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Maintenance of Paternal Methylation and Repression of the Imprinted H19 Gene Requires MBD3
Reese, Kimberly J
Lin, Shu
Verona, Raluca I
Schultz, Richard M
Bartolomei, Marisa S
PLoS Genetics
Paternal repression of the imprinted H19 gene is mediated by a differentially methylated domain (DMD) that is essential to imprinting of both H19 and the linked and oppositely imprinted Igf2 gene. The mechanisms by which paternal-specific methylation of the DMD survive the period of genome-wide demethylation in the early embryo and are subsequently used to govern imprinted expression are not known. Methyl-CpG binding (MBD) proteins are likely candidates to explain how these DMDs are recognized to silence the locus, because they preferentially bind methylated DNA and recruit repression complexes with histone deacetylase activity. MBD RNA and protein are found in preimplantation embryos, and chromatin immunoprecipitation shows that MBD3 is bound to the H19 DMD. To test a role for MBDs in imprinting, two independent RNAi-based strategies were used to deplete MBD3 in early mouse embryos, with the same results. In RNAi-treated blastocysts, paternal H19 expression was activated, supporting the hypothesis that MBD3, which is also a member of the Mi-2/NuRD complex, is required to repress the paternal H19 allele. RNAi-treated blastocysts also have reduced levels of the Mi-2/NuRD complex protein MTA-2, which suggests a role for the Mi-2/NuRD repressive complex in paternal-specific silencing at the H19 locus. Furthermore, DNA methylation was reduced at the H19 DMD when MBD3 protein was depleted. In contrast, expression and DNA methylation were not disrupted in preimplantation embryos for other imprinted genes. These results demonstrate new roles for MBD3 in maintaining imprinting control region DNA methylation and silencing the paternal H19 allele. Finally, MBD3-depleted preimplantation embryos have reduced cell numbers, suggesting a role for MBD3 in cell division.
Author Summary
Genomic imprinting is a specialized system of gene regulation whereby only one copy of a gene is used, either the maternal or the paternal copy. Misregulation of imprinting in humans results in developmental disorders such as Beckwith-Wiedemann Syndrome, and is implicated in many cancers. Study of imprinted genes in mice can lead to a greater understanding of these diseases as well as insight into gene regulation. Many imprinted genes are associated with methylation on the silenced allele. The imprinted gene H19 is maternally expressed and paternally methylated in a region upstream of the promoter known as the differentially methylated domain. This region is required for proper imprinted expression of H19 and its upstream imprinted neighbor Igf2. Our studies have explored the requirement for methyl-CpG binding protein 3 (MBD3) in silencing of the paternal allele. MBD3 is known to be part of a repressive complex that resides at silenced genes. In our experiments, we have shown that MBD3 is required for imprinting of H19, and is also required for the maintenance of methylation on the paternal allele. Finally, the MBD3 protein can be found at the differentially methylated domain. The identification of a protein required for silencing of the paternal allele of H19 is an important step in understanding regulation of this gene.
Related Articles
The interval between Ins2 and Ascl2 is dispensable for imprinting centre function in the murine Beckwith-Wiedemann region
Lefebvre, Louis
Mar, Lynn
Bogutz, Aaron
Oh-McGinnis, Rosemary
Mandegar, Mohammad
Paderova, Jana
Gertsenstein, Marina
Squire, Jeremy A.
Nagy, Andras
Human molecular genetics
Imprinted genes are commonly clustered in domains across the mammalian genome, suggesting a degree of coregulation via long-range coordination of their monoallelic transcription. The distal end of mouse chromosome 7 contains two clusters of imprinted genes within a ~1 Mb domain. This region is conserved on human 11q15.5 where it is implicated in the Beckwith-Wiedemann syndrome. In both species, imprinted regulation requires two critical cis-acting imprinting centres, carrying different germline epigenetic marks and mediating imprinted expression in the proximal and distal sub-domains. The clusters are separated by a region containing the gene for tyrosine hydroxylase (Th) as well as a high density of short repeats and retrotransposons in the mouse. We have used the Cre-loxP recombination system in vivo to engineer an interstitial deletion of this ~280-kb intervening region previously proposed to participate in the imprinting mechanism or to act as a boundary between the two sub-domains. The deletion allele, Del7AI, is silent with respect to epigenetic marking at the two flanking imprinting centres. Reciprocal inheritance of Del7AI demonstrates that the deleted region, which represents more than a quarter of the previously defined imprinted domain, is associated with intrauterine growth restriction in maternal heterozygotes. In homozygotes, the deficiency behaves as a Th null allele and can be rescued pharmacologically by bypassing the metabolic requirement for TH in utero. Our results show that the deleted interval is not required for normal imprinting on distal Chr 7 and uncover a new imprinted growth phenotype.
Related Articles
Germline Mutation in NLRP2 (NALP2) in a Familial Imprinting Disorder (Beckwith-Wiedemann Syndrome)
Meyer, Esther
Lim, Derek
Pasha, Shanaz
Tee, Louise J.
Rahman, Fatimah
Yates, John R. W.
Woods, C. Geoffrey
Reik, Wolf
Maher, Eamonn R.
PLoS Genetics
Beckwith-Wiedemann syndrome (BWS) is a fetal overgrowth and human imprinting disorder resulting from the deregulation of a number of genes, including IGF2 and CDKN1C, in the imprinted gene cluster on chromosome 11p15.5. Most cases are sporadic and result from epimutations at either of the two 11p15.5 imprinting centres (IC1 and IC2). However, rare familial cases may be associated with germline 11p15.5 deletions causing abnormal imprinting in cis. We report a family with BWS and an IC2 epimutation in which affected siblings had inherited different parental 11p15.5 alleles excluding an in cis mechanism. Using a positional-candidate gene approach, we found that the mother was homozygous for a frameshift mutation in exon 6 of NLRP2. While germline mutations in NLRP7 have previously been associated with familial hydatidiform mole, this is the first description of NLRP2 mutation in human disease and the first report of a trans mechanism for disordered imprinting in BWS. These observations are consistent with the hypothesis that NLRP2 has a previously unrecognised role in establishing or maintaining genomic imprinting in humans.
Author Summary
A small set of genes (imprinted genes) are expressed in a "parent-of-origin" manner, a phenomenon known as genomic imprinting. Research in human disorders associated with aberrant genomic imprinting provided insights into the molecular mechanisms of genomic imprinting and the role of imprinted genes in normal growth and development. Beckwith-Wiedemann syndrome (BWS) is a congenital overgrowth syndrome associated with developmental abnormalities and a predisposition to embryonic tumours. BWS results from alterations in expression or function of imprinted genes in the imprinted gene cluster at chromosome 11p15. Although BWS may be caused by a variety of molecular mechanisms, to date, all the genetic and epigenetic defects associated with BWS have been limited to 11p15.5. We report a family with two children affected with BWS and an epigenetic defect at 11p15.5 in which the primary genetic defect mapped outside the imprinted gene cluster. Using autozygosity mapping, we found an extended homozygous region on chromosome 19q13.4 (containing NLRP2 and NLRP7 genes) in the mother. Homozygous inactivating mutations in NLRP7 in women have been associated previously with abnormal imprinting and recurrent hydatidiform moles. We identified a homozygous frameshift mutation in NLRP2 in the mother of the two children with BWS implicating NLRP2 in the establishment and/or maintenance of genomic imprinting/methylation.
Related Articles
ZAC, LIT1 (KCNQ1OT1) and p57KIP2 (CDKN1C) are in an imprinted gene network that may play a role in Beckwith–Wiedemann syndrome
Arima, Takahiro
Kamikihara, Tetsuya
Hayashida, Toshirou
Kato, Kiyoko
Inoue, Toshiaki
Shirayoshi, Yasuaki
Oshimura, Mitsuo
Soejima, Hidenobu
Mukai, Tunehiro
Wake, Norio
Nucleic Acids Research
Loss of genomic imprinting is involved in a number of developmental abnormalities and cancers. ZAC is an imprinted gene expressed from the paternal allele of chromosome 6q24 within a region known to harbor a tumor suppressor gene for several types of neoplasia. p57KIP2 (CDKN1C) is a maternally expressed gene located on chromosome 11p15.5 which encodes a cyclin-dependent kinase inhibitor that may also act as a tumor suppressor gene. Mutations in ZAC and p57KIP2 have been implicated in transient neonatal diabetes mellitus (TNDB) and Beckwith–Wiedemann syndrome, respectively. Patients with these diseases share many characteristics. Here we show that mouse Zac1 and p57Kip2 have a strikingly similar expression pattern. ZAC, a sequence-specific DNA-binding protein, binds within the CpG island of LIT1 (KCNQ1OT1), a paternally expressed, anti-sense RNA thought to negatively regulate p57KIP2 in cis. ZAC induces LIT1 transcription in a methylation-dependent manner. Our data suggest that ZAC may regulate p57KIP2 through LIT1, forming part of a novel signaling pathway regulating cell growth. Mutations in ZAC may, therefore, contribute to Beckwith–Wiedemann syndrome. Furthermore, we find changes in DNA methylation at the LIT1 putative imprinting control region in two patients with TNDB.
Related Articles
Epigenotype-phenotype correlations in Beckwith-Wiedemann syndrome
Engel, J.
Smallwood, A.
Harper, A.
Higgins, M.
Oshimura, M.
Reik, W.
Schofield, P.
Maher, E.
