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.     

Epimutations of the <Emphasis Type="Italic">KCNQ1OT1</Emphasis> imprinting center of chromosome 11 in early human embryolethality

Disturbance of the epigenetic status of the H19 and KCNQ1OT1 imprinting centers of chromosome 11 and the CDKN1C imprinted gene in early human embryolethality have been studied. This is the first study to detect hypomethylation of KCNQ1OT1 in the maternal homolog in placental tissues from some (9.5%) spontaneous abortions with impaired cell proliferation during the first trimester of pregnancy. Tissue specificity of the aberrant methylation status of the imprinting center has been found. A hypothesis on the postimplantation origin of epimutations in somatic cells of the developing embryo is put forward. The selective role of epimutations of imprinted genes in early human ontogeny as compared to uniparental disomy is considered; estimation of the epigenetic risk entailed in using assisted reproductive technologies is discussed.     

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

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

Comparison of multimarker logistic regression models, with application to a genomewide scan of schizophrenia

Background

Imprinted genes show expression from one parental allele only and are important for development and behaviour. This extreme mode of allelic imbalance has been described for approximately 56 human genes. Imprinting status is often disrupted in cancer and dysmorphic syndromes. More subtle variation of gene expression, that is not parent-of-origin specific, termed 'allele-specific gene expression' (ASE) is more common and may give rise to milder phenotypic differences. Using two allele-specific high-throughput technologies alongside bioinformatics predictions, normal term human placenta was screened to find new imprinted genes and to ascertain the extent of ASE in this tissue.

Results

Twenty-three family trios of placental cDNA, placental genomic DNA (gDNA) and gDNA from both parents were tested for 130 candidate genes with the Sequenom MassArray system. Six genes were found differentially expressed but none imprinted. The Illumina ASE BeadArray platform was then used to test 1536 SNPs in 932 genes. The array was enriched for the human orthologues of 124 mouse candidate genes from bioinformatics predictions and 10 human candidate imprinted genes from EST database mining. After quality control pruning, a total of 261 informative SNPs (214 genes) remained for analysis. Imprinting with maternal expression was demonstrated for the lymphocyte imprinted gene ZNF331 in human placenta. Two potential differentially methylated regions (DMRs) were found in the vicinity of ZNF331. None of the bioinformatically predicted candidates tested showed imprinting except for a skewed allelic expression in a parent-specific manner observed for PHACTR2, a neighbour of the imprinted PLAGL1 gene. ASE was detected for two or more individuals in 39 candidate genes (18%).

Conclusions

Both Sequenom and Illumina assays were sensitive enough to study imprinting and strong allelic bias. Previous bioinformatics approaches were not predictive of new imprinted genes in the human term placenta. ZNF331 is imprinted in human term placenta and might be a new ubiquitously imprinted gene, part of a primate-specific locus. Demonstration of partial imprinting of PHACTR2 calls for re-evaluation of the allelic pattern of expression for the PHACTR2-PLAGL1 locus. ASE was common in human term placenta.     

Variable allelic expression of imprinted genes in human pluripotent stem cells during differentiation into specialized cell types in vitro

Genomic imprinting is an epigenetic phenomenon by which a subset of genes is asymmetrically expressed in a parent-of-origin manner. However, little is known regarding the epigenetic behaviors of imprinted genes during human development. Here, we show dynamic epigenetic changes in imprinted genes in hESCs during in vitro differentiation into specialized cell types. Out of 9 imprinted genes with single nucleotide polymorphisms, mono-allelic expression for three imprinted genes (H19, KCNQ1OT1, and IPW), and bi- or partial-allelic expression for three imprinted genes (OSBPL5, PPP1R9A, and RTL1) were stably retained in H9-hESCs throughout differentiation, representing imprinting stability. Three imprinted genes (KCNK9, ATP10A, and SLC22A3) showed a loss and a gain of imprinting in a lineage-specific manner during differentiation. Changes in allelic expression of imprinted genes were observed in another hESC line during in vitro differentiation. These findings indicate that the allelic expression of imprinted genes may be vulnerable in a lineage-specific manner in human pluripotent stem cells during differentiation.     

