Genomic imprinting mechanisms in embryonic and extraembryonic mouse tissues.

Imprinted genes in mice and humans mainly occur in clusters that are associated with differential DNA methylation of an imprint control element (ICE) and at least one nonprotein-coding RNA (ncRNA). Imprinted gene silencing is achieved by parental-specific insulator activity of the unmethylated ICE mediated by CTCF (CCCTC-binding factor) binding, or by ncRNA expression from a promoter in the unmethylated ICE. In many imprinted clusters, some genes, particularly those located furthest away from the ICE, show imprinted expression only in extraembryonic tissues. Recent research indicates that genes showing imprinted expression only in extraembryonic tissues may be regulated by different epigenetic mechanisms compared with genes showing imprinted expression in extraembryonic tissues and in embryonic/adult tissues. The study of extraembryonic imprinted expression, thus, has the potential to illuminate novel epigenetic strategies, but is complicated by the need to collect tissue from early stages of mouse development, when extraembryonic tissues may be contaminated by maternal cells or be present in limited amounts. Research in this area would be advanced by the development of an in vitro model system in which genetic experiments could be conducted in less time and at a lower cost than with mouse models. Here, we summarize what is known about the mechanisms regulating imprinted expression in mouse extraembryonic tissues and explore the possibilities for developing an in vitro model.

Genetic analysis of the organic cation transporter genes Orct2/Slc22a2 and Orct3/Slc22a3 reduces the critical region for the t haplotype mutant t(w73) to 200 kb.

Here we report an analysis of two candidate genes for the t(w73) implantation mutation. The t(w73) gene maps to a 20-cM region of mouse Chromosome (Chr) 17 known as the t-complex, which exists in a wild-type and t haplotype form in present-day mice. The t haplotype variants contain several mutant alleles affecting male fertility and embryonic viability and offer the opportunity to identify genes critical for these processes. t(w73) homozygous embryos are defective in trophoblast production and fail to implant adequately, with death occurring at approximately 7.5 days post coitum (pc). Two recently described organic cation transporter genes, Slc22a2 (Orct2) and Slc22a3 (Orct3), fulfill criteria predicted for t(w73) candidate genes, since both map to the previously defined 500-kb t(w73) minimal region and both are also expressed in 7.5 days pc post-implantation embryos. The genomic locus of the Orct2 gene appears similar in wild-type and t(w73) chromosomes. In contrast, the genomic locus of Orct3 is amplified and displays an altered expression profile in all t haplotype variant chromosomes tested. In addition, Orct3 shows a t(w73) specific polymorphism. To test whether either Orct2 or Orct3 is involved in the t(w73) phenotype, we have performed a genetic rescue experiment using YAC transgenes overexpressing Orct2, and genetic complementation with an allele in which the Orct3 gene was inactivated by homologous recombination. The results eliminate both Orct2 and Orct3 as candidates and further reduce the critical region containing the t(w73) mutant from 500 kb to 200 kb.

Bidirectional action of the Igf2r imprint control element on upstream and downstream imprinted genes.

Imprinting of the maternally-expressed Igf2r gene is controlled by an intronic imprint control element (ICE) known as Region2 that contains the promoter of the noncoding Air RNA, whose transcript overlaps the silenced paternal Igf2r promoter in an antisense orientation. Two novel imprinted genes, Slc22a2 and Slc22a3 are described here that lie 110 and 155 kb 3' to Igf2r and that are not overlapped by the Air transcript but are regulated by the Igf2r-ICE, as previously shown for Igf2r. These results identify a new cluster of imprinted genes whose repression by the bidirectional action of the Region2-ICE is independent of transcript overlap by the Air RNA.

Investigation of elements sufficient to imprint the mouse Air promoter.

Imprinted maternal-allele-specific expression of the mouse insulin-like growth-factor type 2 receptor (Igf2r) gene depends on a 3.7-kb element named region 2, located in the second intron of the gene. Region 2 carries a maternal-allele-specific methylation imprint and contains an imprinted CpG island promoter (Air) that expresses a noncoding antisense RNA from the paternal inherited allele only. Here, we use transgenes to test the minimal requirements for imprinting of Air and to test if the action of region 2 is restricted to Igf2r. Transgenes up to 9 kb with Air as a single promoter are expressed but not imprinted. When coupled to the Igf2r CpG island promoter on a 44-kb transgene, Air was imprinted in one of three lines. However, Air on a 4.6-kb fragment is also imprinted in 2 of 14 lines when inserted in an intron of an adenine phosphoribosyltransferase (Aprt) transgene, and in one line, the imprinted methylation and expression of Air have been transferred onto the Aprt CpG island promoter. These data suggest that a dual CpG island promoter setting may facilitate Air imprinting as a short transgene and also show that Air can transfer imprinting onto other genes. However, for reliable Air imprinting, elements are necessary that are located outside a 44-kb region spanning the Air-Igf2r promoters.