Journal of Medical Genetics
Beckwith-Wiedemann syndrome (BWS) is a model imprinting disorder resulting from mutations or epigenetic events involving imprinted genes at chromosome 11p15.5. Thus, germline mutations in CDKN1C, uniparental disomy (UPD), and loss of imprinting of IGF2 and other imprinted genes have been implicated. Many familial BWS cases have germline CDKN1C mutations. However, most BWS cases are sporadic and UPD or putative imprinting errors predominate in this group. We have identified previously a subgroup of sporadic cases with loss of imprinting (LOI) of IGF2 and epigenetic silencing of H19 proposed to be caused by a defect in a distal 11p15.5 imprinting control element (designated BWSIC1). However, many sporadic BWS patients show biallelic IGF2 expression in the presence of normal H19 methylation and expression patterns. This and other evidence suggested the existence of a further imprinting control element (BWSIC2) at 11p15.5. Recently, we showed that a subgroup of BWS patients have loss of methylation (LOM) at a differentially methylated region (KvDMR1) within the KCNQ1 gene centromeric to the IGF2 and H19 genes. We have now analysed a large series of sporadic cases to define the frequency and phenotypic correlates of epigenetic abnormalities in BWS. LOM at KvDMR1 was detected by Southern analysis or a novel PCR based method in 35 of 69 (51%) sporadic BWS without UPD. LOM at KvDMR1 was often, but not invariably associated with LOI of IGF2. KvDMR1 LOM was not detected in BWS patients with putative BWSIC1 defects and cases with KvDMR1 LOM (that is, putative BWSIC2 defects) invariably had a normal H19 methylation pattern. The incidence of exomphalos in putative BWSIC2 defect patients was not significantly different from that in patients with germline CDKN1C mutations (20/29 and 13/15 respectively), but was significantly greater than that in patients with putative BWSIC1 defects (0/5, p=0.007) and UPD (0/22, p<0.0001). These findings are consistent with the hypothesis that LOM of KvDMR1 (BWSIC2 defect) results in epigenetic silencing of CDKN1C and variable LOI of IGF2. BWS patients with embryonal tumours have UPD or a BWSIC1 defect but not LOM of KvDMR1. This study has further shown how (1) variations in phenotypic expression of BWS may be linked to specific molecular subgroups and (2) molecular analysis of BWS can provide insights into mechanisms of imprinting regulation. Keywords: Beckwith-Wiedemann syndrome; epigenotype-phenotype correlations; imprinting
Related Articles
The Evolution of the DLK1-DIO3 Imprinted Domain in Mammals
Edwards, Carol A
Mungall, Andrew J
Matthews, Lucy
Ryder, Edward
Gray, Dionne J
Pask, Andrew J
Shaw, Geoffrey
Graves, Jennifer A.M
Rogers, Jane
Dunham, Ian
Renfree, Marilyn B
Ferguson-Smith, Anne C
PLoS Biology
A comprehensive, domain-wide comparative analysis of genomic imprinting between mammals that imprint and those that do not can provide valuable information about how and why imprinting evolved. The imprinting status, DNA methylation, and genomic landscape of the Dlk1-Dio3 cluster were determined in eutherian, metatherian, and prototherian mammals including tammar wallaby and platypus. Imprinting across the whole domain evolved after the divergence of eutherian from marsupial mammals and in eutherians is under strong purifying selection. The marsupial locus at 1.6 megabases, is double that of eutherians due to the accumulation of LINE repeats. Comparative sequence analysis of the domain in seven vertebrates determined evolutionary conserved regions common to particular sub-groups and to all vertebrates. The emergence of Dlk1-Dio3 imprinting in eutherians has occurred on the maternally inherited chromosome and is associated with region-specific resistance to expansion by repetitive elements and the local introduction of noncoding transcripts including microRNAs and C/D small nucleolar RNAs. A recent mammal-specific retrotransposition event led to the formation of a completely new gene only in the eutherian domain, which may have driven imprinting at the cluster.
Author Summary
Mammals have two copies of each gene in their somatic cells, and most of these gene pairs are regulated and expressed simultaneously. A fraction of mammalian genes, however, is subject to imprinting—a chemical modification that marks a gene according to its parental origin, so that one parent's copy is expressed while the other parent's copy is silenced. How and why this process evolved is the subject of much speculation. Here we have shown that all the genes in one genomic region, Dlk1-Dio3, which are imprinted in placental mammals such as mouse and human, are not imprinted in marsupial (wallaby) or monotreme (platypus) mammals. This is in contrast to a small number of other imprinted genes that are imprinted in marsupials and other therian mammals and indicates that imprinting arose at each genomic domain at different stages of mammalian evolution. We have compared the sequence of the Dlk1-Dio3 region between seven vertebrate species and identified sequences that are differentially represented in mammals that imprint compared to those that do not. Our data indicate that once imprinted gene regulation is acquired in a domain, it becomes evolutionarily constrained to remain unchanged.
A comparative analysis of genomic imprinting between mammals that imprint and those that don't has provided insights into how and why imprinting evolved.
Related Articles
Loss of imprinting at the Dlk1-Gtl2 locus caused by insertional mutagenesis in the Gtl2 5' region
Steshina, Ekaterina Y
Carr, Michael S
Glick, Elena A
Yevtodiyenko, Aleksey
Appelbe, Oliver K
Schmidt, Jennifer V
BMC Genetics
The Dlk1 and Gtl2 genes define a region of mouse chromosome 12 that is subject to genomic imprinting, the parental allele-specific expression of a gene. Although imprinted genes play important roles in growth and development, the mechanisms by which imprinting is established and maintained are poorly understood. Differentially methylated regions (DMRs), which carry methylation on only one parental allele, are involved in imprinting control at many loci. The Dlk1-Gtl2 region contains three known DMRs, the Dlk1 DMR in the 3' region of Dlk1, the intergenic DMR 15 kb upstream of Gtl2, and the Gtl2 DMR at the Gtl2 promoter. Three mouse models are analyzed here that provide new information about the regulation of Dlk1-Gtl2 imprinting.
A previously existing insertional mutation (Gtl2lacZ), and a targeted deletion in which the Gtl2 upstream region was replaced by a Neo cassette (Gtl2Δ5'Neo), display partial lethality and dwarfism upon paternal inheritance. Molecular characterization shows that both mutations cause loss of imprinting and changes in expression of the Dlk1, Gtl2 and Meg8/Rian genes. Dlk1 levels are decreased upon paternal inheritance of either mutation, suggesting Dlk1 may be causative for the lethality and dwarfism. Loss of imprinting on the paternal chromosome in both Gtl2lacZ and Gtl2Δ5'Neo mice is accompanied by the loss of paternal-specific Gtl2 DMR methylation, while maternal loss of imprinting suggests a previously unknown regulatory role for the maternal Gtl2 DMR. Unexpectedly, when the Neo gene is excised, Gtl2Δ5' animals are of normal size, imprinting is unchanged and the Gtl2 DMR is properly methylated. The exogenous DNA sequences integrated upstream of Gtl2 are therefore responsible for the growth and imprinting effects.
These data provide further evidence for the coregulation of the imprinted Dlk1 and Gtl2 genes, and support a role for Dlk1 as an important neonatal growth factor. The ability of the Gtl2lacZ and Gtl2Δ5'Neo mutations to cause long-range changes in imprinting and gene expression suggest that regional imprinting regulatory elements may lie in proximity to the integration site.
Related Articles
Evolution of the CDKN1C-KCNQ1 imprinted domain
Ager, Eleanor I
Pask, Andrew J
Gehring, Helen M
Shaw, Geoff
Renfree, Marilyn B
BMC Evolutionary Biology
Genomic imprinting occurs in both marsupial and eutherian mammals. The CDKN1C and IGF2 genes are both imprinted and syntenic in the mouse and human, but in marsupials only IGF2 is imprinted. This study examines the evolution of features that, in eutherians, regulate CDKN1C imprinting.
Despite the absence of imprinting, CDKN1C protein was present in the tammar wallaby placenta. Genomic analysis of the tammar region confirmed that CDKN1C is syntenic with IGF2. However, there are fewer LTR and DNA elements in the region and in intron 9 of KCNQ1. In addition there are fewer LINEs in the tammar compared with human and mouse. While the CpG island in intron 10 of KCNQ1 and promoter elements could not be detected, the antisense transcript KCNQ1OT1 that regulates CDKN1C imprinting in human and mouse is still expressed.
CDKN1C has a conserved function, likely antagonistic to IGF2, in the mammalian placenta that preceded its acquisition of imprinting. CDKN1C resides in synteny with IGF2, demonstrating that imprinting of the two genes did not occur concurrently to balance maternal and paternal influences on the growth of the placenta. The expression of KCNQ1OT1 in the absence of CDKN1C imprinting suggests that antisense transcription at this locus preceded imprinting of this domain. These findings demonstrate the stepwise accumulation of control mechanisms within imprinted domains and show that CDKN1C imprinting cannot be due to its synteny with IGF2 or with its placental expression in mammals.
Related Articles
Distinct Methylation Changes at the IGF2-H19 Locus in Congenital Growth Disorders and Cancer
Murrell, Adele
Ito, Yoko
Verde, Gaetano
Huddleston, Joanna
Woodfine, Kathryn
Silengo, Margherita Cirillo
Spreafico, Filippo
Perotti, Daniela
De Crescenzo, Agostina
Sparago, Angela
Cerrato, Flavia
Riccio, Andrea
PLoS ONE
Differentially methylated regions (DMRs) are associated with many imprinted genes. In mice methylation at a DMR upstream of the H19 gene known as the Imprint Control region (IC1) is acquired in the male germline and influences the methylation status of DMRs 100 kb away in the adjacent Insulin-like growth factor 2 (Igf2) gene through long-range interactions. In humans, germline-derived or post-zygotically acquired imprinting defects at IC1 are associated with aberrant activation or repression of IGF2, resulting in the congenital growth disorders Beckwith-Wiedemann (BWS) and Silver-Russell (SRS) syndromes, respectively. In Wilms tumour and colorectal cancer, biallelic expression of IGF2 has been observed in association with loss of methylation at a DMR in IGF2. This DMR, known as DMR0, has been shown to be methylated on the silent maternal IGF2 allele presumably with a role in repression. The effect of IGF2 DMR0 methylation changes in the aetiology of BWS or SRS is unknown.