Placental 5-methylcytosine and 5-hydroxymethylcytosine patterns associate with size at birth

Altered placental function as a consequence of aberrant imprinted gene expression may be one mechanism mediating the association between low birth weight and increased cardiometabolic disease risk. Imprinted gene expression is regulated by epigenetic mechanisms, particularly DNA methylation (5mC) at differentially methylated regions (DMRs). While 5-hydroxymethylcytosine (5hmC) is also present at DMRs, many techniques do not distinguish between 5mC and 5hmC. Using human placental samples, we show that the expression of the imprinted gene CDKN1C associates with birth weight. Using specific techniques to map 5mC and 5hmC at DMRs controlling the expression of CDKN1C and the imprinted gene IGF2, we show that 5mC enrichment at KvDMR and DMR0, and 5hmC enrichment within the H19 gene body, associate positively with birth weight. Importantly, the presence of 5hmC at imprinted DMRs may complicate the interpretation of DNA methylation studies in placenta; future studies should consider using techniques that distinguish between, and permit quantification of, both modifications.     

Imprinting Status of 11p15 Genes in Beckwith–Wiedemann Syndrome Patients with CDKN1C Mutations

Beckwith–Wiedemann syndrome (BWS) is an imprinting disorder characterized by somatic overgrowth, congenital malformations, and predisposition to childhood tumors. Aberrant expression of multiple imprinted genes, including H19, IGF2, KCNQ1OT1, and CDKN1C, has been observed in BWS patients. It has been estimated that mutations in CDKN1C occur in 12–17% of BWS patients. We have screened 10 autosomal dominant pedigrees and 65 sporadic BWS cases by PCR/heteroduplex analysis and DNA sequencing and have identified four mutations, two of which were associated with biallelic IGF2 expression and normal H19 and KCNQ1OT1 imprinting. One patient demonstrated phenotypic expression of paternally transmitted mutation in this maternally expressed gene, a second proband is the child of one of a pair of monozygotic twin females who carry the mutation de novo, and a third patient exhibited unusual skeletal changes more commonly found in other overgrowth syndromes. When considered with other studies published to date, this work reveals the frequency of CDKN1C mutations in BWS to be only 4.9%. This is the first report of an analysis of the imprinting status of genes in the 11p15 region where CDKN1C mutations were associated with loss of IGF2 imprinting and maintenance of H19 and KCNQ1OT1 imprinting.     

Altered gene expression and methylation of the human chromosome 11 imprinted region in small for gestational age (SGA) placentae

Imprinted genes are known to be crucial for placental development and fetal growth in mammals, but no primary epigenetic abnormality in placenta has been documented to compromise human fetal growth. Imprinted genes demonstrate parent-of-origin-specific allelic expression that is epigenetically regulated i.e. extrinsic to the primary DNA sequence. To undertake an epigenetic analysis of poor fetal growth in placentae and cord blood tissues, we first established the tissue-specific patterns of methylation and imprinted gene expression for two imprinting clusters (KvDMR and H19 DMR) on chromosome 11p15 in placentae and neonatal blood for 20 control cases and 24 Small for Gestational Age (SGA) cases. We confirmed that, in normal human placenta, the H19 promoter is unmethylated. In contrast, most other human tissues show paternal methylation. In addition, we showed that the IGF2 DMR2, also paternally methylated in most human tissues, exhibits hypomethylation in placentae. However, in neonatal blood DNA, these two regions maintain the differential methylation status seen in most other tissues. Significantly, we have been able to demonstrate that placenta does maintain differential methylation at the imprinting control regions H19 DMR and KvDMR. Of note, in one SGA placenta, we found a methylation alteration at the H19 DMR and concomitant biallelic expression of the H19 gene, suggesting that loss of imprinting at H19 is one cause of poor fetal growth in humans. Of particular interest, we demonstrated also a decrease in IGF2 mRNA levels in all SGA placentae and showed that the decrease is, in most cases, independent of H19 regulation.     

