Imprinting and the initiation of gene silencing in the germ line.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
M A Surani
Publications and source records attributed to M A Surani.
Identification of the Meg1/Grb10 imprinted gene on mouse proximal chromosome 11, a candidate for the Silver-Russell syndrome gene.
In a systematic screen for maternally expressed imprinted genes using subtraction hybridization with androgenetic and normal fertilized mouse embryos, seven candidate maternally expressed genes (Megs) have been isolated, including the H19 and p57(Kip2) genes that are known to be maternally expressed. Herein, we demonstrate that an imprinted gene, Meg1, is apparently identical to Grb10 (growth factor receptor-bound protein 10), which is located on mouse proximal chromosome 11. Grb10 protein was reported to bind to the insulin receptor and/or the insulin-like growth factor (IGF) I receptor via its src homology 2 domain and to inhibit the associated tyrosine kinase activity that is involved in the growth promoting activities of insulin and IGFs (IGF-I and -II). Thus, it is probable that Meg1/Grb10 is responsible for the imprinted effects of prenatal growth retardation or growth promotion caused by maternal or paternal duplication of proximal chromosome 11 with reciprocal deficiencies (MatDp.prox11 or PatDp.prox11), respectively. In the human, it has been reported that the maternal uniparental disomy 7 is responsible for the Silver-Russell syndrome (SRS) whose effects include pre- and postnatal growth retardation and other dysmorphologies. The human homologue GRB10 on chromosome 7q11.2-12 is a candidate gene for Silver-Russell syndrome.
Animals↗
Imprinting and gene silencing in mice and Drosophila.
H19 and Igf2 are located within a large imprinting domain that confers monoallelic silencing of parental alleles. The silent paternal allele of H19 is hypermethylated and relatively resistant to nucleases. Using a 130 kb yeast artificial chromosome clone, appropriate imprinting of both H19 and Igf2 was observed at single insert loci in transgenic mice. Imprinting was also observed for H19-lacZ transgenes containing 4 kb of upstream sequence, but only at multicopy loci. The H19 RNA is therefore not essential for imprinting. When the H19-lacZ transgene was introduced into Drosophila, a 1.2 kb region was identified within the 4 kb upstream flank that functioned as a bi-directional silencer. This cis element is located within a region that is apparently necessary for imprinting in mice. These studies suggest an evolutionarily conserved mechanism for gene silencing in Drosophila and imprinting in mice. We propose a new model for imprinting of H19 and Igf2 in mice in which silencing of H19 is the default state, and activation of the maternal allele requires a specific activator element.
Animals↗
Epigenotype switching of imprintable loci in embryonic germ cells.
Expression of imprinted genes is dependent on their parental origin. This is reflected in the heritable differential methylation of parental alleles. The gametic imprints are however reversible as they do not endure for more than one generation. To investigate if the epigenetic changes in male and female germ line are similar or not, we derived embryonic germ (EG) cells from primordial germ cells (PGCs) of day 11.5 and 12.5 male and female embryos. The results demonstrate that they have an equivalent epigenotype. First, chimeras made with EG cells derived from both male and female embryos showed comparable fetal overgrowth and skeletal abnormalities, which are similar to but less severe than those induced by androgenetic embryonic stem (ES) cells. Thus, EG cells derived from female embryos resemble androgenetic ES cells more than parthenogenetic cells. Furthermore, the methylation status of both alleles of a number of loci in EG cells was similar to that of the paternal allele in normal somatic cells. Hence, both alleles of Igf2r region 2, Peg1/Mest, Peg3, Nnat were consistently unmethylated in EG cells as well as in the primary embryonic fibroblasts (PEFs) rescued from chimeras. More strikingly, both alleles of p57kip2 that were also unmethylated in EG cells, underwent de novo methylation in PEFs to resemble a paternal allele in somatic cells. The exceptions were the H19 and Igf2 genes that retained the methylation pattern in PEFs as seen in normal somatic tissues. These studies suggest that the initial epigenetic changes in germ cells of male and female embryos are similar.
Animals↗
Mechanism of imprinting on mouse distal chromosome 7.
