Mechanisms Of Genomic Imprinting Research Articles

Functional association of CTCF with the insulator upstream of the H19 gene is parent of origin-specific and methylation-sensitive.

In mammals, a subset of genes inherit gametic marks that establish parent of origin-dependent expression patterns in the soma ([1] and references therein). The currently most extensively studied examples of this phenomenon, termed genomic imprinting, are the physically linked Igf2 (insulin-like growth factor II) and H19 genes, which are expressed mono-allelically from opposite parental alleles [1] [2]. The repressed status of the maternal Igf2 allele is due to cis elements that prevent the H19 enhancers [3] from accessing the Igf2 promoters on the maternal chromosome [4] [5]. A differentially methylated domain (DMD) in the 5' flank of H19 is maintained paternally methylated and maternally unmethylated [6] [7]. We show here by gel-shift and chromatin immunopurification analyses that binding of the highly conserved multivalent factor CTCF ([8] [9] and references therein) to the H19 DMD is methylation-sensitive and parent of origin-dependent. Selectively mutating CTCF-contacting nucleotides, which were identified by methylation interference within the extended binding sites initially revealed by nuclease footprinting, abrogated the H19 DMD enhancer-blocking property. These observations suggest that molecular mechanisms of genomic imprinting may use an unusual ability of CTCF to interact with a diverse spectrum of variant target sites, some of which include CpGs that are responsible for methylation-sensitive CTCF binding in vitro and in vivo.

The Genes for Major Psychosis: Aberrant Sequence or Regulation?

A number of recent clinical and molecular observations in major psychosis indicate that epigenetic factors may be operational in the origin of major mental illness. This article further develops the idea that epigenetic factors may play an etiopathogenic role in schizophrenia and bipolar affective disorder. The putative role of epigenetic factors is shown by the epigenetic interpretation of genetic association studies of the genes for serotonin 2A (HTR2A) and the dopamine D3 (DRD3) receptors in schizophrenia. The idea of epigenetic polymorphism of genetic alleles is introduced, and it is argued that epigenetic variation may explain a number of controversial and unclear findings in allelic and genotypic association studies of HTR2A and DRD3. In linkage analyses of multiplex families with bipolar affective disorder (BPAD), different loci on chromosome 18 indicated co-segregation of alleles of one parental sex with the disease phenotype, and this finding implies that the epigenetic mechanism of genomic imprinting may be involved. Evidence for genomic imprinting provides the background for epigenetic cloning of BPAD risk factors by searching for differentially modified genes on chromosome 18. Finally, epigenetic studies could be relevant to the better understanding of the molecular action of antipsychotic medications. In addition to this, if epimutations are detected in major psychosis, epigenetic treatment directed at correction of epigenetic status of a specific brain gene may eventually be developed.

Beckwith-Wiedemann syndrome: imprinting in clusters revisited.

Genomic imprinting, a process that causes genes to be expressed according to their parental origin, affects a minority of human genes, probably less than 1,000. Nevertheless, this class of epigenetic modification acts on many genes with critical roles in growth and development, and disordered imprinting is implicated in genetic disease and in many cancers. Various theories have been proposed to explain the evolution of imprinting in mammals. The most popular is the Haig parental conflict model, which holds that imprinting evolved as a result of opposing interests of the maternal and paternal genomes. Thus, in polygamous species, paternally derived genes will favor fetal growth at the expense of depleting maternal resources and disadvantaging further offspring. In contrast, the maternal genes will oppose the paternal effect and conserve resources to ensure the fitness of the mother and future offspring. This model predicts that although paternally expressed imprinted genes should promote growth, maternally expressed genes should have opposite effects. Many, but not all, imprinted genes identified to date conform to these predictions (1). The mechanisms by which imprinting is established and maintained are the subject of intense investigation (2). The process must involve a reversible epigenetic marking, and a number of features of imprinted genes have been described. Thus, differential methylation of maternal and paternal alleles is a major characteristic of imprinted genes, and defects in methylation processes (e.g., methyltransferase deficiency) disrupt normal genomic imprinting. Parental allele-specific alterations in the chromosome environment of imprinted genes are revealed by the presence of asynchronous DNA replication and differences in chromatin structure and modification (e.g., histone acetylation). A striking feature of imprinted genes in mammals is their tendency to cluster in the genome. The arrangement of coordinately or oppositely imprinted genes within a cluster offers insights into the mechanisms by which cells establish and maintain appropriate imprints on functionally related genes. Here, we discuss the organization and function of a major imprinted gene cluster, occurring on human chromosome 11p15.5, that has been implicated in the imprinting disorder Beckwith-Wiedemann syndrome (BWS) and in a variety of human cancers including Wilms’ tumor (3, 4). Because the regulation of imprinted genes in the homologous region in the mouse (distal region of chromosome 7) is broadly conserved between mice and humans, experiments in mice have complemented molecular analyses of BWS pathology and have contributed greatly to our current knowledge of mammalian genomic imprinting mechanisms. In this review, we consider the mechanisms and role of genomic imprinting in human development and in BWS pathogenesis, drawing on research on the human and the mouse clusters of imprinted genes.

