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1.  Maintenance of Paternal Methylation and Repression of the Imprinted H19 Gene Requires MBD3 
PLoS Genetics  2007;3(8):e137.
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.
doi:10.1371/journal.pgen.0030137
PMCID: PMC1950162  PMID: 17708683
2.  The interval between Ins2 and Ascl2 is dispensable for imprinting centre function in the murine Beckwith-Wiedemann region 
Human molecular genetics  2009;18(22):10.1093/hmg/ddp379.
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.
doi:10.1093/hmg/ddp379
PMCID: PMC3817080  PMID: 19684026 CAMSID: cams3629
3.  Germline Mutation in NLRP2 (NALP2) in a Familial Imprinting Disorder (Beckwith-Wiedemann Syndrome) 
PLoS Genetics  2009;5(3):e1000423.
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.
doi:10.1371/journal.pgen.1000423
PMCID: PMC2650258  PMID: 19300480
4.  ZAC, LIT1 (KCNQ1OT1) and p57KIP2 (CDKN1C) are in an imprinted gene network that may play a role in Beckwith–Wiedemann syndrome 
Nucleic Acids Research  2005;33(8):2650-2660.
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.
doi:10.1093/nar/gki555
PMCID: PMC1097765  PMID: 15888726
5.  Epigenotype-phenotype correlations in Beckwith-Wiedemann syndrome 
Journal of Medical Genetics  2000;37(12):921-926.
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
doi:10.1136/jmg.37.12.921
PMCID: PMC1734494  PMID: 11106355
6.  The Evolution of the DLK1-DIO3 Imprinted Domain in Mammals 
PLoS Biology  2008;6(6):e135.
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.
doi:10.1371/journal.pbio.0060135
PMCID: PMC2408620  PMID: 18532878
7.  Loss of imprinting at the Dlk1-Gtl2 locus caused by insertional mutagenesis in the Gtl2 5' region 
BMC Genetics  2006;7:44.
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1471-2156-7-44
PMCID: PMC1609179  PMID: 17014736
8.  DNA sequence polymorphisms in a panel of eight candidate bovine imprinted genes and their association with performance traits in Irish Holstein-Friesian cattle 
BMC Genetics  2010;11:93.
Background
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).
Results
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).
Conclusions
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.
doi:10.1186/1471-2156-11-93
PMCID: PMC2965127  PMID: 20942903
9.  Evolution of the CDKN1C-KCNQ1 imprinted domain 
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1471-2148-8-163
PMCID: PMC2427030  PMID: 18510768
10.  Uncoupling Antisense-Mediated Silencing and DNA Methylation in the Imprinted Gnas Cluster 
PLoS Genetics  2011;7(3):e1001347.
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.
doi:10.1371/journal.pgen.1001347
PMCID: PMC3063750  PMID: 21455290
11.  Distinct Methylation Changes at the IGF2-H19 Locus in Congenital Growth Disorders and Cancer 
PLoS ONE  2008;3(3):e1849.
Background
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.
Conclusions/Significance
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.
doi:10.1371/journal.pone.0001849
PMCID: PMC2268001  PMID: 18365005
12.  Genomic imprinting and environmental disease susceptibility. 
Environmental Health Perspectives  2000;108(3):271-278.
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.
PMCID: PMC1637980  PMID: 10706535
13.  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 
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1423-0127-19-95
PMCID: PMC3533950  PMID: 23153226
KvDMR1; H19/IGF2 ICR; KCNQ1OT1; CDKN1C; PLAGL1; Beckwith-Wiedemann syndrome; Methylation; Genomic imprinting; Epigenetics; Bovine
14.  Genomic imprinting of IGF2 in marsupials is methylation dependent 
BMC Genomics  2008;9:205.
Background-
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.
Results-
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.
Conclusion-
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.
doi:10.1186/1471-2164-9-205
PMCID: PMC2386826  PMID: 18454865
15.  Genomic Imprinting in the Arabidopsis Embryo Is Partly Regulated by PRC2 
PLoS Genetics  2013;9(12):e1003862.