Methodology/Principal Findings
We analysed the methylation status of the DMR0 in BWS, SRS and Wilms tumour patients by conventional bisulphite sequencing and pyrosequencing. We show here that, contrary to previous reports, the IGF2 DMR0 is actually methylated on the active paternal allele in peripheral blood and kidney. This is similar to the IC1 methylation status and is inconsistent with the proposed silencing function of the maternal IGF2 allele. Beckwith-Wiedemann and Silver-Russell patients with IC1 methylation defects have similar methylation defects at the IGF2 DMR0, consistent with IC1 regulating methylation at IGF2 in cis. In Wilms tumour, however, methylation profiles of IC1 and IGF2 DMR0 are indicative of methylation changes occurring on both parental alleles rather than in cis.
These results support a model in which DMR0 and IC1 have opposite susceptibilities to global hyper and hypomethylation during tumorigenesis independent of the parent of origin imprint. In contrast, during embryogenesis DMR0 is methylated or demethylated according to the germline methylation imprint at the IC1, indicating different mechanisms of imprinting loss in neoplastic and non-neoplastic cells.
Related Articles
The Parental Non-Equivalence of Imprinting Control Regions during Mammalian Development and Evolution
Schulz, Reiner
Proudhon, Charlotte
Bestor, Timothy H.
Woodfine, Kathryn
Lin, Chyuan-Sheng
Lin, Shau-Ping
Prissette, Marine
Oakey, Rebecca J.
Bourc'his, Déborah
PLoS Genetics
In mammals, imprinted gene expression results from the sex-specific methylation of imprinted control regions (ICRs) in the parental germlines. Imprinting is linked to therian reproduction, that is, the placenta and imprinting emerged at roughly the same time and potentially co-evolved. We assessed the transcriptome-wide and ontology effect of maternally versus paternally methylated ICRs at the developmental stage of setting of the chorioallantoic placenta in the mouse (8.5dpc), using two models of imprinting deficiency including completely imprint-free embryos. Paternal and maternal imprints have a similar quantitative impact on the embryonic transcriptome. However, transcriptional effects of maternal ICRs are qualitatively focused on the fetal-maternal interface, while paternal ICRs weakly affect non-convergent biological processes, with little consequence for viability at 8.5dpc. Moreover, genes regulated by maternal ICRs indirectly influence genes regulated by paternal ICRs, while the reverse is not observed. The functional dominance of maternal imprints over early embryonic development is potentially linked to selection pressures favoring methylation-dependent control of maternal over paternal ICRs. We previously hypothesized that the different methylation histories of ICRs in the maternal versus the paternal germlines may have put paternal ICRs under higher mutational pressure to lose CpGs by deamination. Using comparative genomics of 17 extant mammalian species, we show here that, while ICRs in general have been constrained to maintain more CpGs than non-imprinted sequences, the rate of CpG loss at paternal ICRs has indeed been higher than at maternal ICRs during evolution. In fact, maternal ICRs, which have the characteristics of CpG-rich promoters, have gained CpGs compared to non-imprinted CpG-rich promoters. Thus, the numerical and, during early embryonic development, functional dominance of maternal ICRs can be explained as the consequence of two orthogonal evolutionary forces: pressure to tightly regulate genes affecting the fetal-maternal interface and pressure to avoid the mutagenic environment of the paternal germline.
Author Summary
In mammals, a subset of genes is expressed from only one chromosomal copy, depending on its parental origin. This process, known as genomic imprinting, results from DNA methylation marks deposited in gametes at regulatory sequences called imprinting control regions (ICRs). Most of the DNA methylation controlling imprinting is established in the oocyte, while very few ICRs are methylated in the sperm. We provided insight into the impact and origins of the parental imbalance in genomic imprinting control. We defined the transcriptome-wide effect of imprinting, during the transition period when the embryo becomes dependent upon maternal resources. We found that maternal ICRs have a vital effect on developmental pathways related to the mother-to-fetus exchanges, while paternal ICRs have a dispersed and non-significant effect at that stage. We evidenced that paternal ICRs are lost at a much faster rate than maternal ICRs during mammalian evolution, probably as a mechanistic consequence of different kinetics of the parental germlines. Our results support the notion that two independent evolutionary forces have led to the numerical and functional dominance of maternal ICRs: a selective advantage of parent-specific regulation of genes important for the fetal-maternal interface and pressure to avoid the mutagenic environment of the paternal germline.
Related Articles
Uncoupling Antisense-Mediated Silencing and DNA Methylation in the Imprinted Gnas Cluster
Williamson, Christine M.
Ball, Simon T.
Dawson, Claire
Mehta, Stuti
Beechey, Colin V.
Fray, Martin
Teboul, Lydia
Dear, T. Neil
Kelsey, Gavin
Peters, Jo
PLoS Genetics
There is increasing evidence that non-coding macroRNAs are major elements for silencing imprinted genes, but their mechanism of action is poorly understood. Within the imprinted Gnas cluster on mouse chromosome 2, Nespas is a paternally expressed macroRNA that arises from an imprinting control region and runs antisense to Nesp, a paternally repressed protein coding transcript. Here we report a knock-in mouse allele that behaves as a Nespas hypomorph. The hypomorph mediates down-regulation of Nesp in cis through chromatin modification at the Nesp promoter but in the absence of somatic DNA methylation. Notably there is reduced demethylation of H3K4me3, sufficient for down-regulation of Nesp, but insufficient for DNA methylation; in addition, there is depletion of the H3K36me3 mark permissive for DNA methylation. We propose an order of events for the regulation of a somatic imprint on the wild-type allele whereby Nespas modulates demethylation of H3K4me3 resulting in repression of Nesp followed by DNA methylation. This study demonstrates that a non-coding antisense transcript or its transcription is associated with silencing an overlapping protein-coding gene by a mechanism independent of DNA methylation. These results have broad implications for understanding the hierarchy of events in epigenetic silencing by macroRNAs.
Author Summary
Genomic imprinting is a process resulting in expression of genes according to parental origin. Some imprinted genes are expressed when paternally derived and others when maternally derived. Thus imprinted genes are monoallelically expressed and one copy has to be silenced. There is evidence that some long non-coding RNAs, acting in cis, have a role in silencing. We investigated the role of Nespas, a gene for a non-coding RNA that is only expressed from the paternally derived chromosome in the Gnas cluster and runs antisense to its sense counterpart, Nesp. Expression of Nespas is associated with silencing of Nesp and a repressive methylation mark on the Nesp DNA. We generated a Nespas mutant with reduced levels of activity and showed that it down-regulated its sense counterpart Nesp, in the absence of a DNA methylation mark, but in the presence of an altered chromatin mark. We conclude that Nespas can repress Nesp by a mechanism independent of DNA methylation, by modulating a chromatin mark.
Related Articles
Expression of KCNQ1OT1, CDKN1C, H19, and PLAGL1 and the methylation patterns at the KvDMR1 and H19/IGF2 imprinting control regions is conserved between human and bovine
Robbins, Katherine Marie
Chen, Zhiyuan
Wells, Kevin Dale
Rivera, Rocío Melissa
Journal of Biomedical Science
Beckwith-Wiedemann syndrome (BWS) is a loss-of-imprinting pediatric overgrowth syndrome. The primary features of BWS include macrosomia, macroglossia, and abdominal wall defects. Secondary features that are frequently observed in BWS patients are hypoglycemia, nevus flammeus, polyhydramnios, visceromegaly, hemihyperplasia, cardiac malformations, and difficulty breathing. BWS is speculated to occur primarily as the result of the misregulation of imprinted genes associated with two clusters on chromosome 11p15.5, namely the KvDMR1 and H19/IGF2. A similar overgrowth phenotype is observed in bovine and ovine as a result of embryo culture. In ruminants this syndrome is known as large offspring syndrome (LOS). The phenotypes associated with LOS are increased birth weight, visceromegaly, skeletal defects, hypoglycemia, polyhydramnios, and breathing difficulties. Even though phenotypic similarities exist between the two syndromes, whether the two syndromes are epigenetically similar is unknown. In this study we use control Bos taurus indicus X Bos taurus taurus F1 hybrid bovine concepti to characterize baseline imprinted gene expression and DNA methylation status of imprinted domains known to be misregulated in BWS. This work is intended to be the first step in a series of experiments aimed at determining if LOS will serve as an appropriate animal model to study BWS.
The use of F1 B. t. indicus x B. t. taurus tissues provided us with a tool to unequivocally determine imprinted status of the regions of interest in our study. We found that imprinting is conserved between the bovine and human in imprinted genes known to be associated with BWS. KCNQ1OT1 and PLAGL1 were paternally-expressed while CDKN1C and H19 were maternally-expressed in B. t. indicus x B. t. taurus F1 concepti. We also show that in bovids, differential methylation exists at the KvDMR1 and H19/IGF2 ICRs.
Based on these findings we conclude that the imprinted gene expression of KCNQ1OT1, CDKN1C, H19, and PLAGL1 and the methylation patterns at the KvDMR1 and H19/IGF2 ICRs are conserved between human and bovine. Future work will determine if LOS is associated with misregulation at these imprinted loci, similarly to what has been observed for BWS.