Mechanisms of genomic imprinting

Mechanisms of genomic imprinting

Mechanisms of genomic imprinting

A small number of mammalian genes undergo the process of genomic imprinting whereby the expression level of the alleles of a gene depends upon their parental origin. In the past year, attention has focused on the mechanisms that determine parental-specific expression patterns. Many imprinted genes are located in conserved clusters and, although it is apparent that imprinting of adjacent genes is jointly regulated, multiple mechanisms among and within clusters may operate. Recent developments have also refined the timing of the gametic imprints and further defined the mechanism by which DNA methyltransferases confer allelic methylation patterns.

Genomic imprinting: mom and dad (epi)genetics

Genomic imprinting is an epigenetic chromosomal modification in the gametes or zygotes that results in a non-random monoallelic expression of specific autosomal genes depending upon their parent of origin. A gene could be maternally imprinted (only paternal copy expressed) or paternally imprinted (only maternal copy expressed). The exact mechanism of genomic imprinting is not clear. However, allele-specific methylation of CpG residues at the 5' end of imprinted genes is thought to be responsible for the maintenance of imprinting in somatic cells.

The mechanisms of genomic imprinting.

Genomic imprinting can be regarded as one of many variants of epigenetic modes of gene regulation in eukaryotic cells. There is no a priori reason, therefore, to invoke fundamentally novel mechanisms to explain the imprinting phenomenon in mammals. For example, the different factors involved in imprinting, such as the stable propagation of different chromatin states that repress or permit gene transcription, are well-known entities in a wide range of eukaryotic cells (see Pirotta Chap. 10, and Geramisova and Corces Chap. 11, this vol.). From this point of view, we should perhaps consider the mechanism(s) of imprinting as being the unusual result of combinatory events that occur regularly on an evolutionary scale.

Genetic conflicts in genomic imprinting.

The expression pattern of genes in mammals and plants can depend upon the parent from which the gene was inherited, evidence for a mechanism of parent-specific genomic imprinting. Kinship considerations are likely to be important in the natural selection of many such genes, because coefficients of relatedness will usually differ between maternally and paternally derived genes. Three classes of gene are likely to be involved in genomic imprinting: the imprinted genes themselves, trans-acting genes in the parents, which affect the application of the imprint, and trnas-acting genes in the offspring, which recognize and affect the expression of the imprint. We show that coefficients of relatedness will typically differ among these three classes, thus engendering conflicts of interest between Imprinter genes, imprinted genes, and imprint-recognition genes, with probable consequences for the evolution of the imprinting machinery.

Genomic imprinting and carcinogenesis.

The Mendelian inheritance is based on the fundamental rule in which mammalian genes are expressed equally from two homologous biparental alleles. Recently a small number of genes have been identified to show an exception to this rule in that homologous alleles can function differently in somatic cells depending on whether they come from the mother or the father. This intriguing biological phenomenon is called as genomic imprinting which does not conform classical Mendelian inheritance and has potentially far reaching implications for genetics, evolution, developmental biology and pathology including cancer. The gene encoding insulin-like growth factor 2 (IGF2) harbors at 11p15.5 and serves as paradigm for an imprinted gene. The IGF2 gene has been demonstrated to be imprinted with the paternal allele expressed and the maternal being silent which is evolutionally conserved between mice and human. Loss of imprinting (LOI) of IGF2 has been demonstrated in a dozen of tumor types including Wilms tumor (WT) with a promise of many more to come. The LOI of IGF2 may induce increased or deregulated IGF2 expression which could initiate the onset of WT. Thus the LOI of IGF2 may provide a novel mechanism of gene activation and play a role in the development of a wide range of tumors. This review also discusses other imprinted genes on 11p15 which may have a role in WT or other diseases. Finally molecular mechanisms of genomic imprinting are discussed.

