Paternal expression of WT1 in human fibroblasts and lymphocytes

The Wilms' tumor suppressor gene ( WT1 ) was previously identified as being imprinted, with frequent maternal expression in human placentae and fetal brains. We examined the allele-specific expression of WT1 in cultured human fibroblasts from 15 individuals. Seven of 15 fibroblast lines were heterozygous for polymorphic alleles, and the expression patterns were variable, i.e., equal, unequal or monoallelic paternal expression in three, two and two cases, respectively. Exclusive paternal expression of WT1 was also shown in non-cultured peripheral lymphocytes from the latter two individuals. The allele-specific expression profiles of other imprinted genes, IGF2 and H19, on human chromosome 11 were constant and consistent with those in other tissues. Our unexpected observations of paternal or biallelic expression of WT1 in fibroblasts and lymphocytes, together with the previous findings of maternal or biallelic expression in placentae and brains, suggest that the allele-specific regulatory system of WT1 is unique and may be controlled by a putative tissue- and individual-specific modifier.     

Chromosome 11p15.5 regional imprinting: comparative analysis of KIP2 and H19 in human tissues and Wilms' tumors

The imprinted H19 gene is frequently inactivated in Wilms' tumors (WTs) either by chromosome 11p15.5 loss of heterozygosity (LOH) or by hypermethylation of the maternal allele and it is possible that there might be coordinate disruption of imprinting of multiple 11p15.5 genes in these tumors. To test this we have characterized total and allele-specific mRNA expression levels and DNA methylation of the 11p15.5 KIP2 gene in normal human tissues, WTs and embryonal rhabdomyosarcoma (RMS). Both KIP2 alleles are expressed but there is a bias with the maternal allele contributing 70-90% of mRNA. Tumors with LOH show moderate to marked reductions in KIP2 mRNA relative to control tissues and residual mRNA expression is from the imprinted paternal allele. Among WTs without LOH most cases with H19 inactivation also have reduced KIP2 expression and most cases with persistent H19 expression have high levels of KIP2 mRNA. In contrast to the extensive hypermethylation of the imprinted H19 allele, both KIP2 alleles are hypomethylated and WTs with biallelic H19 hypermethylation lack comparable hypermethylation of KIP2 DNA. 5-aza-2'-deoxycytidine (aza-C) increases H19 expression in RD RMS cells but does not activate KIP2 expression. These data indicate coordinately reduced expression of two linked paternally imprinted genes in most WTs and also suggest mechanistic differences in the maintenance of imprinting at these two loci.     

Microalbuminuria in IDDM is associated with increased expression of monocyte procoagulant activity

Insulin-like growth factor II (IGF-II) plays a key role in mammalian growth, influencing foetal cell division and differentiation and possibly metabolic regulation. The mature 67 amino acid peptide shares sequence homology with both insulin and IGF-I. The liver is the main endocrine source of IGFs, but autocrine/paracrine activity is found in most tissues. The type 1 receptor mediates most of the biological effects of IGF-I and IGF-II; the type 2 receptor is involved with IGF-II degradation. Binding proteins may both localise IGFs to the receptors and regulate their activities. The IGF2 gene is maternally imprinted in mouse and human. Relaxation of IGF2 imprinting occurs in the Beckwith-Wiedemann syndrome of somatic overgrowth, sporadic Wilms' tumour and a number of other cancers. In the general adult population, the IGF2-INS gene cluster may also influence body weight, in which case IGF-II function could become a target for therapeutic intervention in obesity.     

