In vitro fertilization may increase the risk of Beckwith-Wiedemann syndrome related to the abnormal imprinting of the KCN1OT gene.
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Animals↗
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Results for “Long noncoding RNA”
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Loss of CpG methylation is strongly correlated with loss of histone H3 lysine 9 methylation at DMR-LIT1 in patients with Beckwith-Wiedemann syndrome.
To clarify the chromatin-based imprinting mechanism of the p57(KIP2)/LIT1 subdomain at chromosome 11p15.5 and the mouse ortholog at chromosome 7F5, we investigated the histone-modification status at a differentially CpG methylated region of Lit1/LIT1 (DMR-Lit1/LIT1), which is an imprinting control region for the subdomain and is demethylated in half of patients with Beckwith-Wiedemann syndrome (BWS). Chromatin-immunoprecipitation assays revealed that, in both species, DMR-Lit1/LIT1 with the CpG-methylated, maternally derived inactive allele showed histone H3 Lys9 methylation, whereas the CpG-unmethylated, paternally active allele was acetylated on histone H3/H4 and methylated on H3 Lys4. We have also investigated the relationship between CpG methylation and histone H3 Lys9 methylation at DMR-LIT1 in patients with BWS. In a normal individual and in patients with BWS with normal DMR-LIT1 methylation, histone H3 Lys9 methylation was detected on the maternal allele; however, it disappeared completely in the patients with the DMR-LIT1 imprinting defect. These findings suggest that the histone-modification status at DMR-Lit1/LIT1 plays an important role in imprinting control within the subdomain and that loss of histone H3 Lys9 methylation, together with CpG demethylation on the maternal allele, may lead to the BWS phenotype.
Base Sequence↗
Paradoxical NSD1 mutations in Beckwith-Wiedemann syndrome and 11p15 anomalies in Sotos syndrome.
Sotos syndrome is an overgrowth syndrome characterized by pre- and postnatal overgrowth, macrocephaly, advanced bone age, variable degrees of mental retardation, and typical facial features. Defects of the NSD1 gene account for >or=60% of cases of Sotos syndrome, whereas the disease-causing mechanism of other cases remains unknown. Beckwith-Wiedemann syndrome (BWS) is a distinct overgrowth condition characterized by macroglossia, abdominal-wall defects, visceromegaly, embryonic tumors, hemihyperplasia, ear anomalies, renal anomalies, and neonatal hypoglycemia. Deregulation of imprinted growth-regulatory genes within the 11p15 region is the major cause of BWS, whereas the molecular defect underlying a significant proportion of sporadic BWS cases remains unknown. Owing to clinical overlaps between the two syndromes, we investigated whether unexplained cases of Sotos syndrome could be related to 11p15 anomalies and, conversely, whether unexplained BWS cases could be related to NSD1 deletions or mutations. Two 11p15 anomalies were identified in a series of 20 patients with Sotos syndrome, and two NSD1 mutations were identified in a series of 52 patients with BWS. These results suggest that the two disorders may have more similarities than previously thought and that NSD1 could be involved in imprinting of the chromosome 11p15 region.
Beckwith-Wiedemann Syndrome↗
Spinocerebellar ataxia type 8: molecular genetic comparisons and haplotype analysis of 37 families with ataxia.
We reported elsewhere that an untranslated CTG expansion causes the dominantly inherited neurodegenerative disorder spinocerebellar ataxia type 8 (SCA8). SCA8 shows a complex inheritance pattern with extremes of incomplete penetrance, in which often only one or two affected individuals are found in a given family. SCA8 expansions have also been found in control chromosomes, indicating that separate genetic or environmental factors increase disease penetrance among SCA8-expansion-carrying patients with ataxia. We describe the molecular genetic features and disease penetrance of 37 different families with SCA8 ataxia from the United States, Canada, Japan, and Mexico. Haplotype analysis using 17 STR markers spanning an approximately 1-Mb region was performed on the families with ataxia, on a group of expansion carriers in the general population, and on psychiatric patients, to clarify the genetic basis of the reduced penetrance and to investigate whether CTG expansions among different populations share a common ancestral background. Two major ancestrally related haplotypes (A and A') were found among white families with ataxia, normal controls, and patients with major psychosis, indicating a common ancestral origin of both pathogenic and nonpathogenic SCA8 expansions among whites. Two additional and distinct haplotypes were found among a group of Japanese families with ataxia (haplotype B) and a Mexican family with ataxia (haplotype C). Our finding that SCA8 expansions on three independently arising haplotypes are found among patients with ataxia and cosegregate with ataxia when multiple family members are affected further supports the direct role of the CTG expansion in disease pathogenesis.
Case-Control Studies↗
Hypomethylation of the H19 gene causes not only Silver-Russell syndrome (SRS) but also isolated asymmetry or an SRS-like phenotype.
The H19 differentially methylated region (DMR) controls the allele-specific expression of both the imprinted H19 tumor-suppressor gene and the IGF2 growth factor. Hypermethylation of this DMR--and subsequently of the H19 promoter region--is a major cause of the clinical features of gigantism and/or asymmetry seen in Beckwith-Wiedemann syndrome or in isolated hemihypertrophy. Here, we report a series of patients with hypomethylation of the H19 locus. Their main clinical features of asymmetry and growth retardation are the opposite of those seen in patients with hypermethylation of this region. In addition, we show that complete hypomethylation of the H19 promoter is found in two of three patients with the full clinical spectrum of Silver-Russell syndrome. This syndrome is also characterized by growth retardation and asymmetry, among other clinical features. We conclude that patients with these clinical features should be analyzed for H19 hypomethylation.