Epigenetic reprogramming of the human H19 gene in mouse embryonic cells does not erase the primary parental imprint.

Genomic imprinting in mammals is thought to result from epigenetic modifications to chromosomes during gametogenesis, which leads to differential allelic expression during development. There is a requirement for an appropriate experimental system to enable the analysis of the mechanisms of genomic imprinting during embryogenesis. To develop a novel in vitro system for studying the molecular basis of genomic imprinting, we constructed mouse cell lines containing either a paternal or maternal human chromosome 11, by microcell-mediated chromosome transfer. Allele-specific expression and DNA methylation studies revealed that the imprinting status of the human H19 gene was maintained in mouse A9 mono-chromosomal hybrids. Each parental human chromosome was introduced independently into mouse near-diploid immortal fibroblasts (m5S) and two embryonal carcinoma (EC) cell lines (OTF9-63 and P19). The paternal allele of human H19 remained in a repressed state in m5S cells, but was de-repressed in both EC cells. The paternal H19 allele was demethylated extensively in OTF9-63 cells, whereas the only alteration in P19 hybrids was de novo methylation on both alleles in the 3' region. Following in vitro differentiation, the expressed paternal H19 allele was selectively repressed in differentiated derivatives of EC hybrids. These results indicated that human imprint marks could function effectively in mouse cells, and that the imprinting process was epigenetically reprogrammed in embryonal carcinoma cells, without erasure of the primary imprint that marked the parental origin. Therefore, these mono-chromosomal hybrids could provide a valuable in vitro system to study the mechanisms involved in the regulation of imprinted gene expression.

Tissue-specific imprinting of the mouse insulin-like growth factor II receptor gene correlates with differential allele-specific DNA methylation.

Imprinted genes may be expressed uniparentally in a tissue- and development-specific manner. The insulin-like growth factor II receptor gene (Igf2r), one of the first imprinted genes to be identified, is an attractive candidate for studying the molecular mechanism of genomic imprinting because it is transcribed monoallelically in the mouse but biallelically in humans. To identify the factors that control genomic imprinting, we examined allelic expression of Igf2r at different ages in interspecific mice. We found that Igf2r is not always monoallelically expressed. Paternal imprinting of Igf2r is maintained in peripheral tissues, including liver, kidney, heart, spleen, intestine, bladder, skin, bone, and skeletal muscle. However, in central nervous system (CNS), Igf2r is expressed from both parental alleles. Southern analysis of the Igf2r promoter (region 1) revealed that, outside of the CNS where Igf2r is monoallelically expressed, the suppressed paternal allele is fully methylated while the expressed maternal allele is completely unmethylated. In CNS, however, both parental alleles are unmethylated in region 1. The importance of DNA methylation in the maintenance of the genomic imprint was also confirmed by the finding that Igf2r imprinting was relaxed by 5-azacytidine treatment. The correlation between genomic imprinting and allelic Igf2r methylation in CNS and other tissues thus suggests that the epigenetic modification in the promoter region may function as one of the major factors in maintaining the monoallelic expression of Igf2r.

The IPL gene on chromosome 11p15.5 is imprinted in humans and mice and is similar to TDAG51, implicated in Fas expression and apoptosis.

We searched for novel imprinted genes in a region of human chromosome 11p15.5, which contains several known imprinted genes. Here we describe the cloning and characterization of the IPL ( I mprinted in P lacenta and L iver) gene, which shows tissue-specific expression and functional imprinting, with the maternal allele active and the paternal allele relatively inactive, in many human and mouse tissues. Human IPL is highly expressed in placenta and shows low but detectable expression in fetal and adult liver and lung. Mouse Ipl maps to the region of chromosome 7 which is syntenic with human 11p15.5 and this gene is expressed in placenta and at higher levels in extraembryonic membranes (yolk sac), fetal liver and adult kidney. Mouse and human IPL show sequence similarity to TDAG51 , a gene which was shown to be essential for Fas expression and susceptibility to apoptosis in a T lymphocyte cell line. Like several other imprinted genes, mouse and human IPL genes are small and contain small introns. These data expand the repertoire of known imprinted genes and will be helpful in testing the mechanism of genomic imprinting and the role of imprinted genes in growth regulation.

DNA methylation in genomic imprinting.

