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Polymorphism studies of chromosome-15 hybrids. (a) Monoallelic imprinted gene expression in hybrid 55R-16 with biallelic SNRPN DNA methylation. Primers amplify an HphI polymorphism in the IPW gene in exon 3. Digestion of amplified genomic DNA (lane 2) reveals the presence of two alleles in hybrid 55R-16, only one of which is expressed (lane 3). Lane 1 contains the 123-bp ladder size marker. (b) Microsatellite analysis of hybrid cell line GAR-1 and lineage. GAR-1 contains a maternal chromosome 15 from cell line GM04305, in accordance with complete methylation at the SNRPN promoter (see Fig. 2a).
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Somatic-cell hybrids have been shown to maintain the correct epigenetic chromatin states to study developmental globin gene expression as well as gene expression on the active and inactive X chromosomes. This suggests the potential use of somatic-cell hybrids containing either a maternal or a paternal human chromosome as a model system to study kno...
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... two hybrids, 15A and 55R-16, both a methylated and an unmethylated allele were detected ( Fig. 2a; data not shown), and both hybrids express the SNRPN transcript (Fig. 3a), suggesting either a relaxation of the imprint in tissue culture or the presence of both a maternal and a paternal chromosome in these cell lines. To distinguish between these possibilities, we PCR-amplified across a previously described HphI polymorphism in exon 3 of the IPW gene (ref. 22; see Fig. 1) and digested the products to show the presence of two alleles in the DNA of each hybrid, only one of which is present in the cDNA from each hybrid ( Fig. 4a; data not shown). Furthermore, fluorescence in situ hybridization using a commercial SNRPN cosmid and cells from hybrid 15A con- firmed the presence of an intact chromosome 15 in most cells and a second human fragment containing SNRPN in about 10% of cells (National Institute of General Medical Sciences catalog, 1995; J. M. Amos-Landgraf and R.D.N., unpublished data). These data prove the presence of two chromosomes 15 in hybrids 15A and 55R-16. Therefore, monoallelic gene expression has been maintained in the entire panel of chromosome-15 hybrids, some of which are more than 10 yr old and have endured more than 100 passages in tissue ...
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... chromosomes in the hybrids as either maternal or paternal was initially done on the basis of previously established imprinting patterns; however, it was a formal possibility that during the process of fusing cell lines, or after many passages in tissue culture, genomic imprints might have been reversed. To address this possibility, an informative microsatellite marker was typed in the hybrid cell line GAR-1 as well as in the hybrid cell line donor and the parents of the hybrid cell line donor (Fig. 4b). The haplotype analysis shows that the chromosome 15 contained in the GAR-1 hybrid is of maternal origin, corroborating the methylation and expression data. A second hybrid cell line containing a t (15:19) translocation previously determined to be of paternal origin (27) likewise was shown to maintain the correct paternal methylation pattern ( Fig. 2a; Table 1). Therefore, it is likely that all of the hybrids that are unmethylated and express SNRPN contain a single human chromosome 15 of paternal origin, whereas all of the hybrids that are methylated at SNRPN and do not express the transcript contain a single human chro- mosome 15 of maternal ...
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... The primers used for the target human 47S rRNA allow the amplification of the sequence (between +302 and +548) spanning the first cleavage site positioned at +414 within the 5′ external transcribed spacer region: 5′-GTGCGTGTCAGGCGTTCT-3′ and 5′-GGGAGAGGAGCAGACGAG-3′ (Popov et al., 2013). The human β-actin (ACTB) was used as a reference gene and the following primers were used for its amplification: 5′-TGCGTCTGGACCTGGCTG-GC-3′ and 5′-GCCTCAGGGCAGCGGAACC-3′ (Gabriel et al., 1998). The cycling parameters used were those from the manufacturer's instructions. ...