Genomic imprinting results in monoallelic gene expression in a parent-of-origin-dependent manner and is regulated by the differential epigenetic marking of the parental alleles. In plants, genomic imprinting has been primarily described for genes expressed in the endosperm, a tissue nourishing the developing embryo that does not contribute to the next generation. In Arabidopsis, the genes MEDEA (MEA) and PHERES1 (PHE1), which are imprinted in the endosperm, are also expressed in the embryo; whether their embryonic expression is regulated by imprinting or not, however, remains controversial. In contrast, the maternally expressed in embryo 1 (mee1) gene of maize is clearly imprinted in the embryo. We identified several imprinted candidate genes in an allele-specific transcriptome of hybrid Arabidopsis embryos and confirmed parent-of-origin-dependent, monoallelic expression for eleven maternally expressed genes (MEGs) and one paternally expressed gene (PEG) in the embryo, using allele-specific expression analyses and reporter gene assays. Genetic studies indicate that the Polycomb Repressive Complex 2 (PRC2) but not the DNA METHYLTRANSFERASE1 (MET1) is involved in regulating imprinted expression in the embryo. In the seedling, all embryonic MEGs and the PEG are expressed from both parents, suggesting that the imprint is erased during late embryogenesis or early vegetative development. Our finding that several genes are regulated by genomic imprinting in the Arabidopsis embryo clearly demonstrates that this epigenetic phenomenon is not a unique feature of the endosperm in both monocots and dicots.
Author Summary
In most cells nuclear genes are present in two copies, with one maternal and one paternal allele. Usually, the two alleles share the same fate regarding their activity, with both copies being active or both being silent. An exception to this rule are genes that are regulated by genomic imprinting, where only one allele is expressed and the other one remains silent depending on the parent it was inherited from. The two alleles are equal in terms of their DNA sequence but carry different epigenetic marks distinguishing them. Genomic imprinting evolved independently in mammals and flowering plants. In mammals, genes regulated by genomic imprinting are expressed in a wide range of tissues including the embryo and the placenta. In plants, genomic imprinting has been primarily described for genes expressed in the endosperm, a nutritive tissue in the seed with a function similar to that of the mammalian placenta. Here, we describe that some genes are also regulated by genomic imprinting in the embryo of the model plant Arabidopsis thaliana. An epigenetic silencing complex, the Polycomb Repressive Complex 2 (PRC2), partly regulates genomic imprinting in the embryo. Interestingly, embryonic imprints seem to be erased during late embryo or early seedling development.
doi:10.1371/journal.pgen.1003862
PMCID: PMC3854695  PMID: 24339783
16.  The Parental Non-Equivalence of Imprinting Control Regions during Mammalian Development and Evolution 
PLoS Genetics  2010;6(11):e1001214.
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.
doi:10.1371/journal.pgen.1001214
PMCID: PMC2987832  PMID: 21124941
17.  An Unexpected Function of the Prader-Willi Syndrome Imprinting Center in Maternal Imprinting in Mice 
PLoS ONE  2012;7(4):e34348.
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.
doi:10.1371/journal.pone.0034348
PMCID: PMC3319576  PMID: 22496793
18.  An in vitro ES cell imprinting model shows that imprinted expression of the Igf2r gene arises from an allele-specific expression bias 
Development (Cambridge, England)  2009;136(3):437-448.
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.
doi:10.1242/dev.032060
PMCID: PMC2846269  PMID: 19141673
Embryonic stem (ES) cells; Epigenetics; Genomic imprinting; ES in vitro imprinting model; ncRNA; Histone modifications; DNA methylation; Allele-specific expression; Airn (Air)
19.  Epigenetic modification and uniparental inheritance of H19 in Beckwith-Wiedemann syndrome. 
Journal of Medical Genetics  1997;34(5):353-359.
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.
Images
PMCID: PMC1050940  PMID: 9152830
20.  Identification of an Imprinted Gene Cluster in the X-Inactivation Center 
PLoS ONE  2013;8(8):e71222.
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.
doi:10.1371/journal.pone.0071222
PMCID: PMC3735490  PMID: 23940725
21.  An imprinted GFP insertion reveals long-range epigenetic regulation in embryonic lineages 
Developmental biology  2009;336(1):10.1016/j.ydbio.2009.09.023.
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.
doi:10.1016/j.ydbio.2009.09.023
PMCID: PMC3819293  PMID: 19778534 CAMSID: cams3609
genomic imprinting; transgene; GFP; DNA methylation; Ins2; placenta; mouse Chr 7
22.  Identification of the Imprinted KLF14 Transcription Factor Undergoing Human-Specific Accelerated Evolution  
PLoS Genetics  2007;3(5):e65.
