucleases at these promoters, we have observed parental methylation differences in a region several kilobases upstream of the first exon. The differential activity of the parental Igf2 alleles could be achieved through epigenetic modifications situated outside the promoters or by subtle and yet unidentified modifications at the promoters.

The GABAA receptor beta 3 subunit gene: characterization of a human cDNA from chromosome 15q11q13 and mapping to a region of conserved synteny on mouse chromosome 7.

A cDNA encoding the human GABAA receptor beta 3 subunit has been isolated from a brain cDNA library and its nucleotide sequence has been determined. This gene, GABRB3, has recently been mapped to human chromosome 15q11q13, the region deleted in Angelman and Prader-Willi syndromes. The association of distinct phenotypes with maternal versus paternal deletions of this region suggests that one or more genes in this region show parental-origin-dependent expression (genetic imprinting). Comparison of the inferred human beta 3 subunit amino acid sequence with beta 3 subunit sequences from rat, cow, and chicken shows a very high degree of evolutionary conservation. We have used this cDNA to map the mouse beta 3 subunit gene, Gabrb-3, in recombinant inbred strains. The gene is located on mouse chromosome 7, very closely linked to Xmv-33 between Tam-1 and Mtv-1, where two other genes from human 15q11q13 have also been mapped. This provides further evidence for a region of conserved synteny between human chromosome 15q11q13 and mouse chromosome 7. Proximal and distal regions of mouse chromosome 7 show genetic imprinting effects; however, the region of homology with human chromosome 15q11q13 has not yet been associated with these effects.

The syntenic relationship between the critical deletion region for the Prader-Willi/Angelman syndromes and proximal mouse chromosome 7.

The deleted region of the proximal long arm of human chromosome 15, common to a large group of patients with the Prader-Willi and Angelman syndromes, has recently been defined. We have mapped to the mouse genome segments homologous to human probes found within and flanking this deletional region. These probes define a region of conserved synteny on proximal chromosome 7 of the mouse. Because the Prader-Willi and Angelman syndromes are postulated to result from genomic imprinting within the common deletion, these probes may define a genomically imprinted region on mouse chromosome 7.

Close linkage of a transgene insertion site to the steel (Sl) locus on mouse chromosome 10.

The characterization of the insertion sites of exogenous sequences in transgenic mice can identify loci that are potentially useful for the genetic analysis of the mammalian genome. We have found that the transgene insertion site in the transgenic line TG.EB is tightly linked with the Steel (Sl) locus on mouse chromosome 10. In a backcross between doubly heterozygous transgenic Sl (Tg.EB +/+ Sl) mice and wild-type mice, only one recombinant was found in 135 progeny (recombination percentage = 0.7 +/- 0.7). The recombination frequency of the transgene with marker loci known to flank Sl was consistent with a map position close to Sl. Genomic sequences that are adjacent to the transgene insertion site were cloned and found to be tightly linked with the Sl locus in interspecific crosses using nontransgenic mice. Recombination analysis utilizing the transgene insertion site locus was used to show that a recently identified hematopoietic growth factor is encoded at Sl. The cloned sequences from the transgene insertion site are polymorphic in inbred strains of mice and can be utilized to determine the genotype at Sl during early embryonic development. Further, they may be useful in characterizing the genomic region near Sl that is affected in Sl deletion mutants.