The class I phosphoinositide 3-kinase (PI3K) catalytic subunits p110α and p110β are ubiquitously expressed but differently targeted in tumours. In cancer, PIK3CB (encoding p110β) is seldom mutated compared with PIK3CA (encoding p110α) but can contribute to tumorigenesis in certain PTEN-deficient tumours. The underlying molecular mechanisms are, however, unclear. We have previously reported that p110β is highly expressed in endometrial cancer (EC) cell lines and at the mRNA level in primary patient tumours. Here, we show that p110β protein levels are high in both the cytoplasmic and nuclear compartments in EC cells. Moreover, high nuclear:cytoplasmic staining ratios were detected in high-grade primary tumours. High levels of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5) P 3 ] were measured in the nucleus of EC cells, and pharmacological and genetic approaches showed that its production was partly dependent upon p110β activity. Using immunofluorescence staining, p110β and PtdIns(3,4,5) P 3 were localised in the nucleolus, which correlated with high levels of 47S pre-rRNA. p110β inhibition led to a decrease in both 47S rRNA levels and cell proliferation. In conclusion, these results present a nucleolar role for p110β that may contribute to tumorigenesis in EC.
This article has an associated First Person interview with Fatemeh Mazloumi Gavgani, joint first author of the paper.
... Our screening for hemimethylated CpGs in human orthologues of known mouse IGs, with no imprinting data in humans, retrieved 13 genes; six of them are localized within imprinted clusters in mice, and are within or close to imprinted clusters in human as well-CD81, NAP1L4, USP29, ZIM3, BEGAIN, and MIR379. CD81 gene is listed as non-imprinted in Geneimprint database, but the only evidence against its imprinting status comes from a report of biallelic expression in somatic cell hybrids [38]. In mice, Nap1l4 has controversial imprinting status between databases (Table S2); NAP1L4 is biallelically expressed in embryonic and trophoblast stem cells [39], being a weak candidate in humans. ...
Large-scale transcriptome and methylome data analyses obtained by high-throughput technologies have been enabling the identification of novel imprinted genes. We investigated genome-wide DNA methylation patterns in multiple human tissues, using a high-resolution microarray to uncover hemimethylated CpGs located in promoters overlapping CpG islands, aiming to identify novel candidate imprinted genes. Using our approach, we recovered ~30% of the known human imprinted genes, and a further 168 candidates were identified, 61 of which with at least three hemimethylated CpGs shared by more than two tissue types. Thirty-four of these candidate genes are members of the protocadherin cluster on 5q31.3; in mice, protocadherin genes have non-imprinted random monoallelic expression, which might also be the case in humans. Among the remaining 27 genes, ZNF331 was recently validated as an imprinted gene, and six of them have been reported as candidates, supporting our prediction. Five candidates (CCDC166, ARC, PLEC, TONSL, and VPS28) map to 8q24.3, and might constitute a novel imprinted cluster. Additionally, we performed a comprehensive compilation of known human and mice imprinted Epigenomes 2017, 2, 13 2 of 15 genes from literature and databases, and a comparison among high-throughput imprinting studies in humans. The screening for hemimethylated CpGs shared by multiple human tissues, together with the extensive review, appears to be a useful approach to reveal candidate imprinted genes.
... A similar lack of conservation of imprinting between the human and mouse genome was previously reported (47)(48)(49)(50). ...
During pregnancy, placental trophoblasts at the feto-maternal interface produce a broad repertoire of microRNA (miRNA) species. These species include miRNA from the primate-specific chromosome 19 miRNA cluster (C19MC), which is expressed nearly exclusively in the placenta. Trafficking of these miRNAs among the maternal, placental, and fetal compartments is unknown. To determine miRNA expression and trafficking patterns during pregnancy, we sequenced miRNAs in triads of human placenta and of maternal and fetal blood and found large subject-to-subject variability, with C19MC exhibiting compartment-specific expression. We therefore created humanized mice that transgenically express the entire 160-kb human C19MC locus or lentivirally express C19MC miRNA members selectively in the placenta. C19MC transgenic mice expressed a low level of C19MC miRNAs in diverse organs. When pregnant, female C19MC mice exhibited a strikingly elevated (>40-fold) expression of C19MC miRNA in the placenta, compared with other organs, that resembled C19MC miRNAs patterns in humans. Our mouse models showed that placental miRNA traffic primarily to the maternal circulation and that maternal miRNA can traffic to the placenta and even into the fetal compartment. These findings define an extraordinary means of nonhormonal, miRNA-based communication between the placenta and feto-maternal compartments.-Chang, G., Mouillet, J.-F., Mishima, T., Chu, T., Sadovsky, E., Coyne, C. B., Parks, W. T., Surti, U., Sadovsky, Y. Expression and trafficking of placental microRNAs at the feto-maternal interface.