Imprinted genes are expressed in a parent-of-origin manner and are located in clusters throughout the genome. Aberrations in the expression of imprinted genes on human Chromosome 7 have been suggested to play a role in the etiologies of Russell-Silver Syndrome and autism. We describe the imprinting of KLF14, an intronless member of the Krüppel-like family of transcription factors located at Chromosome 7q32. We show that it has monoallelic maternal expression in all embryonic and extra-embryonic tissues studied, in both human and mouse. We examine epigenetic modifications in the KLF14 CpG island in both species and find this region to be hypomethylated. In addition, we perform chromatin immunoprecipitation and find that the murine Klf14 CpG island lacks allele-specific histone modifications. Despite the absence of these defining features, our analysis of Klf14 in offspring from DNA methyltransferase 3a conditional knockout mice reveals that the gene's expression is dependent upon a maternally methylated region. Due to the intronless nature of Klf14 and its homology to Klf16, we suggest that the gene is an ancient retrotransposed copy of Klf16. By sequence analysis of numerous species, we place the timing of this event after the divergence of Marsupialia, yet prior to the divergence of the Xenarthra superclade. We identify a large number of sequence variants in KLF14 and, using several measures of diversity, we determine that there is greater variability in the human lineage with a significantly increased number of nonsynonymous changes, suggesting human-specific accelerated evolution. Thus, KLF14 may be the first example of an imprinted transcript undergoing accelerated evolution in the human lineage.
Author Summary
Imprinted genes are expressed in a parent-of-origin manner, where one of the two inherited copies of the imprinted gene is silenced. Aberrations in the expression of these genes, which generally regulate growth, are associated with various developmental disorders, emphasizing the importance of their discovery and analysis. In this study, we identify a novel imprinted gene, named KLF14, on human Chromosome 7. It is predicted to bind DNA and regulate transcription and was shown to be expressed from the maternally inherited chromosome in all human and mouse tissues examined. Surprisingly, we did not identify molecular signatures generally associated with imprinted regions, such as DNA methylation. Additionally, the identification of numerous DNA sequence variants led to an in-depth analysis of the gene's evolution. It was determined that there is greater variability in KLF14 in the human lineage, when compared to other primates, with a significantly increased number of polymorphisms encoding for changes at the protein level, suggesting human-specific accelerated evolution. As the first example of an imprinted transcript undergoing accelerated evolution in the human lineage, we propose that the accumulation of polymorphisms in KLF14 may be aided by the silencing of the inactive allele, allowing for stronger selection.
doi:10.1371/journal.pgen.0030065
PMCID: PMC1865561  PMID: 17480121
23.  Retinoblastoma and Its Binding Partner MSI1 Control Imprinting in Arabidopsis  
PLoS Biology  2008;6(8):e194.
Parental genomic imprinting causes preferential expression of one of the two parental alleles. In mammals, differential sex-dependent deposition of silencing DNA methylation marks during gametogenesis initiates a new cycle of imprinting. Parental genomic imprinting has been detected in plants and relies on DNA methylation by the methyltransferase MET1. However, in contrast to mammals, plant imprints are created by differential removal of silencing marks during gametogenesis. In Arabidopsis, DNA demethylation is mediated by the DNA glycosylase DEMETER (DME) causing activation of imprinted genes at the end of female gametogenesis. On the basis of genetic interactions, we show that in addition to DME, the plant homologs of the human Retinoblastoma (Rb) and its binding partner RbAp48 are required for the activation of the imprinted genes FIS2 and FWA. This Rb-dependent activation is mediated by direct transcriptional repression of MET1 during female gametogenesis. We have thus identified a new mechanism required for imprinting establishment, outlining a new role for the Retinoblastoma pathway, which may be conserved in mammals.
Author Summary
Imprinting in plants and mammals involves a process whereby one of the two inherited gene variants (alleles) is inactivated. During imprinting, the transcriptional silencing of one allele is mediated by histone modifications or DNA methylation. The expressed parental allele is activated during gametogenesis by poorly understood mechanisms that remove silencing marks. In Arabidopsis, we studied genes expressed only from the maternal allele because the paternal allele is silenced by DNA methylation. We report that the expression of the maternal allele requires the repression of transcription of the major DNA methyltransferase by the sustained activity of the Arabidopsis homologs of the Retinoblastoma pathway. Repression is confined to the female gamete and is essential for the expression of imprinted genes in plants. The conserved transcriptional repression of DNA methyltransferases by the Retinoblastoma pathway suggests that this new regulation of imprinting might be also active in mammals.
A new regulation of imprinting discovered in Arabidopsis involves the Retinoblastoma gene.
doi:10.1371/journal.pbio.0060194
PMCID: PMC2504488  PMID: 18700816
24.  The KCNQ1OT1 imprinting control region and non-coding RNA: new properties derived from the study of Beckwith–Wiedemann syndrome and Silver–Russell syndrome cases 
Human Molecular Genetics  2011;21(1):10-25.
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.
doi:10.1093/hmg/ddr419
PMCID: PMC3235007  PMID: 21920939
25.  Cdkn1c (p57Kip2) is the major regulator of embryonic growth within its imprinted domain on mouse distal chromosome 7 
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1471-213X-7-53
PMCID: PMC1891291  PMID: 17517131

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