As a reversible epigenetic modification which can affect gene expression, DNA methylation has been an attractive candidate for the biochemical mechanism of genomic imprinting. Many correlations in mice and humans link allele-specific DNA methylation to the allele-restricted RNA expression which is the hallmark of imprinted genes. Moreover, abnormal DNA methylation accompanies the pathological functional imprinting of certain human genes on chromosome 11p15.5 in Wilms' tumors and in the Beckwith-Weidemann syndrome and on chromosome 15q11-13 in the Prader-Willi and Angelman syndromes. A role for DNA methylation in maintaining the transcriptional silence of imprinted alleles at some loci has been supported by pharmacological manipulation with 5-aza-2'-deoxycytidine and by experiments with methyltransferase deletion mice. Gametic differences in DNA methylation could also account for the initiation of imprints, but this remains unproven. Comprehensive physical models for the role of DNA methylation in imprinting must account not only for local allele-restricted gene expression but also for the existence of large chromosomal domains containing multiple coordinately imprinted genes.

Mouse U2af1-rs1 is a neomorphic imprinted gene.

The mouse U2af1-rs1 gene is an endogenous imprinted gene on the proximal region of chromosome 11. This gene is transcribed exclusively from the unmethylated paternal allele, while the methylated maternal allele is silent. An analysis of genome structure of this gene revealed that the whole gene is located in an intron of the Murr1 gene. Although none of the three human U2af1-related genes have been mapped to chromosome 2, the human homolog of Murr1 is assigned to chromosome 2. The mouse Murr1 gene is transcribed biallelically, and therefore it is not imprinted in neonatal mice. Allele-specific methylation is limited to a region around U2af1-rs1 in an intron of Murr1. These results suggest that in chromosomal homology and genomic imprinting, the U2af1-rs1 gene is distinct from the genome region surrounding it. We have proposed the neomorphic origin of the U2af1-rs1 gene by retrotransposition and the particular mechanism of genomic imprinting of ectopic genes.

Beck-Wiedemann syndrome and Wilms' tumour.

Patients with rare overgrowth disorders, such as Beck-Wiedemann syndrome and Simpson-Golabi-Behmel syndrome, are predisposed to embryonal tumours, including Wilms' tumour of the kidney. Therefore, these disorders offer a link between hyperplastic growth and cancer. Genetic lesions at chromosome 11p15 have been associated with Beck-Wiedemann syndrome and Wilms' tumour for several years and the presence of the gene encoding insulin-like growth factor-II (IGF-II) in this region has given rise to much speculation over the involvement of this factor in these growth defects. This speculation was heightened by genetic evidence for the involvement of genomic imprinting in Beck-Wiedemann syndrome and Wilms' tumour, combined with the discovery that the IGF-II gene is imprinted. Although there is a wealth of evidence linking the IGF signalling pathway with overgrowth and cancer, recent progress in the study of 11p15 and developments in our understanding of the mechanism of genomic imprinting indicate that additional imprinted genes located in this region also contribute to these growth disorders.

Preconceptual Programming and Sexual Orientation: a Hypothesis

To date, biological explanations of sexual orientation have broadly focused on genes and/or prenatal hormonal environments which are thought to act on the brain to provide the neural circuitry on which sexual orientation is inscribed. The proposed models are open to criticism when applied to a healthy population at large because of the implied reference to developmental anomalies. For this reason the present paper challenges the traditional viewpoint and hypothesizes that the foundation of adult sexual orientation may be the result of adaptive programming beginning before conception. According to this hypothesis the continuum spanning human sexuality has its etiology defined in terms of male and female-mediated forms of selective preconceptual marking. The hypothesis also assumes that, via the mechanism of genomic imprinting, the imprinted gene is able to switch through different states of potential activity from the incomplete to the fully penetrant state resulting in a continuum of orientations ranging from asexual, through graded bisexual to homosexual. An adaptive preconceptual program has biological significance as it ensures a generational preparedness for the prevailing conditions. The physiological aspects and circumstantial evidences which were important in developing the new hypothesis are also described.

Epigenetic control of sexual phenotype in a dioecious plant, Melandrium album.

