Stochastic Loss of Silencing of the Imprinted Ndn/NDN Allele,in a Mouse Model and Humans with Prader-Willi Syndrome,Has Functional Consequences

Genomic imprinting is a process that causes genes to be expressed from one allele only according to parental origin, the other allele being silent. Diseases can arise when the normally active alleles are not expressed. In this context, low level of expression of the normally silent alleles has been considered as genetic noise although such expression has never been further studied. Prader-Willi Syndrome (PWS) is a neurodevelopmental disease involving imprinted genes, including NDN, which are only expressed from the paternally inherited allele, with the maternally inherited allele silent. We present the first in-depth study of the low expression of a normally silent imprinted allele, in pathological context. Using a variety of qualitative and quantitative approaches and comparing wild-type, heterozygous and homozygous mice deleted for Ndn, we show that, in absence of the paternal Ndn allele, the maternal Ndn allele is expressed at an extremely low level with a high degree of non-genetic heterogeneity. The level of this expression is sex-dependent and shows transgenerational epigenetic inheritance. In about 50% of mutant mice, this expression reduces birth lethality and severity of the breathing deficiency, correlated with a reduction in the loss of serotonergic neurons. In wild-type brains, the maternal Ndn allele is never expressed. However, using several mouse models, we reveal a competition between non-imprinted Ndn promoters which results in monoallelic (paternal or maternal) Ndn expression, suggesting that Ndn allelic exclusion occurs in the absence of imprinting regulation. Importantly, specific expression of the maternal NDN allele is also detected in post-mortem brain samples of PWS individuals. Our data reveal an unexpected epigenetic flexibility of PWS imprinted genes that could be exploited to reactivate the functional but dormant maternal alleles in PWS. Overall our results reveal high non-genetic heterogeneity between genetically identical individuals that might underlie the variability of the phenotype.     

Imprinting regulation of the murine Meg1/Grb10 and human GRB10 genes; roles of brain-specific promoters and mouse-specific CTCF-binding sites

The imprinted mouse gene Meg1/Grb10 is expres sed from maternal alleles in almost all tissues and organs, except in the brain, where it is expressed biallelically, and the paternal allele is expressed preferentially in adulthood. In contrast, the human GRB10 gene shows equal biallelic expression in almost all tissues and organs, while it is almost always expressed paternally in the fetal brain. To elucidate the molecular mechanisms of the complex imprinting patterns among the different tissues and organs of humans and mice, we analyzed in detail both the genomic structures and tissue-specific expression profiles of these species. Experiments using 5′-RACE and RT–PCR demonstrated the existence in both humans and mice of novel brain- specific promoters, in which only the paternal allele was active. The promoters were located in the primary differentially methylated regions. Interest ingly, CTCF-binding sites were found only in the mouse promoter region where CTCF showed DNA methylation-sensitive binding activity. Thus, the insulator function of CTCF might cause reciprocal maternal expression of the Meg1/Grb10 gene from another upstream promoter in the mouse, whereas the human upstream promoter is active in both parental alleles due to the lack of the corresponding insulator sequence in this region.     

Rescue of placental phenotype in a mechanistic model of Beckwith-Wiedemann syndrome

Background  

Several imprinted genes have been implicated in the process of placentation. The distal region of mouse chromosome 7 (Chr 7) contains at least ten imprinted genes, several of which are expressed from the maternal homologue in the placenta. The corresponding paternal alleles of these genes are silenced in cis by an incompletely understood mechanism involving the formation of a repressive nuclear compartment mediated by the long non-coding RNA Kcnq1ot1 initiated from imprinting centre 2 (IC2). However, it is unknown whether some maternally expressed genes are silenced on the paternal homologue via a Kcnq1ot1-independent mechanism. We have previously reported that maternal inheritance of a large truncation of Chr7 encompassing the entire IC2-regulated domain (DelTel7 allele) leads to embryonic lethality at mid-gestation accompanied by severe placental abnormalities. Kcnq1ot1 expression can be abolished on the paternal chromosome by deleting IC2 (IC2KO allele). When the IC2KO mutation is paternally inherited, epigenetic silencing is lost in the region and the DelTel7 lethality is rescued in compound heterozygotes, leading to viable DelTel7/IC2KO mice.     