Allelic-expression imbalance of the insulin-like growth factor 2 gene in hepatocellular carcinoma and underlying disease

It has been well documented that the liver is an exceptional organ in which the monoallelic expression of insulin-like growth factor 2 (IGF2) due to genomic imprinting is relaxed during the postnatal period, resulting in biallelic expression thereafter. In the present study, changes in the status of genomic imprinting were examined in 15 hepatocellular carcinomas (HCCs) as well as in 29 liver biopsies of chronic hepatitis or liver cirrhosis without clinical evidence of HCC, following screening for heterozygotes with an ApaI polymorphism in IGF2 in 34 HCCs and 80 such non-HCC cases. Extreme allelic-expression imbalance, leading to restoration of monoallelic IGF2 expression, was observed in 15 (100%) of 15 informative HCCs for the polymorphism with this monoallelic IGF2 expression appearing to be non-random from the paternal allele. Interestingly, the same allelic-expression imbalance was also present in a significant fraction of noncancerous liver specimens of patients with underlying disease known to be associated with HCC development. In contrast, the status of genomic imprinting of H19, another gene closely mapped at 11p15 under opposite imprinting, was strictly maintained in seven (100%) of seven cases informative for an RsaI polymorphism of H19. Together with the previous reports on altered genomic imprinting of IGF2 and H19 in embryonal lesions such as Wilms tumors as well as in lung cancers, the results suggest that perturbations of imprinting status occur as locus and tumor-type specific events in the development of human cancers.     

IGF2 is parentally imprinted during human embryogenesis and in the Beckwith-Wiedemann syndrome

The phenomenon of parental imprinting involves the preferential expression of one parental allele of a subset of chromosomal genes and has so far only been documented in the mouse. We show here, by exploiting sequence polymorphisms in exon nine of the human insulin-like growth factor 2 (IGF2) gene, that only the paternally-inherited allele is active in embryonic and extra-embryonic cells from first trimester pregnancies. In addition, only the paternal allele is expressed in tissues from a patient who suffered from Beckwith-Wiedemann syndrome. Thus the parental imprinting of IGF2 appears to be evolutionarily conserved from mouse to man and has implications for the generation of the Beckwith-Wiedemann syndrome.     

Igf2r and Igf2 gene expression in androgenetic, gynogenetic, and parthenogenetic preimplantation mouse embryos: absence of regulation by genomic imprinting

Genomic imprinting in mammals is believed to result from modifications to chromosomes during gametogenesis that inactivate the paternal or maternal allele. The genes encoding the insulin-like growth factor type 2 (Igf2) and its receptor (Igf2r) are reciprocally imprinted and expressed from the paternal and maternal genomes, respectively, in the fetal and adult mouse. We find that both genes are expressed in androgenetic, gynogenetic, and parthenogenetic preimplantation mouse embryos. These results indicate that inactivation of imprinted genes occurs postfertilization (most likely postimplantation) and that genomic imprinting and gene inactivation are separate processes. We propose that imprinting marks the chromosome so that regulatory factors expressed in cells at later times can recognize the imprint and selectively inactivate the maternal or paternal allele. For these genes, this finding invalidates models of genomic imprinting that require them to be inactive from the time of fertilization.     

Using the evidence. A need for quantity, not quality?

The gene for alpha-stimulating guanine-nucleotide binding polypeptide, Gnas, has been considered as a candidate for the imprinting effects ascribed to distal mouse Chromosome (Chr) 2. Its human homologue (GNAS1) appears, from clinical and biochemical studies of patients with Albright hereditary osteodystrophy, to be paternally imprinted. GNAS1 maps to 20q13, a region that shows linkage conservation with distal mouse Chr 2. We have mapped Gnas within the imprinting region on distal Chr 2 by linkage analysis. To establish if Gnas is imprinted, we have looked for expression differences in tissues taken from mice carrying maternal duplication/paternal deficiency for distal Chr 2 (MatDp2) and its reciprocal (PatDp2). RNA in situ hybridization revealed high levels of Gnas mRNA in glomeruli of PatDp2 embryos at late gestation and lower levels in glomeruli of MatDp2 embryos. These results strongly suggest that Gnas is maternally imprinted and suggest that the mouse gene may be imprinted in a manner opposite that predicted in human.     