Bisphenol A Exposure Disrupts Genomic Imprinting in the Mouse

Exposure to endocrine disruptors is associated with developmental defects. One compound of concern, to which humans are widely exposed, is bisphenol A (BPA). In model organisms, BPA exposure is linked to metabolic disorders, infertility, cancer, and behavior anomalies. Recently, BPA exposure has been linked to DNA methylation changes, indicating that epigenetic mechanisms may be relevant. We investigated effects of exposure on genomic imprinting in the mouse as imprinted genes are regulated by differential DNA methylation and aberrant imprinting disrupts fetal, placental, and postnatal development. Through allele-specific and quantitative real-time PCR analysis, we demonstrated that maternal BPA exposure during late stages of oocyte development and early stages of embryonic development significantly disrupted imprinted gene expression in embryonic day (E) 9.5 and 12.5 embryos and placentas. The affected genes included Snrpn, Ube3a, Igf2, Kcnq1ot1, Cdkn1c, and Ascl2; mutations and aberrant regulation of these genes are associated with imprinting disorders in humans. Furthermore, the majority of affected genes were expressed abnormally in the placenta. DNA methylation studies showed that BPA exposure significantly altered the methylation levels of differentially methylated regions (DMRs) including the Snrpn imprinting control region (ICR) and Igf2 DMR1. Moreover, exposure significantly reduced genome-wide methylation levels in the placenta, but not the embryo. Histological and immunohistochemical examinations revealed that these epigenetic defects were associated with abnormal placental development. In contrast to this early exposure paradigm, exposure outside of the epigenetic reprogramming window did not cause significant imprinting perturbations. Our data suggest that early exposure to common environmental compounds has the potential to disrupt fetal and postnatal health through epigenetic changes in the embryo and abnormal development of the placenta.     

Imprinting status of 11p15 genes in Beckwith-Wiedemann syndrome patients with CDKN1C mutations.

Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder characterized by somatic overgrowth, congenital malformations, and predisposition to childhood tumors. Aberrant expression of multiple imprinted genes, including H19, IGF2, KCNQ1OT1, and CDKN1C, has been observed in BWS patients. It has been estimated that mutations in CDKN1C occur in 12-17% of BWS patients. We have screened 10 autosomal dominant pedigrees and 65 sporadic BWS cases by PCR/heteroduplex analysis and DNA sequencing and have identified four mutations, two of which were associated with biallelic IGF2 expression and normal H19 and KCNQ1OT1 imprinting. One patient demonstrated phenotypic expression of paternally transmitted mutation in this maternally expressed gene, a second proband is the child of one of a pair of monozygotic twin females who carry the mutation de novo, and a third patient exhibited unusual skeletal changes more commonly found in other overgrowth syndromes. When considered with other studies published to date, this work reveals the frequency of CDKN1C mutations in BWS to be only 4.9%. This is the first report of an analysis of the imprinting status of genes in the 11p15 region where CDKN1C mutations were associated with loss of IGF2 imprinting and maintenance of H19 and KCNQ1OT1 imprinting.     

Imprinted genes and the epigenetic regulation of placental phenotype

Imprinted genes are expressed in a parent-of-origin manner by epigenetic modifications that silence either the paternal or maternal allele. They are widely expressed in fetal and placental tissues and are essential for normal placental development. In general, paternally expressed genes enhance feto-placental growth while maternally expressed genes limit conceptus growth, consistent with the hypothesis that imprinting evolved in response to the conflict between parental genomes in the allocation of maternal resources to fetal growth. Using targeted deletion, uniparental duplication, loss of imprinting and transgenic approaches, imprinted genes have been shown to determine the transport capacity of the definitive mouse placenta by regulating its growth, morphology and transporter abundance. Imprinted genes in the placenta are also responsive to environmental challenges and adapt placental phenotype to the prevailing nutritional conditions, in part, by varying their epigenetic status. In addition, interplay between placental and fetal imprinted genes is important in regulating resource partitioning via the placenta both developmentally and in response to environmental factors. By balancing the opposing parental drives on resource allocation with the environmental signals of nutrient availability, imprinted genes, like the Igf2-H19 locus, may act as nutrient sensors and optimise the fetal acquisition of nutrients for growth. These genes, therefore, have a major role in the epigenetic regulation of placental phenotype with long term consequences for the developmental programming of adult health and disease.     

