Differential activity of maternally and paternally derived chromosome regions in mice

Although both parental sexes contribute equivalent genetic information to the zygote, in mammals this information is not necessarily functionally equivalent. Diploid parthenotes possessing two maternal genomes are generally inviable, embryos possessing two paternal genomes in man may form hydatidiform moles, and nuclear transplantation experiments in mice have shown that both parental genomes are necessary for complete embryogenesis. Not all of the genome is involved in these parental effects, however, because zygotes with maternal or paternal disomy for chromosomes 1, 4, 5, 9, 13, 14 and 15 of the mouse survive normally. On the other hand, only the maternal X chromosome is active in mouse extraembryonic membranes, maternal disomy 6 is lethal, while non-complementation of maternal duplication/paternal deficiency or its reciprocal for regions of chromosome 2, 8 and 17 has been recognized. We report that animals with maternal duplication/paternal deficiency and its reciprocal for each of two particular chromosome regions show anomalous phenotypes which depart from normal in opposite directions, suggesting a differential functioning of gene loci within these regions. A further example of non-complementation lethality is also reported.     

CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus

The Insulin-like growth factor 2 (Igf2) and H19 genes are imprinted, resulting in silencing of the maternal and paternal alleles, respectively. This event is dependent upon an imprinted-control region two kilobases upstream of H19 (refs 1, 2). On the paternal chromosome this element is methylated and required for the silencing of H19 (refs 2-4). On the maternal chromosome the region is unmethylated and required for silencing of the Igf2 gene 90 kilobases upstream. We have proposed that the unmethylated imprinted-control region acts as a chromatin boundary that blocks the interaction of Igf2 with enhancers that lie 3' of H19 (refs 5, 6). This enhancer-blocking activity would then be lost when the region was methylated, thereby allowing expression of Igf2 paternally. Here we show, using transgenic mice and tissue culture, that the unmethylated imprinted-control regions from mouse and human H19 exhibit enhancer-blocking activity. Furthermore, we show that CTCF, a zinc finger protein implicated in vertebrate boundary function, binds to several sites in the unmethylated imprinted-control region that are essential for enhancer blocking. Consistent with our model, CTCF binding is abolished by DNA methylation. This is the first example, to our knowledge, of a regulated chromatin boundary in vertebrates.     

The mouse insulin-like growth factor type-2 receptor is imprinted and closely linked to the Tme locus.

T-associated maternal effect (Tme) is the only known maternal-effect mutation in the mouse. The defect is nuclear-encoded and embryos that inherit a deletion of the Tme locus from their mother die at day 15 of gestation. There are many genomically imprinted regions known in the mouse genome but so far no imprinted genes have been cloned. The Tme locus is absent in two chromosome-17 deletion mutants, Thp and the tLub2, and its position has been localized using these deletions to a 1-cM region. We report here that the genes for insulin-like growth factor type-2 receptor (Igf2r) and mitochondrial superoxide dismutase-2 (Sod-2) are absent from both deletions. Probes for these genes and for plasminogen (Plg) and T-complex peptide 1 (Tcp-1) were used in pulsed-field gel mapping to show that Tme must lie within a region of 800-1,100 kb. We also demonstrate that embryos express Igf2r only from the maternal chromosome, and that Tcp-1, Plg and Sod-2 are expressed from both chromosomes. Therefore Igf2r is imprinted and closely linked or identical to Tme.     