Melandrium album (syn. Silene latifolia) is a model dioecious species in which the Y chromosome, present only in heterogametic males, plays both a male-determining and a strict female-suppressing role. We showed that treatment with 5-azacytidine (5-azaC) induces a sex change to androhermaphroditism (an-dromonoecy) in about 21% of male plants, while no apparent phenotypic effect was observed in females. All of these bisexual androhermaphrodites (with the standard male 24, AA + XY karyotype) were mosaics possessing both male and hermaphrodite flowers and, moreover, the hermaphrodite flowers displayed various degrees of gynoecium development and seed setting. Southern hybridization analysis with a repetitive DNA probe showed that the 5-azacytidine-treated plants were significantly hypomethylated in CG doubles, but only to a minor degree in CNG triplets. The bisexual trait was transmitted to two successive generations, but only when androhermaphrodite plants were used as pollen donors. The sex reversal was inherited with incomplete penetrance and varying expressivity. Based on the uniparental inheritance pattern of androhermaphroditism we conclude that it originated either by 5-azaC induced inhibition of Y-linked female-suppressing genes or by a heritable activation of autosomal female-determining/promoting genes which can be reversed, on passage through female meiosis, by a genomic imprinting mechanism. The data presented indicate that female sex suppression in M. album XY males is dependent on methylation of specific DNA sequences and can be heritably modified by hypomethylating drugs.

Genomic Imprinting in Mice: Its Function and Mechanism1

Genomic imprinting is an epigenetic phenomenon by which the two parental alleles of a gene are differentially expressed. Although the function of genomic imprinting is not clear, it has been proposed that it evolved in mammals to regulate intrauterine growth. This proposal is consistent with experiments that were designed to reveal the mechanism and impact of genomic imprinting in a region of mouse chromosome 7 that contains four imprinted genes: Mash-2 (a transcription factor) and H19 (a noncoding RNA) are maternally expressed, whereas Insulin-2 (Ins-2) and Insulin-like growth factor 2 (Igf-2) are paternally expressed. Two targeted disruptions at the locus were generated in mice; these support the hypothesis that the function of the H19 gene is to set up the imprinting of both Igf-2 and Ins-2. H19 transcription on the maternal chromosome precludes transcription of the other two genes by a mechanism that involves competition for a common set of enhancers. On the paternal chromosome the H19 gene is silenced by DNA methylation, thus permitting the use of enhancers by the other genes.

7 Mechanisms of Genomic Imprinting in Mammals

This chapter can be summarized by the following main points: Genomic imprinting results in the functional nonequivalence of the maternal and paternal genomes, thereby preventing the development of viable parthenogenotes and androgenotes in eutherian mammals. Imprinting may have arisen as a result of the specialized evolutionary requirements of the parental genomes or may have been an obligatory step in the development of placentation. A substantial proportion of transgenes and a smaller number of endogenous genes demonstrate imprinted pattern of expression in mice and humans. An analysis of DNA methylation in somatic tissues and germ cells during embryonic and postnatal development reveals dynamic changes, particularly during gametogenesis and early embryogenesis. The nature and timing of these changes suggest that DNA methylation may be involved in genomic imprinting. Imprinted genes display complex methylation patterns. Many aspects of these patterns are consistent with a role for methylation in the imprinted phenotype, although it is currently unclear whether methylation functions in the establishment of imprinting or plays a secondary role in the maintenance of the imprinted pattern of expression. Studies underway to identify new imprinted genes may help elucidate both the function and mechanism of genomic imprinting.

Expression of a lacZ transgene reveals floor plate cell morphology and macromolecular transfer to commissural axons

The floor plate is situated at the ventral midline of the neural tube and is an important intermediate target for commissural axons. During elongation, these axons converge bilaterally on the ventral midline neural tube and after crossing the floor plate make an abrupt rostral turn. Ample evidence indicates that the initial projection of commissural axons to the floor plate is guided by a chemotropic factor secreted by floor plate cells. However, the way in which the subsequent interaction of these axons with the floor plate leads them to make further trajectory changes remains undefined. In an effort to gain further understanding of the structure and function of floor plate cells, we have taken advantage of a line of transgenic mice in which these cells express beta-galactosidase and thus can be stained by histochemical means. In this line, a genomic imprinting mechanism restricts the expression of the lacZ transgene to only a proportion of the floor plate cells, allowing their morphology to be appreciated with particular clarity. Our analysis revealed that the basal processes of floor plate cells are flattened in their rostrocaudal dimension and possess fine lateral branches which are aligned with commissural axons. Unexpectedly, beta-galactosidase activity was also detected within longer transverse linear profiles traversing the floor plate whose ultrastructural appearance was not that of floor plate cells but instead corresponded to that of commissural axons. Enzyme activity was not detected in more proximal axonal segments or in the neuronal cell bodies from which these axons originated. Therefore, we propose that the transgene product, and potentially other proteins synthesized by floor plate cells, can be transferred to decussating axons.