The effects of culture on genomic imprinting profiles in human embryonic and fetal mesenchymal stem cells

Human embryonic stem (hES) cells and fetal mesenchymal stem cells (fMSC) offer great potential for regenerative therapy strategies. It is therefore important to characterize the properties of these cells in vitro. One major way the environment impacts on cellular physiology is through changes to epigenetic mechanisms. Genes subject to epigenetic regulation via genomic imprinting have been characterized extensively. The integrity of imprinted gene expression therefore provides a measurable index for epigenetic stability. Allelic expression of 26 imprinted genes and DNA methylation at associated differentially methylated regions (DMRs) was measured in fMSC and hES cell lines. Both cell types exhibited monoallelic expression of 13 imprinted genes, biallelic expression of six imprinted genes, and there were seven genes that differed in allelic expression between cell lines. fMSC s exhibited the differential DNA methylation patterns associated with imprinted expression. This was unexpected given that gene expression of several imprinted genes was biallelic. However, in hES cells, differential methylation was perturbed. These atypical methylation patterns did not correlate with allelic expression. Our results suggest that regardless of stem cell origin, in vitro culture affects the integrity of imprinted gene expression in human cells. We identify biallelic and variably expressed genes that may inform on overall epigenetic stability. As differential methylation did not correlate with imprinted expression changes we propose that other epigenetic effectors are adversely influenced by the in vitro environment. Since DMR integrity was maintained in fMSC but not hES cells, we postulate that specific hES cell derivation and culturing practices result in changes in methylation at DMRs.Key words: genomic imprinting, embryonic stem cells, mesenchymal stem cells, differentiation, methylation, epigenetic stability     

KCNQ1OT1 regulates the retinoblastoma cell proliferation,migration and SIRT1/JNK signaling pathway by targeting miR-124/SP1 axis

Objective: Long non-coding RNA (lncRNA) KCNQ1OT1 was reported to be tightly associated with tumorigenesis and progression of multiple cancers. However, the expression and biological functions of KCNQ1OT1 in retinoblastoma (RB) are still unknown. We aim to elucidate the potential function and underlying mechanism of KCNQ1OT1 in regulating the progression of RB. Methods: The levels of KCNQ1OT1 were assayed by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) analysis. The cell proliferation of RB cells (Y79 and WERI-Rb-1) were evaluated through Cell Counting Kit 8 (CCK-8) assay. Meanwhile, Y79 and WERI-Rb-1 cell apoptosis and cell cycle were assessed by Flow Cytometry analysis. Dual luciferase reporter assay were performed to illustrate the interaction between KCNQ1OT1, miR-124, and SP1. Results: We found that KCNQ1OT1 was up-regulated and miR-124 was down-regulated in RB tissues and cells. Moreover, knockdown of KCNQ1OT1 reduced the proliferation, migration, and cell cycle, as well as promoted cell apoptosis of Y79 and WERI-Rb-1 cells. Western blot analysis consistently proved cell cycle and apoptosis related protein expression levels. More importantly, KCNQ1OT1 was a sponge of microRNA (miR)-124. MiR-124 inhibition strongly reversed the effect on cell proliferation, cycle arrest, and apoptosis by KCNQ1OT1 knockdown mediation. In addition, KCNQ1OT1 regulated expression of SP1, a direct target of miR-124 in RB. On the other hand, miR-124 inhibitor abrogated the active effect of KCNQ1OT1 silencing on silent information regulator 1 (SIRT1)/c-Jun N-terminal kinase (JNK) signaling pathway. The function of KCNQ1OT1 was verified in vivo. Conclusions: These findings implied that KCNQ1OT1 silencing inhibited RB progression and activated SIRT1/JNK signaling pathway partially by modulating the miR-124/SP1 axis.