KvDMR1; H19/IGF2 ICR; KCNQ1OT1; CDKN1C; PLAGL1; Beckwith-Wiedemann syndrome; Methylation; Genomic imprinting; Epigenetics; Bovine
Related Articles
Genomic imprinting of IGF2 in marsupials is methylation dependent
Lawton, Betty R
Carone, Benjamin R
Obergfell, Craig J
Ferreri, Gianni C
Gondolphi, Christina M
VandeBerg, John L
Imumorin, Ikhide
O'Neill, Rachel J
O'Neill, Michael J
BMC Genomics
Parent-specific methylation of specific CpG residues is critical to imprinting in eutherian mammals, but its importance to imprinting in marsupials and, thus, the evolutionary origins of the imprinting mechanism have been the subject of controversy. This has been particularly true for the imprinted Insulin-like Growth Factor II (IGF2), a key regulator of embryonic growth in vertebrates and a focal point of the selective forces leading to genomic imprinting. The presence of the essential imprinting effector, DNMT3L, in marsupial genomes and the demonstration of a differentially methylated region (DMR) in the retrotransposon-derived imprinted gene, PEG10, in tammar wallaby argue for a role for methylation in imprinting, but several studies have found no evidence of parent-specific methylation at other imprinted loci in marsupials.
We performed the most extensive search to date for allele-specific patterns of CpG methylation within CpG isochores or CpG enriched segments across a 22 kilobase region surrounding the IGF2 gene in the South American opossum Monodelphis domestica. We identified a previously unknown 5'-untranslated exon for opossum IGF2, which is flanked by sequences defining a putative neonatal promoter, a DMR and an active Matrix Attachment Region (MAR). Demethylation of this DMR in opossum neonatal fibroblasts results in abherrant biallelic expression of IGF2.
The demonstration of a DMR and an active MAR in the 5' flank of opossum IGF2 mirrors the regulatory features of the 5' flank of Igf2 in mice. However, demethylation induced activation of the maternal allele of IGF2 in opossum differs from the demethylation induced repression of the paternal Igf2 allele in mice. While it can now be concluded that parent-specific DNA methylation is an epigentic mark common to Marsupialia and Eutheria, the molecular mechanisms of transcriptional silencing at imprinted loci have clearly evolved along independent trajectories.
Related Articles
Identification of an Imprinted Gene Cluster in the X-Inactivation Center
Kobayashi, Shin
Totoki, Yasushi
Soma, Miki
Matsumoto, Kazuya
Fujihara, Yoshitaka
Toyoda, Atsushi
Sakaki, Yoshiyuki
Okabe, Masaru
Ishino, Fumitoshi
PLoS ONE
Mammalian development is strongly influenced by the epigenetic phenomenon called genomic imprinting, in which either the paternal or the maternal allele of imprinted genes is expressed. Paternally expressed Xist, an imprinted gene, has been considered as a single cis-acting factor to inactivate the paternally inherited X chromosome (Xp) in preimplantation mouse embryos. This means that X-chromosome inactivation also entails gene imprinting at a very early developmental stage. However, the precise mechanism of imprinted X-chromosome inactivation remains unknown and there is little information about imprinted genes on X chromosomes. In this study, we examined whether there are other imprinted genes than Xist expressed from the inactive paternal X chromosome and expressed in female embryos at the preimplantation stage. We focused on small RNAs and compared their expression patterns between sexes by tagging the female X chromosome with green fluorescent protein. As a result, we identified two micro (mi)RNAs–miR-374-5p and miR-421-3p–mapped adjacent to Xist that were predominantly expressed in female blastocysts. Allelic expression analysis revealed that these miRNAs were indeed imprinted and expressed from the Xp. Further analysis of the imprinting status of adjacent locus led to the discovery of a large cluster of imprinted genes expressed from the Xp: Jpx, Ftx and Zcchc13. To our knowledge, this is the first identified cluster of imprinted genes in the cis-acting regulatory region termed the X-inactivation center. This finding may help in understanding the molecular mechanisms regulating imprinted X-chromosome inactivation during early mammalian development.
Related Articles
Epigenetic modification and uniparental inheritance of H19 in Beckwith-Wiedemann syndrome.
Catchpoole, D
Lam, W W
Valler, D
Temple, I K
Joyce, J A
Reik, W
Schofield, P N
Maher, E R
Journal of Medical Genetics
Beckwith-Wiedemann syndrome (BWS) is a congenital overgrowth syndrome associated with a characteristic pattern of visceromegaly and predisposition to childhood tumours. BWS is a genetically heterogeneous disorder; most cases are sporadic but approximately 15% are familial and a small number of BWS patients have cytogenetic abnormalities involving chromosome 11p15. Genomic imprinting effects have been implicated in familial and non-familial BWS. We have investigated the molecular pathology of 106 sporadic BWS cases; 17% (14/83) of informative cases had uniparental disomy (UPD) for chromosome 11p15.5. In each case UPD appeared to result from a postzygotic event resulting in mosaicism for segmental paternal isodisomy. The critical region for isodisomy was refined to a 25 cM interval between D11S861 and D11S2071 which contained the IGF2, H19, and p57(KIP2) genes. In three cases isodisomy for 11q markers was detected but this did not extend further than 11q13-q21 suggesting that complete chromosome 11 disomy may not produce a BWS phenotype. The allele specific methylation status of the H19 gene was investigated in 80 sporadic BWS cases. All 13 cases with UPD tested displayed hypermethylation consistent with an excess of paternal H19 alleles. In addition, five of 63 (8%) cases with normal biparental inheritance had H19 hypermethylation consistent with an "imprinting centre" mutation (ICM) or "imprinting error" (IE) lesion. The phenotype of patients with putative ICM/IE mutations was variable and overlapped with that of non-UPD sporadic BWS cases with normal H19 methylation. However, exomphalos was significantly (p < 0.05) more common in the latter group. These findings may indicate differential effects on the expression of imprinted genes in chromosome 11p15 according to the precise molecular pathology. Analysis of H19 methylation is useful for the diagnosis of both UPD or altered imprinting in BWS and shows that a variety of molecular mechanisms may cause relaxation of IGF2 imprinting in BWS.
Related Articles
The KCNQ1OT1 imprinting control region and non-coding RNA: new properties derived from the study of Beckwith–Wiedemann syndrome and Silver–Russell syndrome cases
Chiesa, Nicoletta
De Crescenzo, Agostina
Mishra, Kankadeb
Perone, Lucia
Carella, Massimo
Palumbo, Orazio
Mussa, Alessandro
Sparago, Angela
Cerrato, Flavia
Russo, Silvia
Lapi, Elisabetta
Cubellis, Maria Vittoria
Kanduri, Chandrasekhar
Cirillo Silengo, Margherita
Riccio, Andrea
Ferrero, Giovanni Battista
Human Molecular Genetics
A cluster of imprinted genes at chromosome 11p15.5 is associated with the growth disorders, Silver–Russell syndrome (SRS) and Beckwith–Wiedemann syndrome (BWS). The cluster is divided into two domains with independent imprinting control regions (ICRs). We describe two maternal 11p15.5 microduplications with contrasting phenotypes. The first is an inverted and in cis duplication of the entire 11p15.5 cluster associated with the maintenance of genomic imprinting and with the SRS phenotype. The second is a 160 kb duplication also inverted and in cis, but resulting in the imprinting alteration of the centromeric domain. It includes the centromeric ICR (ICR2) and the most 5′ 20 kb of the non-coding KCNQ1OT1 gene. Its maternal transmission is associated with ICR2 hypomethylation and the BWS phenotype. By excluding epigenetic mosaicism, cell clones analysis indicated that the two closely located ICR2 sequences resulting from the 160 kb duplication carried discordant DNA methylation on the maternal chromosome and supported the hypothesis that the ICR2 sequence is not sufficient for establishing imprinted methylation and some other property, possibly orientation-dependent, is needed. Furthermore, the 1.2 Mb duplication demonstrated that all features are present for correct imprinting at ICR2 when this is duplicated and inverted within the entire cluster. In the individuals maternally inheriting the 160 kb duplication, ICR2 hypomethylation led to the expression of a truncated KCNQ1OT1 transcript and to down-regulation of CDKN1C. We demonstrated by chromatin RNA immunopurification that the KCNQ1OT1 RNA interacts with chromatin through its most 5′ 20 kb sequence, providing a mechanism likely mediating the silencing activity of this long non-coding RNA.
Related Articles
DNA sequence polymorphisms in a panel of eight candidate bovine imprinted genes and their association with performance traits in Irish Holstein-Friesian cattle
Magee, David A
Sikora, Klaudia M
Berkowicz, Erik W
Berry, Donagh P
Howard, Dawn J
Mullen, Michael P
Evans, Ross D
Spillane, Charles
MacHugh, David E
BMC Genetics
Studies in mice and humans have shown that imprinted genes, whereby expression from one of the two parentally inherited alleles is attenuated or completely silenced, have a major effect on mammalian growth, metabolism and physiology. More recently, investigations in livestock species indicate that genes subject to this type of epigenetic regulation contribute to, or are associated with, several performance traits, most notably muscle mass and fat deposition. In the present study, a candidate gene approach was adopted to assess 17 validated single nucleotide polymorphisms (SNPs) and their association with a range of performance traits in 848 progeny-tested Irish Holstein-Friesian artificial insemination sires. These SNPs are located proximal to, or within, the bovine orthologs of eight genes (CALCR, GRB10, PEG3, PHLDA2, RASGRF1, TSPAN32, ZIM2 and ZNF215) that have been shown to be imprinted in cattle or in at least one other mammalian species (i.e. human/mouse/pig/sheep).