Deletion of the H19 differentially methylated domain results in loss of imprinted expression of H19 and Igf2

Differentially methylated sequences associated with imprinted genes are proposed to control genomic imprinting. A 2-kb region located 5' to the imprinted mouse H19 gene is hypermethylated on the inactive paternal allele throughout development. To determine whether this differentially methylated domain (DMD) is required for imprinted expression at the endogenous locus, we have generated mice harboring a 1.6-kb targeted deletion of the DMD and assayed for allelic expression of H19 and the linked, oppositely imprinted Igf2 gene. H19 is activated and Igf2 expression is reduced when the DMD deletion is paternally inherited; conversely, upon maternal transmission of the mutation, H19 expression is reduced and Igf2 is activated. Consistent with the DMD's hypothesized role of setting up the methylation imprint, the mutation also perturbs allele-specific methylation of the remaining H19 sequences. In conclusion, these experiments show that the H19 hypermethylated 5' flanking sequences are required to silence paternally derived H19. Additionally, these experiments demonstrate a novel role for the DMD on the maternal chromosome where it is required for the maximal expression of H19 and the silencing of Igf2. Thus, the H19 differentially methylated sequences are required for both H19 and Igf2 imprinting.     

Malignant potential, major histocompatibility complex antigen expression, and genomic imprinting of rat trophoblast cell lines

Invasive growth, variation in major histocompatibility complex antigen expression, and genomic imprinting are important properties of both trophoblast cells and malignant tumors. This study, undertaken to address these three issues, used cultured trophoblast cell lines derived from Day 11/12 rat placentas of all mating combinations of the DA and WF inbred strains. In addition, genomic imprinting was also examined in intact rat placentas from Days 11-19. There was no correlation in trophoblast cells between class I antigen expression, DNA content, and cell ploidy on the one hand and oncogenic potential on the other hand. The constitutive suppression of class II antigens in the trophoblast cells could not be abrogated by treatment with interferon-gamma, whereas such treatment always maximally induced class I antigen expression regardless of the initial resting levels. The trophoblast cells at Day 11/12 expressed both maternal and paternal class I antigens, and studies in whole placental tissues showed that the imprinting of the maternal class I antigens was manifested by a decreased level of expression rather than an absence of expression. Thus, genomic imprinting in the rat placenta is a quantitative, rather than an all-or-none, phenomenon.     

Genetic conflicts, multiple paternity and the evolution of genomic imprinting

We present nine diallelic models of genetic conflict in which one allele is imprintable and the other is not to examine how genomic imprinting may have evolved. Imprinting is presumed to be either maternal (i.e., the maternally derived gene is inactivated) or paternal. Females are assumed to be either completely monogamous or always bigamous, so that we may see any effect of multiple paternity. In contrast to previous verbal and quantitative genetic models, we find that genetic conflicts need not lead to paternal imprinting of growth inhibitors and maternal imprinting of growth enhancers. Indeed, in some of our models--those with strict monogamy--the dynamics of maternal and paternal imprinting are identical. Multiple paternity is not necessary for the evolution of imprinting, and in our models of maternal imprinting, multiple paternity has no effect at all. Nevertheless, multiple paternity favors the evolution of paternal imprinting of growth inhibitors and hinders that of growth enhancers. Hence, any degree of multiple paternity means that growth inhibitors are more likely to be paternally imprinted, and growth enhancers maternally so. In all of our models, stable polymorphism of imprinting status is possible and mean fitness can decrease over time. Neither of these behaviors have been predicted by previous models.     

The imprinting box of the mouse Igf2r gene

Genomic imprinting is a phenomenon characterized by parent-of-origin-specific expression. The imprint is a mark established during germ-cell development to distinguish between the paternal and maternal copies of the imprinted genes. This imprint is maintained throughout embryo development and erased in the embryonic gonads to set the stage for a new imprint. DNA methylation is essential in this process as shown by the presence of differentially methylated regions (DMRs) in all imprinted genes and by the loss of imprinting in mice that are deficient in DNA methylation or upon deletion of DMRs. Here we show that a DMR in the imprinted Igf2r gene (which encodes the receptor for insulin-like growth factor type-2) that has been shown to be necessary for imprinting includes a 113-base-pair sequence that constitutes a methylation imprinting box. We identify two new cis-acting elements in this box that bind specific proteins: a de novo methylation signal and an allele-discrimination signal. We propose that this regulatory system, which we show to be involved in the establishment of differential methylation in the Igf2r DMR, represents a critical element in the imprinting process.