High diversity due to balancing selection in the promoter region of the Medea gene in Arabidopsis lyrata

Molecular imprinting is the differential expression and/or silencing of alleles according to their parent of origin [1, 2]. Conflicts between parents, or parents and offspring, should cause "arms races," with accelerated evolution of the genes involved in imprinting. This should be detectable in the evolution of imprinting genes' protein sequences and in the promoter regions of imprinted genes. Previous studies, however, found no evidence of more amino acid substitutions in imprinting genes [1, 3]. We have analyzed sequence diversity of the Arabidopsis lyrata Medea (MEA) gene and divergence from the A. thaliana sequence, including the first study of the promoter region. In A. thaliana, MEA is imprinted, with paternal alleles silenced in endosperm cells [4, 5], and also functions in the imprinting machinery [4, 6]; MEA protein binding at the MEA promoter region indicates self-regulated imprinting [7-9]. We find the same paternal MEA allele silencing in A. lyrata endosperm but no evidence for adaptive evolution in the coding region, whereas the 5' flanking region displays high diversity, with distinct haplotypes, suggesting balancing selection in the promoter region.     

Imprinting analysis of the mouse chromosome 7C region in DNMT1-null embryos

The mouse chromosome 7C, orthologous to the human 15q11–q13 has an imprinted domain, where most of the genes are expressed only from the paternal allele. The imprinted domain contains paternally expressed genes, Snurf/Snrpn, Ndn, Magel2, Mkrn3, and Frat3, C/D-box small nucleolar RNAs (snoRNAs), and the maternally expressed gene, Ube3a. Imprinted expression in this large (approximately 3–4 Mb) domain is coordinated by a bipartite cis-acting imprinting center (IC), located upstream of the Snurf/Snrpn gene. The molecular mechanism how IC regulates gene expression of the whole domain remains partially understood. Here we analyzed the relationship between imprinted gene expression and DNA methylation in the mouse chromosome 7C using DNA methyltransferase 1 (DNMT1)-null mutant embryos carrying Dnmt1^ps alleles, which show global loss of DNA methylation and embryonic lethality. In the DNMT1-null embryos at embryonic day 9.5, the paternally expressed genes were biallelically expressed. Bisulfite DNA methylation analysis revealed loss of methylation on the maternal allele in the promoter regions of the genes. These results demonstrate that DNMT1 is necessary for monoallelic expression of the imprinted genes in the chromosome 7C domain, suggesting that DNA methylation in the secondary differentially methylated regions (DMRs), which are acquired during development serves primarily to control the imprinted expression from the maternal allele in the mouse chromosome 7C.     

Imprinting disruption of the CDKN1C/KCNQ1OT1 domain: the molecular mechanisms causing Beckwith-Wiedemann syndrome and cancer

Human chromosomal region 11p15.5, which is homologous to mouse chromosome region 7F5, is a well-known imprinted region. The CDKN1C/KCNQ1OT1 imprinted domain, which is one of two imprinted domains at 11p15.5, includes nine imprinted genes regulated by an imprinting center (IC). The CDKN1C/KCNQ1OT1 IC is a differentially methylated region of KCNQ1OT1(KCNQ1OT-DMR) with DNA methylation on the maternal allele and no methylation on the paternal allele. CDKN1C (alias p57KIP2), an imprinted gene with maternal expression, encoding a cyclin-dependent kinase inhibitor, is a critical gene within the CDKN1C/KCNQ1OT1 domain. In Beckwith-Wiedemann syndrome (BWS), approximately 50% of patients show loss of DNA methylation accompanied by loss of histone H3 Lys9 dimethylation on maternal KCNQ1OT-DMR, namely an imprinting disruption, leading to diminished expression of CDKN1C. In cancer, at least three molecular mechanisms--imprinting disruption, aberrant DNA methylations at the CDKN1C promoter, and loss of heterozygosity (LOH) of the maternal allele--are seen and all three result in diminished expression of CDKN1C. Imprinting disruption of the CDKN1C/KCNQ1OT1 domain is involved in the development of both BWS and cancer and it changes the maternal epigenotype to the paternal type, leading to diminished CDKN1C expression. In this review, we describe recent advances in epigenetic control of the CDKN1C/KCNQ1OT1 imprinted domain in both humans and mice.     