Heterozygosities for all SNPs analysed ranged from 0.09 to 0.46 and significant deviations from Hardy-Weinberg proportions (P ≤ 0.01) were observed at four loci. Phenotypic associations (P ≤ 0.05) were observed between nine SNPs proximal to, or within, six of the eight analysed genes and a number of performance traits evaluated, including milk protein percentage, somatic cell count, culled cow and progeny carcass weight, angularity, body conditioning score, progeny carcass conformation, body depth, rump angle, rump width, animal stature, calving difficulty, gestation length and calf perinatal mortality. Notably, SNPs within the imprinted paternally expressed gene 3 (PEG3) gene cluster were associated (P ≤ 0.05) with calving, calf performance and fertility traits, while a single SNP in the zinc finger protein 215 gene (ZNF215) was associated with milk protein percentage (P ≤ 0.05), progeny carcass weight (P ≤ 0.05), culled cow carcass weight (P ≤ 0.01), angularity (P ≤ 0.01), body depth (P ≤ 0.01), rump width (P ≤ 0.01) and animal stature (P ≤ 0.01).
Of the eight candidate bovine imprinted genes assessed, DNA sequence polymorphisms in six of these genes (CALCR, GRB10, PEG3, RASGRF1, ZIM2 and ZNF215) displayed associations with several of the phenotypes included for analyses. The genotype-phenotype associations detected here are further supported by the biological function of these six genes, each of which plays important roles in mammalian growth, development and physiology. The associations between SNPs within the imprinted PEG3 gene cluster and traits related to calving, calf performance and gestation length suggest that this domain on chromosome 18 may play a role regulating pre-natal growth and development and fertility. SNPs within the bovine ZNF215 gene were associated with bovine growth and body conformation traits and studies in humans have revealed that the human ZNF215 ortholog belongs to the imprinted gene cluster associated with Beckwith-Wiedemann syndrome--a genetic disorder characterised by growth abnormalities. Similarly, the data presented here suggest that the ZNF215 gene may have an important role in regulating bovine growth. Collectively, our results support previous work showing that (candidate) imprinted genes/loci contribute to heritable variation in bovine performance traits and suggest that DNA sequence polymorphisms within these genes/loci represents an important reservoir of genomic markers for future genetic improvement of dairy and beef cattle populations.
Related Articles
Cdkn1c (p57Kip2) is the major regulator of embryonic growth within its imprinted domain on mouse distal chromosome 7
Andrews, Stuart C
Wood, Michelle D
Tunster, Simon J
Barton, Sheila C
Surani, M Azim
John, Rosalind M
BMC Developmental Biology
Cdkn1c encodes an embryonic cyclin-dependant kinase inhibitor that acts to negatively regulate cell proliferation and, in some tissues, to actively direct differentiation. This gene, which is an imprinted gene expressed only from the maternal allele, lies within a complex region on mouse distal chromosome 7, called the IC2 domain, which contains several other imprinted genes. Studies on mouse embryos suggest a key role for genomic imprinting in regulating embryonic growth and this has led to the proposal that imprinting evolved as a consequence of the mismatched contribution of parental resources in mammals.
In this study, we characterised the phenotype of mice carrying different copy number integrations of a bacterial artificial chromosome spanning Cdkn1c. Excess Cdkn1c resulted in embryonic growth retardation that was dosage-dependent and also responsive to the genetic background. Two-fold expression of Cdkn1c in a subset of tissues caused a 10–30% reduction in embryonic weight, embryonic lethality and was associated with a reduction in the expression of the potent, non-imprinted embryonic growth factor, Igf1. Conversely, loss of expression of Cdkn1c resulted in embryos that were 11% heavier with a two-fold increase in Igf1.
We have shown that embryonic growth in mice is exquisitely sensitive to the precise dosage of Cdkn1c. Cdkn1c is a maternally expressed gene and our findings support the prediction of the parental conflict hypothesis that that the paternal genome silences genes that have an inhibitory role in embryonic growth. Within the IC2 imprinted domain, Cdkn1c encodes the major regulator of embryonic growth and we propose that Cdkn1c was the focal point of the selective pressure for imprinting of this domain.
Related Articles
Genomic imprinting and environmental disease susceptibility.
Jirtle, R L
Sander, M
Barrett, J C
Environmental Health Perspectives
Genomic imprinting is one of the most intriguing subtleties of modern genetics. The term "imprinting" refers to parent-of-origin-dependent gene expression. The presence of imprinted genes can cause cells with a full parental complement of functional autosomal genes to specifically express one allele but not the other, resulting in monoallelic expression of the imprinted loci. Genomic imprinting plays a critical role in fetal growth and behavioral development, and it is regulated by DNA methylation and chromatin structure. This paper summarizes the Genomic Imprinting and Environmental Disease Susceptibility Conference held 8-10 October 1998 at Duke University, Durham, North Carolina. The conference focused on the importance of genomic imprinting in determining susceptibility to environmentally induced diseases. Conference topics included rationales for imprinting: parental antagonism and speciation; methods for imprinted gene identification: allelic message display and monochromosomal mouse/human hybrids; properties of the imprinted gene cluster human 11p15.5 and mouse distal 7; the epigenetics of X-chromosome inactivation; variability in imprinting: imprint erasure, non-Mendelian inheritance ratios, and polymorphic imprinting; imprinting and behavior: genetics of bipolar disorder, imprinting in Turner syndrome, and imprinting in brain development and social behavior; and aberrant methylation: methylation and chromatin structure, methylation and estrogen exposure, methylation of tumor-suppressor genes, and cancer susceptibility. Environmental factors are capable of causing epigenetic changes in DNA that can potentially alter imprint gene expression and that can result in genetic diseases including cancer and behavioral disorders. Understanding the contribution of imprinting to the regulation of gene expression will be an important step in evaluating environmental influences on human health and disease.
Related Articles
An Unexpected Function of the Prader-Willi Syndrome Imprinting Center in Maternal Imprinting in Mice
Wu, Mei-Yi
Jiang, Ming
Zhai, Xiaodong
Beaudet, Arthur L.
Wu, Ray-Chang
PLoS ONE
Genomic imprinting is a phenomenon that some genes are expressed differentially according to the parent of origin. Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurobehavioral disorders caused by deficiency of imprinted gene expression from paternal and maternal chromosome 15q11–q13, respectively. Imprinted genes at the PWS/AS domain are regulated through a bipartite imprinting center, the PWS-IC and AS-IC. The PWS-IC activates paternal-specific gene expression and is responsible for the paternal imprint, whereas the AS-IC functions in the maternal imprint by allele-specific repression of the PWS-IC to prevent the paternal imprinting program. Although mouse chromosome 7C has a conserved PWS/AS imprinted domain, the mouse equivalent of the human AS-IC element has not yet been identified. Here, we suggest another dimension that the PWS-IC also functions in maternal imprinting by negatively regulating the paternally expressed imprinted genes in mice, in contrast to its known function as a positive regulator for paternal-specific gene expression. Using a mouse model carrying a 4.8-kb deletion at the PWS-IC, we demonstrated that maternal transmission of the PWS-IC deletion resulted in a maternal imprinting defect with activation of the paternally expressed imprinted genes and decreased expression of the maternally expressed imprinted gene on the maternal chromosome, accompanied by alteration of the maternal epigenotype toward a paternal state spread over the PWS/AS domain. The functional significance of this acquired paternal pattern of gene expression was demonstrated by the ability to complement PWS phenotypes by maternal inheritance of the PWS-IC deletion, which is in stark contrast to paternal inheritance of the PWS-IC deletion that resulted in the PWS phenotypes. Importantly, low levels of expression of the paternally expressed imprinted genes are sufficient to rescue postnatal lethality and growth retardation in two PWS mouse models. These findings open the opportunity for a novel approach to the treatment of PWS.
Related Articles
Postnatal Survival of Mice with Maternal Duplication of Distal Chromosome 7 Induced by a Igf2/H19 Imprinting Control Region Lacking Insulator Function
Han, Li
Szabó, Piroska E.
Mann, Jeffrey R.
PLoS Genetics
The misexpressed imprinted genes causing developmental failure of mouse parthenogenones are poorly defined. To obtain further insight, we investigated misexpressions that could cause the pronounced growth deficiency and death of fetuses with maternal duplication of distal chromosome (Chr) 7 (MatDup.dist7). Their small size could involve inactivity of Igf2, encoding a growth factor, with some contribution by over-expression of Cdkn1c, encoding a negative growth regulator. Mice lacking Igf2 expression are usually viable, and MatDup.dist7 death has been attributed to the misexpression of Cdkn1c or other imprinted genes. To examine the role of misexpressions determined by two maternal copies of the Igf2/H19 imprinting control region (ICR)—a chromatin insulator, we introduced a mutant ICR (ICRΔ) into MatDup.dist7 fetuses. This activated Igf2, with correction of H19 expression and other imprinted transcripts expected. Substantial growth enhancement and full postnatal viability was obtained, demonstrating that the aberrant MatDup.dist7 phenotype is highly dependent on the presence of two unmethylated maternal Igf2/H19 ICRs. Activation of Igf2 is likely the predominant correction that rescued growth and viability. Further experiments involved the introduction of a null allele of Cdkn1c to alleviate its over-expression. Results were not consistent with the possibility that this misexpression alone, or in combination with Igf2 inactivity, mediates MatDup.dist7 death. Rather, a network of misexpressions derived from dist7 is probably involved. Our results are consistent with the idea that reduced expression of IGF2 plays a role in the aetiology of the human imprinting-related growth-deficit disorder, Silver-Russell syndrome.