Genomic imprinting is an epigenetic mode of gene regulation that results in expression of the autosomal 'imprinted' genes from only a single allele, determined exclusively by parental origin. To date over 20 imprinted genes have been identified in mouse and man and these appear to lie in clusters in restricted regions on a subset of chromosomes. This may be a critical feature of imprinting suggesting a domain-type mode of regulation. Imprinted domains are replicated asynchronously, show sex-specific meiotic recombination frequencies and have CpG-rich regions that are differentially methylated, often associated with the imprinted genes themselves. Mouse distal chromosome 7 is one such domain, containing at least nine imprinted genes spanning over 1 Mb of DNA. For the maternally expressed p57Kip2 gene, passage through the female germline is essential to generate the active state, whereas passage through the male germline is needed to force the maternally expressed H19 gene into an inactive state. It is therefore possible that the mouse distal chromosome 7 imprinted domain is actually composed of two or more independently regulated subdomains.
Animals↗
Abnormal maternal behaviour and growth retardation associated with loss of the imprinted gene Mest.
Mest (also known as Peg1), an imprinted gene expressed only from the paternal allele during development, was disrupted by gene targeting in embryonic stem (ES) cells. The targeted mutation is imprinted and reversibly silenced by passage through the female germ line. Paternal transmission activates the targeted allele and causes embryonic growth retardation associated with reduced postnatal survival rates in mutant progeny. More significantly, Mest-deficient females show abnormal maternal behaviour and impaired placentophagia, a distinctive mammalian behaviour. Our results provide evidence for the involvement of an imprinted gene in the control of adult behaviour.
Adult↗
Transient retardation in embryo growth in normal female mice made pregnant by males whose testes had been heated.
In three separate experiments, using three different strains of mice, when normal females were mated by males whose testes had been heated once to 42 degrees C for 20 min, the embryos at 10.5 days post-coitum were approximately 20% smaller than control embryos. In one experiment, the difference was still present, although proportionately less, at 15.5 and 18.5 days and, in another experiment, a difference could be seen in 11.5 and 13.5 day old embryos but not in 12.5, 14.5, 15.5 or 18.5 day old embryos. The frequency of mating and pregnancy rates were unaffected. In one experiment, the time available for mating was restricted to 4 h instead of overnight, without effect on the result. In another experiment, other males were heated for 30 min, and these showed a period of infertility from 10 to 32 days later, preceded and followed by the production of smaller than normal embryos; litter size was also reduced in the period after the return of fertility in these animals. The yolk sacs and the trophoblasts of the embryos sired by the heated males were also slightly smaller than those sired by the controls in the two experiments in which these were measured. The pattern of weight reduction is thus different from that seen in gynogenetic embryos or when the gene for insulin-like growth factor (IGF)-II is disrupted, and suggests a reduction in embryo growth at the earlier stages, with compensatory growth occurring later in pregnancy.
Animals↗
Enhancer-dependent, locus-wide regulation of the imprinted mouse insulin-like growth factor II gene.
The mouse insulin-like growth factor II (IGF-II) gene is subject to parental imprinting and is predominantly expressed from the paternal chromosome. This allele-specific expression is modified further by cell type, developmental stage, and growth conditions. We show that the ratio of the three major IGF-II mRNAs, each produced from a distinct promoter, is consistent in a variety of tissues and cells representing different modes and phases of the complex regulation. Nuclear run-on assays show that the major changes in total IGF-II mRNA level occur at the level of transcription. Moreover, a targeted disruption of the endoderm-specific enhancers, located 90 kb away from the gene, affects all promoters. The dependency of the promoters on distal enhancers is also shown by transgenesis experiments. Our findings suggest that enhancer-dependent, locus-wide mechanisms play a major role in the coordinate regulation of the multiple IGF-II promoters.
Animals↗
Embryonic germ cells induce epigenetic reprogramming of somatic nucleus in hybrid cells.
Genomic reprogramming of primordial germ cells (PGCs), which includes genome-wide demethylation, prevents aberrant epigenetic modifications from being transmitted to subsequent generations. This process also ensures that homologous chromosomes first acquire an identical epigenetic status before an appropriate switch in the imprintable loci in the female and male germ lines. Embryonic germ (EG) cells have a similar epigenotype to PGCs from which they are derived. We used EG cells to investigate the mechanism of epigenetic modifications in the germ line by analysing the effects on a somatic nucleus in the EG-thymic lymphocyte hybrid cells. There were striking changes in methylation of the somatic nucleus, resulting in demethylation of several imprinted and non-imprinted genes. These epigenetic modifications were heritable and affected gene expression as judged by re-activation of the silent maternal allele of Peg1/Mest imprinted gene in the somatic nucleus. This remarkable change in the epigenotype of the somatic nucleus is consistent with the observed pluripotency of the EG-somatic hybrid cells as they differentiated into a variety of tissues in chimeric embryos. The epigenetic modifications observed in EG-somatic cell hybrids in vitro are comparable to the reprogramming events that occur during germ cell development.