Maternal and paternal alleles exhibit differential histone methylation and acetylation at maize imprinted genes

Imprinting is an epigenetically controlled form of gene regulation in which the expression of a gene is based on its parent of origin. This epigenetic regulation is likely to involve allele-specific DNA or histone modifications. The relative abundance of eight different histone modifications was tested at various regions in several imprinted maize (Zea mays) genes using a chromatin immunoprecipitation protocol coupled with quantitative allele-specific single nucleotide polymorphism assays. Histone H3 lysine-27 di- and tri-methylation are paternally enriched at the imprinted loci Mez1, ZmFie1 and Nrp1. In contrast, acetylation of histones H3 and H4 and H3K4 dimethylation are enriched at the maternal alleles of these genes. Di- and tri-methylation of H3 lysine-9, which is generally associated with constitutively silenced chromatin, was not enriched at either allele of imprinted loci. These patterns of enrichment were specific to tissues that exhibit imprinting. In addition, the enrichment of these modifications was dependent upon the parental origin of an allele and not sequence differences between the alleles, as demonstrated by reciprocal crosses. This study presents a detailed view of the chromatin modifications that are associated with the maternal and paternal alleles at imprinted loci and provides evidence for common histone modifications at multiple imprinted loci.     

Epigenetic Resetting of a Gene Imprinted in Plant Embryos

Genomic imprinting resulting in the differential expression of maternal and paternal alleles in the fertilization products has evolved independently in placental mammals and flowering plants. In most cases, silenced alleles carry DNA methylation [1]. Whereas these methylation marks of imprinted genes are generally erased and reestablished in each generation in mammals [2], imprinting marks persist in endosperms [3], the sole tissue of reported imprinted gene expression in plants. Here we show that the maternally expressed in embryo 1 (mee1) gene of maize is imprinted in both the embryo and endosperm and that parent-of-origin-specific expression correlates with differential allelic methylation. This epigenetic asymmetry is maintained in the endosperm, whereas the embryonic maternal allele is demethylated on fertilization and remethylated later in embryogenesis. This report of imprinting in the plant embryo confirms that, as in mammals, epigenetic mechanisms operate to regulate allelic gene expression in both embryonic and extraembryonic structures. The embryonic methylation profile demonstrates that plants evolved a mechanism for resetting parent-specific imprinting marks, a necessary prerequisite for parent-of-origin-dependent gene expression in consecutive generations. The striking difference between the regulation of imprinting in the embryo and endosperm suggests that imprinting mechanisms might have evolved independently in both fertilization products of flowering plants.     

Differential methylation persists at the mouse Rasgrf1 DMR in tissues displaying monoallelic and biallelic expression

A subset of mammalian genes exhibits genomic imprinting, whereby one parental allele is preferentially expressed. Differential DNA methylation at imprinted loci serves both to mark the parental origin of the alleles and to regulate their expression. In mouse, the imprinted gene Rasgrf1 is associated with a paternally methylated imprinting control region which functions as an enhancer blocker in its unmethylated state. Because Rasgrf1 is imprinted in a tissue-specific manner, we investigated the methylation pattern in monoallelic and biallelic tissues to determine if methylation of this region is required for both imprinted and non-imprinted expression. Our analysis indicates that DNA methylation is restricted to the paternal allele in both monoallelic and biallelic tissues of somatic and extraembryonic lineages. Therefore, methylation serves to mark the paternal Rasgrf1 allele throughout development, but additional factors are required for appropriate tissue-specific regulation of expression at this locus.     

Rasgrf1 imprinting is regulated by a CTCF-dependent methylation-sensitive enhancer blocker

Imprinted methylation of the paternal Rasgrf1 allele in mice occurs at a differentially methylated domain (DMD) 30 kbp 5' of the promoter. A repeated sequence 3' of the DMD regulates imprinted methylation, which is required for imprinted expression. Here we identify the mechanism by which methylation controls imprinting. The DMD is an enhancer blocker that binds CTCF in a methylation-sensitive manner. CTCF bound to the unmethylated maternal allele silences expression. CTCF binding to the paternal allele is prevented by repeat-mediated methylation, allowing expression. Optimal in vitro enhancer-blocking activity requires CTCF binding sites. The enhancer blocker can be bypassed in vivo and imprinting abolished by placing an extra enhancer proximal to the promoter. Together, the repeats and the DMD constitute a binary switch that regulates Rasgrf1 imprinting.