Impaired activity of the extraneuronal monoamine transporter system known as uptake-2 in Orct3/Slc22a3-deficient mice.

Two uptake systems that control the extracellular concentrations of released monoamine neurotransmitters such as noradrenaline and adrenaline have been described. Uptake-1 is present at presynaptic nerve endings, whereas uptake-2 is extraneuronal and has been identified in myocardium and vascular and nonvascular smooth muscle cells. The gene encoding the uptake-2 transporter has recently been identified in humans (EMT), rats (OCT3), and mice (Orct3/Slc22a3). To generate an in vivo model for uptake-2, we have inactivated the mouse Orct3 gene. Homozygous mutant mice are viable and fertile with no obvious physiological defect and also show no significant imbalance of noradrenaline or dopamine. However, Orct3-null mice show an impaired uptake-2 activity as measured by accumulation of intravenously administered [(3)H]MPP(+) (1-methyl-4-phenylpyridinium). A 72% reduction in MPP(+) levels was measured in hearts of both male and female Orct3 mutant mice. No significant differences between wild-type and mutant mice were found in any other adult organ or in plasma. When [(3)H]MPP(+) was injected into pregnant females, a threefold-reduced MPP(+) accumulation was observed in homozygous mutant embryos but not in their placentas or amniotic fluid. These data show that Orct3 is the principal component for uptake-2 function in the adult heart and identify the placenta as a novel site of action of uptake-2 that acts at the fetoplacental interface.

Non-imprinted Igf2r expression decreases growth and rescues the Tme mutation in mice.

In the mouse the insulin-like growth factor receptor type 2 gene (Igf2r) is imprinted and maternally expressed. Igf2r encodes a trans-membrane receptor that transports mannose-6-phosphate tagged proteins and insulin-like growth factor 2 to lysosomes. During development the receptor reduces the amount of insulin-like growth factors and thereby decreases embryonic growth. The dosage of the gene is tightly regulated by genomic imprinting, leaving only the maternal copy of the gene active. Although the function of Igf2r in development is well established, the function of imprinting the gene remains elusive. Gene targeting experiments in mouse have demonstrated that the majority of genes are not sensitive to gene dosage, and mice heterozygous for mutations generally lack phenotypic alterations. To investigate whether reduction of Igf2r gene dosage by genomic imprinting has functional consequences for development we generated a non-imprinted allele (R2Delta). We restored biallelic expression to Igf2r by deleting a critical element for repression of the paternal allele (region 2) in mouse embryonic stem cells. Maternal inheritance of the R2Delta allele has no phenotype; however, paternal inheritance results in biallelic expression of Igf2r, which causes a 20% reduction in weight late in embryonic development that persists into adulthood. Paternal inheritance of the R2Delta allele rescues the lethality of a maternally inherited Igf2r null allele and a maternally inherited Tme (T-associated maternal effect) mutation. These data show that the biological function of imprinting Igf2r is to increase birth weight and they also establish Igf2r as the Tme gene.

The extraneuronal monoamine transporter Slc22a3/Orct3 co-localizes with the Maoa metabolizing enzyme in mouse placenta.

Monoamine clearance is a combined function of uptake mechanisms in the plasma membrane with intracellular metabolizing enzymes. Two different uptake mechanisms have been described. Uptake(1) is located in presynaptic neurones, whereas uptake(2) is extraneuronal. Recently, the Slc22a3/Orct3 gene was identified as the extraneuronal monoamine transporter. In mouse embryonic development Orct3 expression is restricted to the placenta, which is also a site of expression of neuronal transporters. We have used RNA blots and in situ hybridization to examine the expression of Orct3 and other members of the monoamine uptake and metabolizing pathways in mouse placenta. The results show that Orct3 expression overlaps that of the monoamine metabolizing enzyme Maoa in the labyrinth layer of the placenta with an expression pattern distinct from that of the neuronal transporters Slc6a2/Net and Slc6a4/Sert.

The imprinted antisense RNA at the Igf2r locus overlaps but does not imprint Mas1.