Alleles↗
The role of the paternal genome in the development of the mouse germ line.
The mouse germ line originates at 6.5 days post coitum (dpc) in the proximal epiblast, apparently in response to signals from the primitive endoderm or the extraembryonic mesoderm [1,2]. Some studies have implied a significant role for imprinted genes in germ-line development [3,4]. These genes, whose expression is determined by their parental origin [5], serve complementary functions during mammalian development [6-9] and exert striking reciprocal phenotypic effects on androgenetic (AG: two paternal genomes) and parthenogenetic (GG/PG: two maternal genomes) cells [3,4,10]. This may include a fundamental effect on germ-cell development because PG but not AG cells can differentiate into viable gametes [3,4,11], suggesting that the maternal genome is obligatory for development of the mammalian germ line. Here we show unequivocally that AG cells can differentiate into germ cells, and that in chimeras with normal cells they produce functional sperm. These studies establish that the paternal and maternal genomes can individually provide both the signal and the response required for the specification of germ cells in mammals.
Animals↗
Structure and expression of the mouse L23mrp gene downstream of the imprinted H19 gene: biallelic expression and lack of interaction with the H19 enhancers.
The human L23 (mitochondrial)-related protein gene, located 40 kb downstream of the imprinted H19 gene, is biallelically expressed. We have cloned and characterized its mouse homolog, L23mrp, which maps to the conserved syntenic region on mouse chromosome 7. The promoter of L23mrp is a CpG island that is transcribed ubiquitously, but at different levels, in different fetal tissues. Allele-specific expression analysis revealed that both parental alleles are equally active. Since the enhancers located between H19 and L23mrp had been shown to be involved in the imprinted expression of Ins-2, Igf-2, and H19, we asked whether they also influence L23mrp. Analysis of mice with a targeted deletion of the enhancers demonstrated that they were not disrupted in the expression of L23mrp. These findings indicate that L23mrp is functionally insulated from the Ins-2/Igf-2/H19 domain in terms of both imprinting and enhancer action.
Alleles↗
Genetic and functional analysis of neuronatin in mice with maternal or paternal duplication of distal Chr 2.
Functional differences between parental genomes are due to differential expression of parental alleles of imprinted genes. Neuronatin (Nnat) is a recently identified paternally expressed imprinted gene that is initially expressed in the rhombomeres and pituitary gland and later more widely in the central and peripheral nervous system mainly in postmitotic and differentiating neuroepithelial cells. Nnat maps to distal chromosome (Chr) 2, which contains an imprinting region that causes morphological abnormalities and early neonatal lethality. More detailed mapping analysis of Nnat showed that it is located between the T26H and T2Wa translocation breakpoints which is, surprisingly, proximal to the reported imprinting region between the T2Wa and T28H translocation breakpoints, suggesting that there may be two distinct imprinting regions on distal chromosome 2. To investigate the potential role of Nnat, we compared normal embryos with those which were PatDp.dist2.T26H (paternal duplication/maternal deficiency of chromosome 2 distal to the translocation breakpoint T26H) and MatDp.dist2.T26H. Expression of Nnat was detected in the PatDp.dist2.T26H embryos, where both copies of Nnat are paternally inherited, and normal embryos but no expression was detected in the MatDp.dist2.T26H embryos with the two maternally inherited copies. The differential expression of Nnat was supported by DNA methylation analysis with the paternally inherited alleles being unmethylated and the maternal alleles fully methylated. Although experimental embryos appeared grossly similar phenotypically in the structures where expression of Nnat was detected, differences in folding of the cerebellum were observed in neonates, and other more subtle developmental or behavioral effects due to gain or loss of Nnat cannot be ruled out.
Animals↗
Mapping of the Fas-associated factor 1 gene, Faf1, to mouse chromosome 4C6 by FISH.
Explore the source record for details and available documents.
Adaptor Proteins, Signal Transducing↗
Mapping of the eukaryotic initiation factor eIF-1A gene, Eif1a, to mouse chromosome 12D-E by FISH.
Explore the source record for details and available documents.
Animals↗
An imprinting element from the mouse H19 locus functions as a silencer in Drosophila.