... Polymorphisms in the GABA A -b3 receptor subunit are associated with ASD (Cook et al., 1998;Martin et al., 2000). Moreover, differential methylations of the GABA A gene (Bittel et al., 2005;Gabriel et al., 1998;Hogart et al., 2007;Meguro et al., 1997) may result in epigenetic modifications and modifications of clinical phenotypes in dup(15)/autism. ...
Postmortem studies of brains of individuals with idiopathic autism and 15q11.2–q13 duplications autism (dup(15)) identify a cluster of neuropathological features differentiating these cohorts. They show a need for both reclassification of autism according to etiology, clinical presentation and neuropathology, and a commonality of clinical and neuropathological traits justifying autism diagnosis. The features differentiating these cohorts include: (a) maternal origin in patients with dup(15); (b) autism in 78% of subjects; (c) more severe clinical phenotypes, with intellectual deficit (100%), early-onset of severe or intractable seizures in 78% of subjects, and increased prevalence (up to 67%) of sudden unexplained death; (d) high prevalence of microcephaly, with mean brain weight 300g less than in idiopathic autism; (e) several-fold increase in the number of developmental abnormalities, including defects of migration and dysplastic changes, especially numerous in the hippocampal formation; and (f) significant increase in the intraneuronal amyloid load, reflecting enhanced amyloid-β precursor protein processing with α-secretase.
... A map of parent of origin-specific epigenetic modifications suggests that this imprinted locus may have links not only with autism but also with other psychiatric phenotypes (35). Differential methylations in the 15q11-q13 region, including the GABA A gene (30,(36)(37)(38), may contribute to epigenetic modifications and a broader clinical phenotypes in dup(15)/autism. Several other genes located in or near the 15q11-q13 region may contribute to a variable phenotype of autism, including a gene for juvenile epilepsy located near D15S165 (39) and a locus for agenesis of the corpus callosum (40). ...
The purposes of this study were to identify differences in patterns of developmental abnormalities between the brains of individuals with autism of unknown etiology and those of individuals with duplications of chromosome 15q11.2-q13 (dup[15]) and autism and to identify alterations that may contribute to seizures and sudden death in the latter. Brains of 9 subjects with dup(15), 10 with idiopathic autism, and 7 controls were examined. In the dup(15) cohort, 7 subjects (78%) had autism, 7 (78%) had seizures, and 6 (67%) had experienced sudden unexplained death. Subjects with dup(15) autism were microcephalic, with mean brain weights 300 g less (1,177 g) than those of subjects with idiopathic autism (1,477 g; p<0.001). Heterotopias in the alveus, CA4, and dentate gyrus and dysplasia in the dentate gyrus were detected in 89% of dup(15) autism cases but in only 10% of idiopathic autism cases (p < 0.001). By contrast, cerebral cortex dysplasia was detected in 50% of subjects with idiopathic autism and in no dup(15) autism cases (p<0.04). The different spectrum and higher prevalence of developmental neuropathologic findings in the dup(15) cohort than in cases with idiopathic autism may contribute to the high risk of early onset of seizures and sudden death.
... Several lines of evidence suggest these genes as candidate loci for neuropsychiatric disorders, including mouse knockout and association studies linking them with autistic phenotypes (Ma et al. 2005) and the observation that maternal, but not paternal, duplications of this region are seen in both autism and schizophrenia (Cook et al. 1997;Webb et al. 2008). While previous studies have yielded evidence suggesting that the GABA A gene cluster in 15q12 is imprinted in humans (Meguro et al. 1997;Bittel et al. 2005), there is also conflicting evidence suggesting that they are not imprinted (Gabriel et al. 1998;Hogart et al. 2007). We demonstrate that this 687-kb critical region contains a novel DMR ;150 kb distal to the 39 end of GABRG3, which we suggest, therefore, likely represents the imprinted element responsible for the increased risk of psychosis in PWS patients with matUPD15 or maternal duplications of 15q12. ...