Author Summary
Parthenogenetic mouse embryos with two maternal genomes die early in development due to the misexpression of imprinted genes. To gain further insight into which misexpressions might be involved, we examined some of the misexpressions that could determine the small size and fetal death of a "partial parthenogenone"—embryos with maternal duplication of distal Chr 7 (MatDup.dist7). We investigated the involvement of two maternal copies of the Igf2/H19 imprinting control region (ICR), which is associated with lack of activity of the Igf2 gene, encoding a growth factor, and over-activity of H19. By introducing a mutant ICR, we activated Igf2 and expected to correct other misexpressions, such as that of H19. The result was substantial increase in growth and full postnatal viability of MatDup.dist7 fetuses, demonstrating the dependency of their abnormal phenotype on two maternal copies of the ICR. Activation of Igf2 was probably the main effector of this rescue. These results are consistent with the idea that reduced expression of IGF2 is causal in the human growth deficit disorder, Silver-Russell syndrome.
Related Articles
Rescue of placental phenotype in a mechanistic model of Beckwith-Wiedemann syndrome
Oh-McGinnis, Rosemary
Bogutz, Aaron B
Lee, Kang Yun
Higgins, Michael J
Lefebvre, Louis
BMC Developmental Biology
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.
Considering the important functions of several IC2-regulated genes in placentation, we set out to determine whether these DelTel7/IC2KO rescued conceptuses develop normal placentae. We report no abnormalities with respect to the architecture and vasculature of the DelTel7/IC2KO rescued placentae. Imprinted expression of several of the IC2-regulated genes critical to placentation is also faithfully recapitulated in DelTel7/IC2KO placentae.
Taken together, our results demonstrate that all the distal chromosome 7 imprinted genes implicated in placental function are silenced by IC2 and Kcnq1ot1 on the paternal allele. Furthermore, our results demonstrate that the methylated maternal IC2 is not required for the regulation of nearby genes. The results show the potential for fully rescuing trans placental abnormalities that are caused by imprinting defects.
Related Articles
An in vitro ES cell imprinting model shows that imprinted expression of the Igf2r gene arises from an allele-specific expression bias
Latos, Paulina A.
Stricker, Stefan H.
Steenpass, Laura
Pauler, Florian M.
Huang, Ru
Senergin, Basak H.
Regha, Kakkad
Koerner, Martha V.
Warczok, Katarzyna E.
Unger, Christine
Barlow, Denise P.
Development (Cambridge, England)
Genomic imprinting is an epigenetic process that results in parental-specific gene expression. Advances in understanding the mechanism that regulates imprinted gene expression in mammals have largely depended on generating targeted manipulations in embryonic stem (ES) cells that are analysed in vivo in mice. However, genomic imprinting consists of distinct developmental steps, some of which occur in post-implantation embryos, indicating that they could be studied in vitro in ES cells. The mouse Igf2r gene shows imprinted expression only in post-implantation stages, when repression of the paternal allele has been shown to require cis-expression of the Airn non-coding (nc) RNA and to correlate with gain of DNA methylation and repressive histone modifications. Here we follow the gain of imprinted expression of Igf2r during in vitro ES cell differentiation and show that it coincides with the onset of paternal-specific expression of the Airn ncRNA. Notably, although Airn ncRNA expression leads, as predicted, to gain of repressive epigenetic marks on the paternal Igf2r promoter, we unexpectedly find that the paternal Igf2r promoter is expressed at similar low levels throughout ES cell differentiation. Our results further show that the maternal and paternal Igf2r promoters are expressed equally in undifferentiated ES cells, but during differentiation expression of the maternal Igf2r promoter increases up to 10-fold, while expression from the paternal Igf2r promoter remains constant. This indicates, contrary to expectation, that the Airn ncRNA induces imprinted Igf2r expression not by silencing the paternal Igf2r promoter, but by generating an expression bias between the two parental alleles.
Embryonic stem (ES) cells; Epigenetics; Genomic imprinting; ES in vitro imprinting model; ncRNA; Histone modifications; DNA methylation; Allele-specific expression; Airn (Air)
Related Articles
An imprinted GFP insertion reveals long-range epigenetic regulation in embryonic lineages
Jones, Meaghan
Lefebvre, Louis
Developmental biology
Imprinted genes are often grouped in clusters at defined chromosomal locations. Long-range regulatory effects are implicated in the control of imprinting and these could be co-opted in the emergence of novel imprinted genes during evolution. We present a detailed analysis of a novel imprinted GFP mouse line. Tel7KI is a new insertion allele near the Ins2 locus within a cluster of imprinted genes on distal mouse Chr 7. The GFP reporter becomes regulated by the host domain in two notable fashions. First, transcription of GFP is imprinted and active exclusively from the maternally inherited allele in the embryo. Second, the expressed maternal allele is subject to position effects reflecting a distinct pattern of expression. The GFP reporter acquires silencing DNA methylation marks on the paternal allele after fertilization. This imprinting is not acquired in the placenta, where GFP is active from both parental alleles, demonstrating key epigenetic differences between embryonic and extra-embryonic lineages. Our analysis shows that imprinted clusters can provide environments conducive to the acquisition of imprinting upon novel inserted transcriptional units. The Tel7KI line offers new powerful avenues to explore both genetic and environmental factors implicated in the acquisition and maintenance of imprinted transcription in mammals.
genomic imprinting; transgene; GFP; DNA methylation; Ins2; placenta; mouse Chr 7
Related Articles
At Least Ten Genes Define the Imprinted Dlk1-Dio3 Cluster on Mouse Chromosome 12qF1
Hagan, John P.
O'Neill, Brittany L.
Stewart, Colin L.
Kozlov, Serguei V.
Croce, Carlo M.
PLoS ONE
Genomic imprinting is an exception to Mendelian genetics in that imprinted genes are expressed monoallelically, dependent on parental origin. In mammals, imprinted genes are critical in numerous developmental and physiological processes. Aberrant imprinted gene expression is implicated in several diseases including Prader-Willi/Angelman syndromes and cancer.
Methodology/Principal Findings
To identify novel imprinted genes, transcription profiling was performed on two uniparentally derived cell lines, androgenetic and parthenogenetic primary mouse embryonic fibroblasts. A maternally expressed transcript termed Imprinted RNA near Meg3/Gtl2 (Irm) was identified and its expression studied by Northern blotting and whole mounts in situ hybridization. The imprinted region that contains Irm has a parent of origin effect in three mammalian species, including the sheep callipyge locus. In mice and humans, both maternal and paternal uniparental disomies (UPD) cause embryonic growth and musculoskeletal abnormalities, indicating that both alleles likely express essential genes. To catalog all imprinted genes in this chromosomal region, twenty-five mouse mRNAs in a 1.96Mb span were investigated for allele specific expression.
Ten imprinted genes were elucidated. The imprinting of three paternally expressed protein coding genes (Dlk1, Peg11, and Dio3) was confirmed. Seven noncoding RNAs (Meg3/Gtl2, Anti-Peg11, Meg8, Irm/"Rian", AK050713, AK053394, and Meg9/Mirg) are characterized by exclusive maternal expression. Intriguingly, the majority of these noncoding RNA genes contain microRNAs and/or snoRNAs within their introns, as do their human orthologs. Of the 52 identified microRNAs that map to this region, six are predicted to regulate negatively Dlk1, suggesting an additional mechanism for interactions between allelic gene products. Since several previous studies relied heavily on in silico analysis and RT-PCR, our findings from Northerns and cDNA cloning clarify the genomic organization of this region. Our results expand the number of maternally expressed noncoding RNAs whose loss may be responsible for the phenotypes associated with mouse pUPD12 and human pUPD14 syndromes.
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subset: commoncrawl


text:
minted 1
adjetivoBritish informal
Having a lot of money; rich: I imagine those kids think I'm minted her minted parents are now splashing out on a diamond ring for her
Más ejemplos en oraciones
- The absolutely minted singer, 25, has dug deep to treat her loved ones on a cruise around the Med.
- Despite people thinking that he's minted now, Elliot insists major deals are not quite the cash grab like they used to be.
- I imagine those kids think I'm minted and drive a Range Rover but honestly I'm not!
For editors and proofreaders
Saltos de línea: mint¦ed
Definición de minted en:
minted 2
Flavoured or seasoned with mint: grilled lamb chops with minted potatoes
Más ejemplos en oraciones
- He visited the family's home in Harrogate to show Mrs Sowerby how to cook a Christmas dinner of salmon, minted potatoes, leeks in a mildly curried cream sauce and braised cos lettuce.
- The main courses arrived after a nice interval, with a side-order of vegetables that included minted new potatoes, roasted carrots, broccoli and green beans.
- Leni gave into her atavistic instincts and had the grilled organic lamb cutlets with minted pea purée, potato gratin, tomato salsa and red wine for €24.95.
For editors and proofreaders
Saltos de línea: mint¦ed
Definición de minted en:
¿Qué te llama la atención de esta palabra o frase?
Los comentarios que no respeten nuestras Normas comunitarias podrían ser moderados o eliminados.
Muy popular en Reino Unido
Muy popular en Australia
= de moda


text:
Whale 'bumps' into tour boat off Maui
A mother and her calf got up-close-and-personal with a boat and its passengers in waters off Lahaina.
Sponsored by:
A mother and her calf got up-close-and-personal with a boat and its passengers in waters off Lahaina.
"All the bus drivers know her. She sits here just like a person does," said commuter Tiona Rainwater.
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But when Sony announced it would release the movie online, his get-rich-quick scheme fell apart.