Birth of parthenogenetic mice that can develop to adulthood

Only mammals have relinquished parthenogenesis, a means of producing descendants solely from maternal germ cells. Mouse parthenogenetic embryos die by day 10 of gestation. Bi-parental reproduction is necessary because of parent-specific epigenetic modification of the genome during gametogenesis. This leads to unequal expression of imprinted genes from the maternal and paternal alleles. However, there is no direct evidence that genomic imprinting is the only barrier to parthenogenetic development. Here we show the development of a viable parthenogenetic mouse individual from a reconstructed oocyte containing two haploid sets of maternal genome, derived from non-growing and fully grown oocytes. This development was made possible by the appropriate expression of the Igf2 and H19 genes with other imprinted genes, using mutant mice with a 13-kilobase deletion in the H19 gene as non-growing oocytes donors. This full-term development is associated with a marked reduction in aberrantly expressed genes. The parthenote developed to adulthood with the ability to reproduce offspring. These results suggest that paternal imprinting prevents parthenogenesis, ensuring that the paternal contribution is obligatory for the descendant.     

Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene

The expression of the insulin-like growth factor 2 (Igf2) and H19 genes is imprinted. Although these neighbouring genes share an enhancer, H19 is expressed only from the maternal allele, and Igf2 only from the paternally inherited allele. A region of paternal-specific methylation upstream of H19 appears to be the site of an epigenetic mark that is required for the imprinting of these genes. A deletion within this region results in loss of imprinting of both H19 and Igf2 (ref. 5). Here we show that this methylated region contains an element that blocks enhancer activity. The activity of this element is dependent upon the vertebrate enhancer-blocking protein CTCF. Methylation of CpGs within the CTCF-binding sites eliminates binding of CTCF in vitro, and deletion of these sites results in loss of enhancer-blocking activity in vivo, thereby allowing gene expression. This CTCF-dependent enhancer-blocking element acts as an insulator. We suggest that it controls imprinting of Igf2. The activity of this insulator is restricted to the maternal allele by specific DNA methylation of the paternal allele. Our results reveal that DNA methylation can control gene expression by modulating enhancer access to the gene promoter through regulation of an enhancer boundary.     

Distinct physiological and behavioural functions for parental alleles of imprinted Grb10

Imprinted genes, defined by their preferential expression of a single parental allele, represent a subset of the mammalian genome and often have key roles in embryonic development, but also postnatal functions including energy homeostasis and behaviour. When the two parental alleles are unequally represented within a social group (when there is sex bias in dispersal and/or variance in reproductive success), imprinted genes may evolve to modulate social behaviour, although so far no such instance is known. Predominantly expressed from the maternal allele during embryogenesis, Grb10 encodes an intracellular adaptor protein that can interact with several receptor tyrosine kinases and downstream signalling molecules. Here we demonstrate that within the brain Grb10 is expressed from the paternal allele from fetal life into adulthood and that ablation of this expression engenders increased social dominance specifically among other aspects of social behaviour, a finding supported by the observed increase in allogrooming by paternal Grb10-deficient animals. Grb10 is, therefore, the first example of an imprinted gene that regulates social behaviour. It is also currently alone in exhibiting imprinted expression from each of the parental alleles in a tissue-specific manner, as loss of the peripherally expressed maternal allele leads to significant fetal and placental overgrowth. Thus Grb10 is, so far, a unique imprinted gene, able to influence distinct physiological processes, fetal growth and adult behaviour, owing to actions of the two parental alleles in different tissues.     

Parental imprinting of the mouse H19 gene.

THE mouse H19 gene encodes one of the most abundant RNAs in the developing mouse embryo. It is expressed at the blastocyst stage of development, and accumulates to high levels in tissues of endodermal and mesodermal origin (H. Kim, unpublished result). After birth the gene is expressed in all tissues except skeletal muscle. It lacks a common open reading frame in the 2.5-kilobase RNA, but has considerable nucleotide sequence similarity between the genes of rodents and humans. Expression of the gene in transgenic mice results in late prenatal lethality, suggesting that the dosage of its gene product is strictly controlled. The H19 gene maps to the distal segment of mouse chromosome 7, in a region that is parentally imprinted, a process by which genes are differentially expressed on the maternal and paternal chromosomes. We have now used an RNase protection assay that can distinguish between H19 alleles in four subspecies of Mus, to demonstrate that the H19 gene is parentally imprinted, with the active copy derived from the mother. This assay will be of general use in assaying allele-specific gene expression.     