The maternal and paternal genomes possess distinct epigenetic marks that distinguish them at imprinted loci. In order to identify imprinted loci, we used a novel method, taking advantage of the fact that uniparental disomy (UPD) provides a system that allows the two parental chromosomes to be studied independently. We profiled the paternal and maternal methylation on chromosome 15 using immunoprecipitation of methylated DNA and hybridization to tiling oligonucleotide arrays. Comparison of six individuals with maternal versus paternal UPD15 revealed 12 differentially methylated regions (DMRs). Putative DMRs were validated by bisulfite sequencing, confirming the presence of parent-of-origin-specific methylation marks. We detected DMRs associated with known imprinted genes within the Prader-Willi/Angelman syndrome region, such as SNRPN and MAGEL2, validating this as a method of detecting imprinted loci. Of the 12 DMRs identified, eight were novel, some of which are associated with genes not previously thought to be imprinted. These include a site within intron 2 of IGF1R at 15q26.3, a gene that plays a fundamental role in growth, and an intergenic site upstream of GABRG3 that lies within a previously defined candidate region conferring an increased maternal risk of psychosis. These data provide a map of parent-of-origin-specific epigenetic modifications on chromosome 15, identifying DNA elements that may play a functional role in the imprinting process. Application of this methodology to other chromosomes for which UPD has been reported will allow the systematic identification of imprinted sites throughout the genome.
... GABRB3 protein expression is reduced in RTT, caused by mutations in MECP2 on Xq28 (Samaco et al., 2005). Although Gabrb3 is biallelically expressed in mouse brain, conflicting data exist regarding the imprinting status of the 15q11-13 GABR genes in humans (Meguro et al., 1997;Bittel et al., 2003;Bittel et al., 2005;Gabriel et al., 1998). A recent study showed that all three GABR genes were biallelically expressed in 21 control brain samples, demonstrating the lack of imprinting in normal human cortex . ...
Symposium 5 focused on research approaches that are aimed at understanding common patterns of immunological and neurological dysfunction contributing to neurodevelopmental disorders such as autism and ADHD. The session focused on genetic, epigenetic, and environmental factors that might act in concert to influence autism risk, severity and co-morbidities, and immunological and neurobiological targets as etiologic contributors. The immune system of children at risk of autism may be therefore especially susceptible to psychological stressors, exposure to chemical triggers, and infectious agents. Identifying early biomarkers of risk provides tangible approaches toward designing studies in animals and humans that yield a better understanding of environmental risk factors, and can help identify rational intervention strategies to mitigate these risks.
... Chromosome 15q11-13 contains a cluster of three GABA A receptor subunit genes, GABRB3, GABRA5, and GABRG3, encoding receptor subunits for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). The mouse orthologues, contained within a syntenic block on chromosome 7qB5, are biallelically expressed in brain (14)(15)(16), however, conflicting data exist on the imprinting status of the human 15q11-13 GABA A receptor subunit genes (17)(18)(19)(20)(21)). An early study of GABRB3 expression in human hydatidiform mole and normal villi tissues suggested preferential maternal gene expression (20). ...
... While imprinting is well characterized for some 15q11-13 genes, such as SNRPN (36) and UBE3A (27,28), the imprinting status of the GABA A receptor cluster has been disputed (18)(19)(20)(21). We directly analyzed allelic expression of 15q11-13 GABA A receptor subunit genes in human brain and report that GABRB3, GABRA5, and GABRG3 are normally biallelically expressed in frontal cortex, supporting previous findings in mouse (14,15). ...