The man sold his girlfriend's mattress set for $100, little did he realize her cat was hiding inside.
Starting Wednesday afternoon, you can watch the controversial Sony Pictures comedy "The Interview" online.
Sony said the movie "will be in a number of theaters on Christmas Day."
Sony Music said that Cocker passed away on Monday "after a hard fought battle with lung cancer."
Elton John and David Furnish planned a celebration at their estate near Windsor Castle west of London.
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Nine years of Comedy Central's "The Colbert Report" came to an end along with its mythical presiding pundit.
The Human Rights Foundation has been working with groups in South Korea to drop balloons into the North that are filled with banned items.
He posted an offer last month on Reddit, and received thousands of emails, including 18 from actual Elizabeth Gallaghers with Canadian passp…
Next month will mark 80 years since the birth of the pop culture icon.
Zuckerberg said they haven't figured out a way to prevent the feature from being a "force for bad."
Facebook is updating their search feature to allow you to find any post shared with you. The new feature adds the ability to search for word…
Mutant horrors, singers and kissing strangers had a big year on YouTube.


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The Modern English Version of the Bible, released in September, is now available for your iPhone. Here's how to get it. read more
Charisma Magazine
Page 12 of 39
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Devastated by an EF-5 tornado last spring, members of Elevate Church in Moore, Oklahoma, received some sunshine and hope via donations from Charisma readers through Charisma Cares. read more
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Here's why it's important for Believers—especially current and future journalism students—to make their voices heard through the media. read more
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There are many versions of the Bible, but a new translation is encouraging Christians to get back to our biblical roots and start a revolution in our hearts. read more
When 'Til Death Do You Part' Becomes a Touching Reality
As Joyce Strader goes on to glory, it's moving to see her husband, Karl, and their children honor their wife and mother. read more
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A Prophetic Look at the Future of the Assemblies of God
As Charisma magazine turns 39 and the AG hits 100, Steve Strang looks back—and ahead—at the denomination in which he was raised. read more
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Prophetic Warning: Confederacy of Dark Forces Planning Olympic Terror Attack
The Lord also showed me He is calling undercover intercessors to press into new realms of prayer to ...
Free Ebook: The Ultimate Guide to Reaching Millennials
Position your church to reach millennials by discovering what makes them tick
Pastor Prophesies a 'Spirit of Assassination and Murder' Will Overtake RNC
Fast and pray that this dark spirit will be thwarted.
5 Truths to Understand When Leading Leaders
If you are in leadership for any length of time, there will probably come a time where you will need to lead other leaders.
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I often tell the guys I mentor: "I wish I had known this when I was your age."
The 3 Stages of Satan's Last Days Strategy Against Us
Here's how we can avoid falling for Satan's setup.
Discerning the Enemy's Mass Weapon of Destruction in This Season
This spirit is running rampant to steal your faith, kill your dreams and destroy your life.
6 Reasons This Popular Meditation Trend Is Dangerous for Christians
This is not what God meant when He spoke to Joshua.
Lester Sumrall's 7 Troubling Prophecies Over America
Have these already happened?
How the Death of This Pastor's Son Sparked Personal Revival
Listen to these podcasts to gain insight on how the head of the Church of God of Prophecy turned ...
2 Ways the Enemy Builds Strongholds in Your Life
There is a spiritual form of a horrible disease that is being lived out way too often in the life ...
Free Ebook: The Ultimate Guide to Reaching Millennials
Position your church to reach millennials by discovering what makes them tick
5 Truths to Understand When Leading Leaders
If you are in leadership for any length of time, there will probably come a time where you will need to lead other leaders.
How to Boldly Enter God's Presence... Right After You Sin
"There is a lie that Satan uses consistently and relentlessly against every believer ... "
What God Taught Me During My 30-Day 'Facebook Fast'
Could you survive 30 days without social media?
A Sobering Prophecy About the Spirit of Judgment and Burning
"The remnant church will not experience the outpouring of the glory required for the awakening ...
7 Ways Your Body Tells You It's Toxic
If you are sick, tired and overweight, toxins could be holding you back from the divine health God ...
Why You Can Be of Good Cheer When Your Heart Is Broken
Do you remember the poem "Footprints in the Sand?"
An Open Letter to Worship Leaders: Stop Trying to Be 'Cool'
"It comes down to who you're aiming to please."
4 Key Scriptures to Help You Keep in Step With the Holy Spirit
A crucial transformation must happen if you want to please God daily—and this is what it ...
God's Power at Work in the Church Today
Charisma's founder, Steve Strang writes about today's hottest topics affecting the Christian faith.
Romans 10:17 Revelation Drives Over 1 Million People to Listen to These Messages
No matter where I travel ministering around the country, people tell me they are avid ...
Can Demons Possess a Born-Again Christian?
Demonic activity is even running rampant in Christian homes and churches and it needs to be ...


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Michigan Wants To Be The King Of Driverless Car Testing
Good Morning! Welcome to The Morning Shift, your roundup of the auto news you crave, all in one place every weekday morning. Here are the important stories you need to know.
Good Morning! Welcome to The Morning Shift, your roundup of the auto news you crave, all in one place every weekday morning. Here are the important stories you need to know.
The FBI sent out a PSA just over a month ago warning us all about the risks of car hacking, but drivers in Michigan may not have to worry about that too much after all. If two new bills pass through the state's Senate, hackers of electronic vehicle systems could face a sentence of up to life in prison.
This semi-truck driver in northwestern Michigan had a decision to make: smash into a little Buick, or crank the steering wheel hard and pray. Well, here's a video showing what happened after he chose the latter option.
Someone claiming to be the owner of Whitney's Tire and Auto in Ann Arbor, Michigan replied to a negative Yelp review with an absolutely disgusting racist rant. The internet has responded by denouncing the business, but the business owner says it's all a hoax.
A local law enforcement officer crashed a police cruiser into a home in Harper Woods, Michigan, destroying the home's front porch and bay window and totalling the vehicle, according to multiple local news reports.
Emails obtained by the Detroit News reveal that top aides to Michigan Governor Rick Snyder were issuing warnings over the water quality in Flint as far back as October 2014—a year before the city switched out its own contaminated water supply and reconnected to Detroit's.
How many old Jeeps does my new hero Ron Hattner have on his northeast Michigan property? He stopped counting around 40 or 50. Some might see Ron as a crazy hoarder; I see him as a guy who's working to preserve one particular aspect of American car culture.
You know what they say: sometimes, you've just got to drive your car onto a roof. And by "they," I mean "literally zero people."
As a car enthusiast, I live on Craigslist. I'm always looking out for great deals on cool cars. I'm an addict. But in spite of what we recently decided on Answers of the Day, I think Michigan might be the single worst place to buy a used car on Craigslist. Here's why.
Good news, citizen! Remember how we all were sad with the news that the trusty work horse that is the Ford Crown Victoria was ceasing production? Worry no more, as Detroit has a new plan to keep their cruisers-a-cruisin'.
It's been a tough road for driverless cars: We recently learned, for example, that all of the crashes involving Google's test-phase autonomous vehicles have been caused by humans. Which is one of the reasons experts have just opened up a testing center in Michigan that's trying to recreate the chaos of the human-built…
Today we bring you an update to yesterday's story about a Good Samaritan motorist who caught a speeding Dodge Challenger on his dash cam, only to watch police arrest and charge the driver of a different Challenger: the wrongfully-arrested driver has been released and the charges have been dropped.
A driver in the Detroit area had an extremely close call on Friday as a black Dodge Challenger raced past him on the right side in excess of 100 mph. He caught the incident on his dash cam video. The police made an arrest, but the car they nabbed wasn't the car that passed him. Do police in Allen Park, Michigan have…
This dashcam footage from a semi-truck rolling down Michigan's I-94 shows how ominous last week's massive highway pileup looked on a sleet-soaked horizon. The wreck almost appears out of nowhere, and all hell breaks loose almost as soon as the camera truck comes to a stop.
One semi truck caught up in the deadly 90-car highway pileup today in Michigan happened to be filled with fireworks. Here's what that looked like.
A family in Michigan called 911 yesterday morning to relay about as horrifying a winter driving situation as I can imagine. They were trapped in their minivan, stuck onto the bumper of a semi truck that didn't know they were there.
When somebody loses control and totally ruins someone's house, it's usually not very hard to find them as they tend to still be an unwanted guest in the house. But one person in Ottawa, Michigan managed to get away, and it's up to us to help find them – with nothing but the parts they left behind. UPDATE!
On Christmas Eve, 1913, 73 people died in a stampede after a false cry of Fire! at a children's Christmas party in northern Michigan. There was no fire and the event marked the turning point in a bitter labor dispute in a large mining community. Two questions always arise: How could such a thing happen and why has…
Michigan Gov. Rick Snyder has signed into law a bill that reinforces laws against the direct-sales model that Tesla Motors employs, effectively barring the automaker from selling its wares in the state.


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Accepting new patients
Morrilton, AR 72110 Phone number & directions
A medical license is required for a doctor to practice medicine in a particular state. Requirements vary by state but most require, at a minimum, post-graduate training in the doctor's specific specialty. An NPI number is a national identifier unique to an individual health care provider and is managed by the Center for Medicare & Medicaid Services (CMS).
Learn more about Dr. Jimmie Magie:
View the full profile to get information on:
- Qualifications
- What's important to you
View full profile
Patient Reviews
Write a Review
Prepare before visiting Dr. Magie
Chronic Dry Eyes Patient Education Learn about symptoms and treatments of chronic dry eyes by reading Vitals patient education guides.Before Visiting Dr. Magie Learn More
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Diabetic Macular Edema
Macular Degeneration
Specialties & Research
Dr. Jimmie Magie, MD is board certified in Ophthalmology.