Genomic imprinting as originally described in Sciara is displayed by many organisms. In mammals, X-inactivation and the parent-of-origin-specific silencing of imprinted genes are examples of this phenomenon. A heritable chromatin structural modification may be the critical mechanism in such instances of chromosome condensation and preferential gene inactivation. H19 is an imprinted gene in which the repressed paternal allele is hypermethylated and the compacted chromatin is relatively resistant to digestion by nucleases. In order to uncover underlying conserved epigenetic mechanisms we have introduced a mouse H19 transgene into Drosophila. We show here that a 1.2-kb H19 upstream sequence functions in cis as a parent-of-origin independent silencing element in Drosophila. Strikingly, this cis-acting element is located within an upstream region that is necessary for H19 imprinting in mice. These results suggest involvement of an evolutionary conserved mechanism in both genes silencing in Drosophila and imprinting in mice.
Animals↗
Genomic structure and parent-of-origin-specific methylation of Peg1.
We previously identified Peg1/Mest as a novel paternally expressed gene in the developing mouse embryo. The human PEG1 gene was recently assigned to 7q32 and shown to be imprinted and paternally expressed. Therefore, PEG1 deficiency could participate in the aetiology of pre- and post-natal growth retardation associated with maternal uniparental disomy 7 in humans. We have now initiated the characterization of the Peg1 locus in order to identify and dissect cis-acting elements implicated in its imprinted monoallelic expression. The genomic structure of Peg1 as well as the DNA sequence of the 5'-end of the gene, including 2.4 kb of promoter sequences and covering the first 2 exons, have been determined. Important sequence elements, such as a CpG island spanning exon 1 and direct repeats, are identified and discussed. To address the role of epigenetic modifications in the imprinting of Peg1, a methylation analysis of the Peg1 gene is presented. Partially methylated cytosine residues in 13.5 d.p.c. embryos and undifferentiated ES cells were identified. Using embryos carrying a targetted mutation at the Peg1 locus, we show that this partial promoter methylation pattern reflects a strict parent-of-origin-specific differential methylation: the expressed paternal allele is unmethylated, whereas the silenced maternal allele is fully methylated at the CpG sites studied. That the gametes carry the epigenetic information necessary to lay down this allele-specific methylation pattern is suggested by analysis of DNA isolated from sperm and parthenogenetic embryos.
Animals↗
Imprinting of Igf2 and H19 from a 130 kb YAC transgene.
A stringent test for imprint control elements is to examine their function at ectopic loci in transgenic experiments. Igf2 and H19 are part of a larger imprinting region and as a first step, we examined these reciprocally imprinted genes in transgenic experiments using a 130 kb YAC clone. After paternal inheritance, H19 was appropriately repressed and Igf2 was expressed, irrespective of copy number or genetic background. After maternal inheritance H19 was consistently expressed, albeit with some variability. The levels of H19 expression per copy of the transgene inversely correlated with Igf2 (-lacZ) expression in cis. The consistent imprinting of H19 from this YAC contrasts with the previously described imprinting of mini-H19 transgenes, which only occurs at multi-copy loci, is inconsistent, and is prone to genetic background effects. We propose a novel model in which silencing of the H19 gene is the default state and its activation after maternal inheritance is the key mechanistic event for imprinting in this region. In addition, in situ analysis of the Igf2-lacZ reporter indicates that additional mesoderm-specific enhancers are present within the YAC clone. No obvious phenotype was detected from the excess gene dosage of H19.
Animals↗
Genomic imprinting and the differential roles of parental genomes in brain development.
Certain genes are expressed either from the maternal or the paternal genome as a result of genomic imprinting, a process that confers functional differences on parental genomes during mammalian development. In this study we focus on the cumulative effects of imprinted genes on brain development by examining the fate of androgenetic (Ag: duplicated paternal genome) and parthenogenetic/gynogenetic (Pg/Gg: duplicated maternal genome) cells in chimeric embryos. Striking cell autonomous differences in the phenotypic properties of the uniparental cells were observed. Ag cells contributed substantially to the hypothalamic structures and not the cortex. By contrast, Pg/Gg cells contributed substantially to the cortex, striatum and hippocampus but not to the hypothalamic structures. Furthermore growth of the brain was enhanced by Pg/Gg and retarded by Ag cells. We propose that genomic imprinting may be responsible for a change in strategy controlling brain development in mammals. In particular, genomic imprinting may have facilitated a rapid non-linear expansion of the brain, especially the cortex, during development over evolutionary time.
Animals↗