Genetic imprinting suggested by maternal heterodisomy in nondeletion Prader-Willi syndrome

Prader-Willi syndrome (PWS) is the most common form of dysmorphic genetic obesity associated with mental retardation. About 60% of cases have a cytological deletion of chromosome 15q11q13 (refs 2, 3). These deletions occur de novo exclusively on the paternal chromosome. By contrast, Angelman syndrome (AS) is a very different clinical disorder and is also associated with deletions of region 15q11q13 (refs 6-8), indistinguishable from those in PWS except that they occur de novo on the maternal chromosome. The parental origin of the affected chromosomes 15 in these disorders could, therefore, be a contributory factor in determining their clinical phenotypes. We have now used cloned DNA markers specific for the 15q11q13 subregion to determine the parental origin of chromosome 15 in PWS individuals not having cytogenetic deletions; these individuals account for almost all of the remaining 40% of PWS cases. Probands in two families displayed maternal uniparental disomy for chromosome 15q11q13. This is the first demonstration that maternal heterodisomy--the presence of two different chromosome 15s derived from the mother--can be associated with a human genetic disease. The absence of a paternal contribution of genes in region 15q11q13, as found in PWS deletion cases, rather than a mutation in a specific gene(s) in this region may result in expression of the clinical phenotype. Thus, we conclude that a gene or genes in region 15q11q13 must be inherited from each parent for normal human development.     

Uniparental paternal disomy in a genetic cancer-predisposing syndrome.

The 11p15.5 region of human chromosome 11 seems to contain a locus or loci involved in congenital overgrowth anomalies as well as in the genesis of many tumours associated with the Beckwith-Wiedemann syndrome (BWS). Given the unusual differential parental allele involvement in the different aetiological forms of BWS and the loss of maternal alleles in associated tumours, we have now used 11p15.5 markers to determine the parental origin of chromosome 11 in eight sporadic cases of BWS. Probands in three informative families had uniparental paternal disomy for region 11p15.5. Further, an overall greatly increased frequency of homozygosity for several 11p15.5 markers in 21 sporadic BWS patients suggests that isodisomy probably accounts for an even higher proportion of BWS sporadic cases. This demonstrates that uniparental paternal disomy can be associated with a genetic cancer-predisposing syndrome.     

Maternal Rnf12/RLIM is required for imprinted X-chromosome inactivation in mice

Two forms of X-chromosome inactivation (XCI) ensure the selective silencing of female sex chromosomes during mouse embryogenesis. Imprinted XCI begins with the detection of Xist RNA expression on the paternal X?chromosome (Xp) at about the four-cell stage of embryonic development. In the embryonic tissues of the inner cell mass, a random form of XCI occurs in blastocysts that inactivates either Xp or the maternal X?chromosome (Xm). Both forms of XCI require the non-coding Xist RNA that coats the inactive X?chromosome from which it is expressed. Xist has crucial functions in the silencing of X-linked genes, including Rnf12 (refs 3, 4) encoding the ubiquitin ligase RLIM (RING finger LIM-domain-interacting protein). Here we show, by targeting a conditional knockout of Rnf12 to oocytes where RLIM accumulates to high levels, that the maternal transmission of the mutant X?chromosome (Δm) leads to lethality in female embryos as a result of defective imprinted XCI. We provide evidence that in Δm female embryos the initial formation of Xist clouds and Xp silencing are inhibited. In contrast, embryonic stem cells lacking RLIM are able to form Xist clouds and silence at least some X-linked genes during random XCI. These results assign crucial functions to the maternal deposit of Rnf12/RLIM for the initiation of imprinted XCI.     