Education & Awards
Dr. Magie graduated from University of Arkansas , Little Rock.
Dr. Magie has 5 hospital affiliations:
- Baptist Health Medical Center Heber Spings
- St Anthony's Healthcare Center
Similar doctors nearby
Dr. Justin Charton
Dr. Stephen Magie
Dr. Cedric Pratt
Dr. Melanie Smith
Dr. Stephen Davis
Dr. David Baker
- Additional Information
- Dr. Jimmie John Magie MD is a male Ophthalmologist and practices in Ophthalmology.
- Where does Dr. Jimmie J. Magie practice?
- Dr. Jimmie Magie has 2 office locations.
Dr. Jimmie Magie practices Ophthalmology near Morrilton, AR.
Additional practice locations include: Conway, AR.
See Morrilton Ophthalmologists.


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I implemented this plug in, after purchasing the Pro features to allow for RETS integration. Support was excellent in the beginning, but tapered off some once "everything" was live and functional. It is not non-existent, just not as good as it was in the very beginning.
One unresolved problem is that there are a few fields in the Innovia MLS but not available for mapping in Realtyna's WPL system, and one is even a required field. I've been back and forth with Realtyna for months with no real answer. They apparently don't want to or don't have the technical ability or knowledge to fix it. The last response from Realtyna was "I couldn't find these 2 fields into the MLS fields so we cannot map them to WPL fields." I don't understand how they are required in the MLS, but not available as an option in WPL. I've sent them yet another email and will update this review if this issue is resolved. For anyone who is curious, the fields we have discovered that are missing are "Buyer Broker Field" and either "Special Assessment" or "Yearly Assessment."
Other than that, the experience has been good, and "everything" is otherwise functional. It is not a perfect system, but is acceptable.
The features of this plugin are quite extensive, even more-so when you add functionality from the pro plugin. That said, there are some things that I don't personally like.
The non-wordpress UI makes things confusing. Everything has custom icons, buttons, etc... sometimes I can't tell what's a button and what's not.
While the features are very sophisticated, they border on complicated, making setting up a property take longer than it should. As I look through nearly a dozen tabs, I wonder where (if at all) I can put the school district, maybe I have to create a custom feature? I don't think I'll bother.
Even though I entered the address and it was found on google maps, I had to enter the country before it allowed me to save the listing (saying that "location is required").
The front-end is overly packed with information and fanciness, where it could easily be more simple. Also the slider squashes my images.
In-all, I think this plugin tries too hard.
The verdict: If you want something that will look fancy out of the box, this may be for you. If you want something that's more lightweight, flexible, and simple, this is overkill.
You must log in to submit a review. You can also log in or register using the form near the top of this page.


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Company Profile
ASSFALG GmbH
73525 Schwäbisch Gmünd
|+49 (0)7171 92505-0
|+49 (0)7171 92505-50
- Products and services
- About us
- Pictures
- Videos
- Files
- Location
- Company focus
- App
- Members
- Events
- Jobs
- Press News
- Social Media
- Business partners
Further products and services
About us
Innovation - Customer Proximity - Internationality
Assfalg sees significantly from these three criteria the sustainable succes of ther company history.
Founded 1932 as a wholesale house for the sales of tools and chamfering- and deburring machines, Assfalg from Schwäbisch Gmünd is now national and international one of the most innovative company both in the field of the chamfering- and deburring technology and in the magnetic clamping and lifting technology.
In the metal working technology the Swabian, traditional company offers high-quality
cutting tools.
The brand new products are presented regularly on leading national exhibitions, like EMO, METAV, AMB, EuroBlech and so on, and on international exhibitions in Moscow, Mumbai and Bangalore. So, Assfalg expands internationally their customer base.
Now, Assfalg, located in the area of Stuttgart, is a wholesale house and manufacturer of own products in extended workbench particularly in India. This development started in 2000, so Assfalg supplies their customers worldwide with patented products guaranteed the highest level of technology. In the magnetic clamping and lifting field Assfalg points as a partner of a traditional, innovative Indian manufacturer
with numerous patented products like the electro-permanent magnetic (EPM)
chuck ASSQUAD with a full steel surface as well as the both-sided magnetic clamping elements DOUBLE MAG and TRIPLE MAG and the EPM battery lifting magnet. In the deburring and chamfering division, Assfalg is manufacturer since a long time of some handheld machines. Again a unique machine could
be developed, for example the milling machine for cutting process of steel bars ZF-1. This machine is able to processing across as alongside steel bars, beginning with cross section 10 x 3 mm and length from 50 mm up to 6 m. Since June 2012 Assfalg has a Joint Venture partner in India selling together the chamfering- and deburring machines.
Assfalg is now managed by the fourth generation of owners:
Dipl.Wirt.-Ing. Ronald and Annette Assfalg and Christian Assfalg, great-grandson of the founder, are actively involved in running the the family firm as managing partners.
The fruits of the success wouldn`t have been possible without the creativity and competence of the partly long-time employees. Therefore Assfalg is especially keen of the qualification of their staff and has traditionally a higherthan average education rate for the trainees and the students of the Dual College.
Since November 2011, Assfalg has the Certificate Management System as per DIN EN ISO 9001:2008.
For ensuring the success for the next years, Assfalg follows the slogan: Establishing a head start by adjusting the own products according to the requirements of
their customers, by developing, manufacturing and providing.
Europe (East / West)
Contact person
| Ronald Assfalg
0 7171 925 05-13
| Annette Assfalg
0 7171 925 05-11
|Human Resources
| Annette Assfalg
0 7171 925 05-11
| Annette Assfalg
0 7171 925 05-11
Other data
|Commercial register
|Legal form
|Number of employees
|Quality certificate (ISO 9000 etc)
|Trade name
|Year of foundation
Branch offices
125499 Moscow
|+7/ 495/ 739-5102-0
Trader Europe OOO Maschinenbau Ul. Pisoreva 18
190121 St. Petersburg
Kukal & Uhlir s.r.o. Tisova 241
Tel +7 / 812 / 495-30-80 http://www.maschinnenbau.ru aW5mb0BtYXNjaGlubmVuYmF1LnJ1
35801 Kraslice
Czech Republic
Tel +42 / 0775 / 866-060 http://www.kukal-uhlir.cz aW5mb0BrdWthbC11aGxpci5jeg==
190121 St. Petersburg
|+7 / 812 / 495-30-80
35801 Kraslice
Czech Republic
|+42 / 0775 / 866-060


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This hotel is conveniently located around 8th Street and Ocean Drive. The staff are really, really nice. They even let us store our luggage there for almost a whole day since we took a really late flight out. The rooms each have a different theme which is fun and the rooms were actually really quiet at least where I was - in the Up, Up in the Air room. The only complaints I have are - (1) the bed was SUPER uncomfortable (it was really, really hard) and (2) we had a lot of issues with the water becoming scolding hot.
- Official Description (provided by the hotel):
- A toy-hotel decorated with taste and no compromises, playing around different styles, recycled furniture and objects in an exquisite design and contemporary flair. At the Pelican, you see dining rooms from the 40's, refrigerator's from the 30's, sideboards stolen from a whorehouse, schools and churches for a paramount visual result, emphasizing the trends of the underground culture, movies (John Waters, Pedro Almodovar) and Disney cartoons. ... more less
- Reservation Options:
- TripAdvisor is proud to partner with Orbitz, Booking.com, Hotwire, Travelocity, Hotels.com, TripOnline SA, Odigeo, Cheap Tickets, Priceline, Expedia and Jetsetter so you can book your Pelican Hotel reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
- Also Known As:
- Pelican Hotel Miami Beach
- Pelican Miami Beach


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At James Madison Intermediate School, we believe that students should:
Start where you are.
Use what you have.
Do what you can.
~Arthur Ashe
Our goal is to develop your child academically, grow them socially and emotionally, and guide them towards independent thinking and problem solving. Students will be encouraged to display good character and to make good choices that will result in successful outcomes. The active participation of students, staff, families and community organizations are essential for our continued success.
High standards and expectations for each student in regard to responsible citizenship and academic effort are the foundation of our school. It is with pride that we hold these high standards, and ask each of our students to commit to maintaining the extraordinary effort of achievement and contribution that will be the legacy of JMI. It is the contribution of our students to our school community that makes JMI an exceptional learning community.
Our staff at JMI is simply amazing. They continually create opportunities for intellectual risk-taking, collaboration, problem solving, and application of classroom learning to real-life situations. Our teachers design instruction to integrate a variety of innovative technological tools and resources to enhance learning. Their goal is to educate the whole child, instilling social and emotional experiences that will support their growth, as they make the journey from third to fifth grade.
Come join us on an extraordinary journey, where we have the opportunity to explore the world through the eyes of our children. Together we can imagine and discover the endless possibilities the world has to offer for the future of our children.


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September 19, 2011 by Jamie Page
Thank you to everyone who entered our competition for a Free Coaching Session with Lee Saxby at the upcoming New York City Barefoot Run . Whilst we would very much like to help everyone and offer all our entrees a session with Lee, we are limited for time over the weekend, so we can only select nine winners. Here are the winners: @parano1dandroid Caity McCartell Tuck @fredmoehn MissZippy1 Jesse Kristopher Wood Succubus AmyL54 If you are on the list please reply to the email or Direct Message, we have sent you, to confirm your slot. For those who entered but... Read More
Barefoot Running, Coaching, Lee Saxby, Nyc Barefoot Run
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