Human chromosome 15q11-13 is a complex locus containing imprinted genes as well as a cluster of three GABA(A) receptor subunit (GABR) genes-GABRB3, GABRA5 and GABRG3. Deletion or duplication of 15q11-13 GABR genes occurs in multiple human neurodevelopmental disorders including Prader-Willi syndrome (PWS), Angelman syndrome (AS) and autism. GABRB3 protein expression is also reduced in Rett syndrome (RTT), caused by mutations in MECP2 on Xq28. Although Gabrb3 is biallelically expressed in mouse brain, conflicting data exist regarding the imprinting status of the 15q11-13 GABR genes in humans. Using coding single nucleotide polymorphisms we show that all three GABR genes are biallelically expressed in 21 control brain samples, demonstrating that these genes are not imprinted in normal human cortex. Interestingly, four of eight autism and one of five RTT brain samples showed monoallelic or highly skewed allelic expression of one or more GABR gene, suggesting that epigenetic dysregulation of these genes is common to both disorders. Quantitative real-time RT-PCR analysis of PWS and AS samples with paternal and maternal 15q11-13 deletions revealed a paternal expression bias of GABRB3, while RTT brain samples showed a significant reduction in GABRB3 and UBE3A. Chromatin immunoprecipitation and bisulfite sequencing in SH-SY5Y neuroblastoma cells demonstrated that MeCP2 binds to methylated CpG sites within GABRB3. Our previous studies demonstrated that homologous 15q11-13 pairing in neurons was dependent on MeCP2 and was disrupted in RTT and autism cortex. Combined, these results suggest that MeCP2 acts as a chromatin organizer for optimal expression of both alleles of GABRB3 in neurons.
... Alpha-fetoprotein, an immature endodermal cell marker, can be retrieved in areas of glandular appearance (D). The nuclei were staned with DAPI (blue disorders linked to maternally or paternally expressed genes (Gabriel et al., 1998 ). Studies of imprinting in phESC require a detailed investigation because of the possible influence upon phESC differentiation and functionality of their derivatives. ...
Parthenogenetic activation of human oocytes may be one way to produce histocompatible cells for cell-based therapy. We report the successful derivation of six pluripotent human embryonic stem cell (hESC) lines from blastocysts of parthenogenetic origin. The parthenogenetic human embryonic stem cells (phESC) demonstrate typical hESC morphology, express appropriate markers, and possess high levels of alkaline phosphatase and telomerase activity. The phESC lines have a normal 46, XX karyotype, except one cell line, and have been cultured from between 21 to 35 passages. The phESC lines form embryoid bodies in suspension culture and teratomas after injection to immunodeficient animals and give differentiated derivatives of all three embryonic germ layers. DNA profiling of all six phESC lines demonstrates that they are MHC matched with the oocyte donors. The study of imprinted genes demonstrated further evidence of the parthenogenetic origin of the phESC lines. Our research has resulted in a protocol for the production of human parthenogenetic embryos and the derivation of stem cell lines from them, which minimizes the presence of animal-derived components, making the derived phESC lines more suitable for potential clinical use.
... Despite the apparent conservation of imprinting among mammals, there are few examples of human transgenes imprinting appropriately in mice [38,39,464748. Furthermore, there are few data available to show that small regions can reliably direct imprinting on transgenes, thus making the precise dissection of these regions quite challenging. ...
Imprinting centers (IC) can be defined as cis-elements that are recognized in the germ line and are epigenetically modified to bring about the full imprinting program in a somatic cell. Two paternally expressed human genes, HYMAI and PLAGL1 (LOT1/ZAC), are located within human chromosome 6q24. Within this region lies a 1-kb CpG island that is differentially methylated in somatic cells, unmethylated in sperm, and methylated in mature oocytes in mice, characteristic features of an IC. Loss of methylation of the homologous region in humans is observed in patients with transient neonatal diabetes mellitus and hypermethylation is associated with a variety of cancers, suggesting that this region regulates the expression of one or more key genes in this region involved in these diseases. We now report that a transgene carrying the human HYMAI/PLAGL1 DMR was methylated in the correct parent-origin-specific manner in mice and this was sufficient to confer imprinted expression from the transgene. Therefore, we propose that this DMR functions as the IC for the HYMAI/PLAGL1 domain.