The gene encoding the insulin-like growth-factor type-2 receptor (Igf2r) is maternally expressed and imprinted. A CpG island in Igf2r intron 2 that carries a maternal-specific methylation imprint was shown in a transgenic model to be essential for Igf2r imprinting and for the production of an antisense RNA from the paternal allele. We report here that the endogenous region2 is the promoter for this antisense RNA (named Air, for antisense Igf2r RNA) and that the 3' end lies 107,796 bp distant in an intron of the flanking, but non-imprinted, gene Mas1.

The uniqueness of the imprinting mechanism.

An epigenetic imprinting mechanism that is based on a gamete-specific methylation imprint restricts expression of a subset of mammalian genes to one parental chromosome. Recent results suggest that imprints may act only indirectly to induce monoallelic expression of coding genes. Instead, atypical non-coding RNAs appear to be a primary target of the imprints, and their parental-specific repression correlates with parental-specific expression of linked coding genes.

Cloning of the mouse and human solute carrier 22a3 (Slc22a3/SLC22A3) identifies a conserved cluster of three organic cation transporters on mouse chromosome 17 and human 6q26-q27.

Here we report the isolation of the mouse and human solute carrier genes Slc22a3/SLC22A3. Slc22a3 is specifically expressed in placenta, but the levels of expression decline toward the end of gestation. A BAC contig spanning the mouse Slc22a3 gene was constructed, and Slc22a3 was mapped between the Igf2r and Plg genes in close association with two additional members of the Slc22a gene family, mouse Slc22a1 and Slc22a2. A partial cDNA sequence of the human SLC22A3 gene was reconstituted from sequenced EST clones. SLC22A3 is expressed in first-trimester and term placenta, but also in skeletal muscle, prostate, aorta, liver, fetal lung, salivary gland, and adrenal gland. Using a somatic cell hybrid panel and a human YAC clone, SLC22A3 was mapped to the syntenic region on human chromosome 6q26-q27, between the IGF2R and APO(a)-like genes. SLC22A1 and SLC22A2 localized to the same locus, demonstrating the conservation of the close physical linkage of these three organic cation transporter genes in mouse and human.

Imprinting of the mouse Igf2r gene depends on an intronic CpG island.

Gametic imprinting is a developmental process that uses cis-acting epigenetic mechanisms to induce parental-specific expression in autosomal and X-linked genes. From the limited knowledge we have of imprinted genes it seems clear that they play a role in regulating the growth and development of the placenta and embryo in utero, and also in the early post-natal phase. The biological function of imprinting in mammals is not yet understood but it may act to regulate the supply of nutrition to the embryo in order to maintain a balance between fetal demands and maternal resources. The molecular basis of imprinting any gene has not yet been disclosed. However. the current model proposes that single or clustered genes should be associated with a specific imprinting element which carries a reversible epigenetic imprinting signal, inherited from one parent and maintained on the same parental chromosome in diploid cells. A common imprinting recognition sequence has not been identified amongst the 20 known imprinted genes, but such an element is thought to exist from observations of mouse transgenes and transgenes of the imprinted H19 gene, which have shown that short DNA sequences can maintain imprinted expression at different chromosome locations. However, it is also clear from gene deletion experiments in the mouse and the analysis of deletions in the human Prader-Willi/Angelman syndromes that imprinted genes are also influenced by distant DNA sequences. We have earlier proposed that CG rich sequences resembling CpG islands, which are associated with many imprinted genes and often subject to parental-specific methylation, could act as a common imprinting element. The mouse insulin-like growth factor type 2 receptor (Igf2r) gene contains a CpG island known as region2, in the second intron. Region2 was proposed as the imprinting element of this gene because it inherited a methylation imprint from the female gamete that was maintained only on the maternal chromosome in diploid cells. We have used YAC (yeast artificial chromosome) transgenes carrying the complete Igf2r locus, to test if imprinting and parental-specific methylation of the mouse Igf2r gene is maintained when transferred to other chromosomal locations and to test whether imprinting is dependent on the intronic CpG island proposed as the imprinting element for this gene. Gametic imprints are epigenetic modifications which are imposed onto the gametic chromosomes and cause parental-specific differences in the expression of a small number of genes in the embryo. As a consequence, correct imposition of the imprints in the parental germlines is a prerequisite for successful development of mammals and any anomaly in the expression of imprinted genes is often accompanied by aberration of embryonic growth. The link between imprinting and growth regulation is best exemplified by the Igf2 and Igf2r genes. Both genes show parental-specific expression patterns in the embryo. Igf2 is a general embryonic mitogen, and mice lacking Igf2 are markedly reduced in size (DeChiara, T.M., Robertson, E.J., Efstratiadis, A., 1991. Parental imprinting of the mouse insulin-like growth factor II gene. Cell 64, 849-859). Igf2r acts to fine tune the amount of growth factor, and embryos lacking this gene show overgrowth and die perinatally (Lau, M.M., Stewart, C.E., Liu, Z., Bhatt, H., Rotwein, P., Stewart, C.L., 1994. Loss of the imprinted IGF2/cation-independent mannose 6-phosphate receptor results in fetal overgrowth and perinatal lethality. Genes Dev. 8, 2953 2963; Wang, Z.Q., Fung, M.R., Barlow, D.P., Wagner, E.F., 1994. Regulation of embryonic growth and lysosomal targeting by the imprinted Igf2/Mpr gene. Nature 372, 464-467). The phenomenon of gametic imprinting is predicted to arise by an unusual regulation of the imposition and erasure of epigenetic modifications. There are three events which are required for imprinting: firstly, the imprint must be imposed in one of the gametes before fertilisat