Genomic imprinting determines methylation of parental alleles in transgenic mice

Mouse embryogenesis relies on the presence of both the maternal and the paternal genome for development to term. It has been proposed that specific modifications are imprinted onto the chromosomes during gametogenesis; these modifications are stably propagated, and their expression results in distinct and complementary contributions of the two parental genomes to the development of the embryo and the extraembryonic membranes. Genetic data further suggest that a substantial proportion of the genome could be subject to chromosomal imprinting, the molecular nature of which is unknown. We used random DNA insertions in transgenic mice to probe the genome for modified regions. The DNA methylation patterns of transgenic alleles were compared after transmission from mother or father in seven mouse strains carrying autosomal insertions of the same transgenic marker. One of these loci showed a clear difference in DNA methylation specific for its parental origin, with the paternally inherited copy being relatively undermethylated. This difference was observed in embryos on day 10 of gestation, but not in their extraembryonic membranes. Moreover, the methylation pattern was faithfully reversed upon each germline transmission to the opposite sex. Our findings provide evidence for heritable molecular differences between maternally and paternally derived alleles on mouse chromosomes.     

Insulin-IGF2 region on chromosome 11p encodes a gene implicated in HLA-DR4-dependent diabetes susceptibility

A class of alleles at the VNTR (variable number of tandem repeat) locus in the 5' region of the insulin gene (INS) on chromosome 11p is associated with increased risk of insulin-dependent diabetes mellitus (IDDM), but family studies have failed to demonstrate linkage. INS is thought to contribute to IDDM susceptibility but this view has been difficult to reconcile with the lack of linkage evidence. We thus investigated polymorphisms of INS and neighbouring loci in random diabetics, IDDM multiplex families and controls. HLA-DR4-positive diabetics showed an increased risk associated with common variants at polymorphic sites in a 19-kilobase segment spanned by the 5' INS VNTR and the third intron of the gene for insulin-like growth factor II (IGF2). As INS is the major candidate gene from this region, diabetic and control sequence were compared to identify all INS polymorphisms that could contribute to disease susceptibility. In multiplex families the IDDM-associated alleles were transmitted preferentially to HLA-DR4-positive diabetic offspring from heterozygous parents. The effect was strongest in paternal meioses, suggesting a possible role for maternal imprinting. Our results strongly support the existence of a gene or genes affecting HLA-DR4 IDDM susceptibility which is located in a 19-kilobase region of INS-IGF2. Our results also suggest new ways to map susceptibility loci in other common diseases.     

Seed development: Early paternal gene activity in Arabidopsis.

Both parental genomes are expressed during embryogenesis, although the time of activation of the paternally inherited genes varies between organisms. Results reported by Vielle-Calzada et al. have suggested that delayed activation of the paternal genome seems to be the rule in plant development. We find, however, that during early embryogenesis in Arabidopsis, paternal genes are expressed and are sufficient for normal development. Our findings indicate that there is no overall maternal control of early embryogenesis, and that the contribution of the parental alleles needs to be assessed for each gene individually.     

Influence of parental chromosomes on spatial specificity in androgenetic----parthenogenetic chimaeras in the mouse

The presence of both parental genomes is essential for development to term in the mouse embryo probably because of germline-specific modifications of homologous chromosomes. Neither androgenetic nor parthenogenetic embryos can by themselves develop to term; any post-implantation embryos they produce have opposite phenotypes, which reflects the presence of complementary information in parental chromosomes. The development of androgenetic----parthenogenetic chimaeras is of considerable interest because in this case both parental chromosomes are available even though they are in separate cells. We demonstrate here that in post-implantation chimaeric fetuses, the expression of parental information results in spatial specificity so that parthenogenetic cells are confined to the embryo but the trophoblast consists almost entirely of androgenetic cells. The yolk sac contains both cell types. However, there is incomplete functional complementation because the chimaeras do not reach term. Although failure to reach term may occur partly because of inadequate intermingling and interactions between embryonic cells, it is more likely that further control of mouse development depends on the presence of both sets of chromosomes within the same cells.