Making sense of imprinting the mouse and human IGF2R loci.

The mouse and human IGF2R genes are similar in terms of expression pattern, gene structure and organization. Both genes have features that are common to imprinted genes. These common features are allele-specific methylation and replication asynchrony, plus the ability to restrict expression to one parental allele in diploid cells despite the presence of two functional parental alleles. In inbred laboratory mice Igf2r is initially expressed from both parental chromosomes in preimplantation embryos, it then shows maternal-specific monoallelic expression in all tissues of the postimplantation embryo and adult. The human gene is similarly monoallelically expressed in preterm postimplantation embryonic tissues (preimplantation embryos have not been examined). The behaviour of the human gene then diverges from that observed in inbred mice because it shows biallelic expression in term embryonic tissues and in the adult. An extra difference displayed by the human gene is that monoallelic expression is polymorphic and only occurs in 50% of individuals. The mechanism of IGF2R imprinting will be discussed with relevance to these similarities and differences between the mouse and human genes.

Imprinted expression of the Igf2r gene depends on an intronic CpG island.

Gametic imprinting is a developmental process that induces parental-specific expression or repression of autosomal and X-chromosome-linked genes. The mouse Igf2r gene (encoding the receptor for insulin- like growth factor type-2) is imprinted and is expressed from the maternal allele after embryonic implantation. We previously proposed that methylation of region 2, a region rich in cytosine-guanine doublets (a 'CpG island') in the second intron of Igf2r, is the imprinting signal that maintains expression of the maternal allele. Here we use mouse transgenes to test the role of region 2 and the influence of chromosome location on Igf2r imprinting. Yeast artificial chromosome transgenes successfully reproduced the imprinted methylation and expression pattern of the endogenous Igf2r gene; deletion of region 2 from these transgenes caused a loss of imprinting and restored biallelic Igf2r expression. These results define a primary role for region 2 and a negligible role for chromosomal location in Igf2r imprinting; they also show that methylation imprints can maintain allelic expression. Short transgenes containing only region 2 and yeast artificial chromosome transgenes with an inactive Igf2r promoter do not attract parental-specific methylation. All transgenes showing paternal-specific repression of Igf2r produced an antisense RNA whose transcription was dependent on region 2. The production of an antisense RNA by the repressed parental allele is reminiscent of the imprinting of the Igf2/H19 gene pair and may indicate that expression competition could play a general role in imprinting.

Characterization of the C3 YAC contig from proximal mouse chromosome 17 and analysis of allelic expression of genes flanking the imprinted Igf2r gene.

The imprinted mouse insulin-like growth factor type 2 receptor (Igf2r) maps to the middle of a gene-rich region in band A2 of mouse chromosome 17. The t(Lub2) chromosome 17 variant contains a small deletion that removes at least seven genes including Igf2r. We have constructed a YAC contig spanning the entire t(Lub2) deletion and created a restriction map that covers 700 kb. The position, transcription orientation, and imprinted status of the genes immediately flanking Igf2r have been assessed. We show here that the Mas gene, which lies 65 kb upstream to Igf2r, contains a novel 5' exon and is not imprinted in adult tissues. We further show that the recently identified Lx1 gene lies immediately downstream and is also expressed from both parental alleles in adult tissues. The remaining genes in this region have previously been shown to be biallelically expressed.

Identification of a male meiosis-specific gene, Tcte2, which is differentially spliced in species that form sterile hybrids with laboratory mice and deleted in t chromosomes showing meiotic drive.

Tcte2 (t complex testes expressed 2) is a male meiosis-specific gene that maps to band 3.3 of mouse chromosome 17. Two distinct male fertility defects, hybrid sterility and transmission ratio distortion, have previously been mapped to this region. Hybrid sterility arises in crosses between different mouse species and the F1 generation males have defects in the first meiotic division and are sterile. Transmission ratio distortion is shown by males heterozygous for the t haplotype form of chromosome 17 and is a type of meiotic drive in which male gametes function unequally at fertilization. The Tcte2 gene expresses a coding mRNA and a number of putative non-ORF transcripts in meiosis I. A deletion of the 5' part of the locus abolishes Tcte2 expression on the t haplotype form of chromosome 17. Additionally, the series of putative non-ORF RNAs at the Tcte2 locus are differentially spliced in species that show hybrid sterility when crossed to laboratory mice. The identification of polymorphisms in t haplotypes and in different mouse species allows alleles of Tcte2 to be proposed as candidates for loci which contribute to both meiotic drive and hybrid sterility phenotypes. While theoretical considerations have previously been used to propose that speciation and meiotic drive involve alleles of the same genes, Tcte2 is the first cloned candidate gene to support this link at a molecular level.

Paternal repression of the imprinted mouse Igf2r locus occurs during implantation and is stable in all tissues of the post-implantation mouse embryo.

The insulin-like growth factor type 2 receptor, also known as the cation independent mannose-6-phosphate receptor (Igf2r) is an imprinted gene, which is repressed on the paternally inherited allele in midgestation mouse embryos. We have used a LacZ reporter gene, targeted into the endogenous gene, to investigate the developmental timing, tissue specificity and stability of the repression of the paternal allele. The LacZ expression pattern in pre- and post-implantation embryos, confirmed that Igf2r shows monoallelic expression only after implantation. We show here, additionally, that Igf2r shows biallelic expression in all cells of the preimplantation embryo at E4.5, and, that monoallelic expression is clearly present in all tissues at E6.5, in the early post-implantation embryo. Imprinted expression is maintained in later embryonic development and no tissue was observed to escape imprinting up to E13.5 of development. Thus, for Igf2r, the onset of monoallelic expression occurs in all cells during the implantation period and paternal repression is maintained in all tissues of the late developing embryo.

Competition--a common motif for the imprinting mechanism?

Imprinted genes, in contrast to the majority of mammalian genes, are able to restrict expression to one of the two parental alleles in somatic diploid cells. Although the silent allele of an imprinted gene appears to be transcriptionally repressed, it often bears little other resemblance to normal genes in an inactive state. The key to the imprinting mechanism may be a form of parental-specific expression-competition between cis-linked genes and not parental-specific expression versus repression. Thus, the imprinting mechanism may be better understood if the chromosomal region containing imprinted genes is viewed as 'active' on both parental chromosomes.

The Lx1 gene maps to mouse chromosome 17 and codes for a protein that is homologous to glucose and polyspecific transmembrane transporters.

A novel mouse gene, provisionally named Lx1, has been cloned and sequenced. Lx1 most likely represents the mouse homolog of the rat gene OCT1, which encodes a polyspecific transmembrane transporter that is possibly involved in drug elimination. The LX1 predicted protein is highly hydrophobic, possesses twelve putative transmembrane domains, and also shares significant homology with members of the sugar transporter family, particularly the novel liver-specific transporter NLT. Lx1 mRNA is expressed at high levels in mouse liver, kidney, and intestine, and at low levels in the adrenals and in lactating mammary glands. The Lx1 gene maps very close to the imprinted Igf2r/Mpr300 gene on mouse Chromosome (Chr) 17, in a region that is syntenic to human Chr 6q. Chr 6q has been previously associated with transient neonatal diabetes mellitus and breast cancer.

Random and imprinted monoallelic expression.

This review has surveyed the current literature on random and imprinted monoallelic expression from the unusual perspective of considering that these two forms of monoallelic expression (MAE) may be regulated by similar mechanisms. The true extent of genes which show MAE is not yet known. Imprinted MAE is thought to involve 0.1-0.2% of mammalian genes, while the number of genes in the random MAE category cannot at present be predicted since very few genes have been examined with assays that distinguish parental transcripts in single cells. The perspective adopted in this review is speculative since only one gene (XIST) is known which displays both random and imprinted MAE. However, a recent report that H19 may also show random allelic expression, suggests that MAE should be further investigated as a gene regulation mechanism in mammals.