Abnormalities, Multiple↗
XIST expression in human oocytes and preimplantation embryos.
During mouse preimplantation development, the exclusive expression of the Xist gene from the paternally inherited allele is thought to play a role in the inactivation of the paternally inherited X chromosome in the extra-embryonic cell lineages of the developing female embryo. Recently, inactivation of the paternally inherited X chromosome has also been shown to occur in the extraembryonic cell lineages of the human female conceptus. In this paper, we determine whether the pattern of XIST expression in human preimplantation embryos is similarly correlated with paternal X inactivation. We developed procedures sensitive to the single cell, for the simultaneous analysis of XIST and HPRT expression and of sexing, initially using human fibroblast cells. Application of these procedures to human cleavage-stage embryos derived by in vitro fertilization revealed a pattern of XIST expression different from that in the mouse. Transcripts of the XIST gene were detected as early as the 1-cell zygote and, with increasing efficiency, through to the 8-cell stage of preimplantation development. In addition, transcripts of XIST were detected in both male (hence from the maternally inherited allele) and female preimplantation embryos. This pattern of expression is not consistent with a role for the early expression of the XIST gene in the choice of paternal X inactivation in the extraembryonic cell lineages of the developing human embryo.
Animals↗
XIST expression and X-chromosome inactivation in human preimplantation embryos.
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Blastocyst↗
Coding mutations in p57KIP2 are present in some cases of Beckwith-Wiedemann syndrome but are rare or absent in Wilms tumors.
The Beckwith-Wiedemann syndrome (BWS) is marked by fetal organ overgrowth and conveys a predisposition to certain childhood tumors, including Wilms tumor (WT). The genetics of BWS have implicated a gene that maps to chromosome 11p15 and is paternally imprinted, and the gene encoding the cyclin-cdk inhibitor p57KIP2 has been a strong candidate. By complete sequencing of the coding exons and intron/exon junctions, we found a maternally transmitted coding mutation in the cdk-inhibitor domain of the KIP2 gene in one of five cases of BWS. The BWS mutation was an in-frame three-amino-acid deletion that significantly reduced but did not fully abrogate growth-suppressive activity in a transfection assay. In contrast, no somatic coding mutations in KIP2 were found in a set of 12 primary WTs enriched for cases that expressed KIP2 mRNA, including cases with and without 11p15.5 loss of heterozygosity. Two other 11p15.5 loci, the linked and oppositely imprinted H19 and IGF2 genes, have been previously implicated in WT pathogenesis, and several of the tumors with persistent KIP2 mRNA expression and absence of KIP2 coding mutations showed full inactivation of H19. These data suggest that KIP2 is a BWS gene but that it is not uniquely equivalent to the 11p15.5 "WT2" tumor-suppressor locus.
Beckwith-Wiedemann Syndrome↗
Growth restricted in vitro culture conditions alter the imprinted gene expression patterns of mouse embryonic stem cells.
Embryonic stem (ES) cell-derived clones and chimeras are often associated with growth abnormalities during fetal development, leading to the production of over/under-weight offspring that show elevated neonatal mortality and morbidity. Due to the role played by imprinted genes in controlling fetal growth, much of the blame is pointed at improper epigenetic reprogramming of cells used in the procedures. We have analyzed the expression pattern of two growth regulatory imprinted genes, namely insulin like growth factor II (Igf2) and H19, in mouse ES cells cultured under growth restricted conditions and after in vitro aging. Culture of cells with serum-depleted media (starvation) and at high cell density (confluence) increased the expression of both imprinted genes and led to aberrant methylation profiles of differentially methylated regions in key regulatory sites of Igf2 and H19. These findings confirm that growth constrained cultures of ES cells are associated with alterations to methylation of the regulatory domains and the expression patterns of imprinted genes, suggesting a possible role of epigenetic factors in the loss of developmental potential.
Animals↗
Identification and characterization of BC1 RNP particles.
Rodent brain-specific small cytoplasmic BC1 RNA is an unusual RNA in several respects. It is an RNA polymerase III transcript expressed specifically in neurons, with regional and developmental regulation. Moreover, it is one of a few RNAs actively transported into dendrites. Three findings indicate that BC1 RNA exists as a ribonucleoprotein complex in vivo. First, the buoyant density of fractions containing BC1 RNA from brain extract on CsCI and Cs2SO4 gradients is 1.45 g/ml and 1.55 g/ml, respectively; this is consistent with the density of RNA-protein complexes. Second, in sucrose gradients, the BC1 particle has a larger S value (8.7S) than naked RNA (6.1S). Third, BC1 RNA from brain extracts migrates with retarded mobility compared to naked BC1 RNA during agarose gel electrophoresis. Additionally, in comparison to the signal recognition particle (SRP), the BC1 RNP is more heat resistant and less Mg(2+)-dependent. The buoyant density of the BC1 RNP suggests the presence of protein(s) with a total mass of about 138kD.
Animals↗
Identification of human autoantigen La/SS-B as BC1/BC200 RNA-binding protein.
Rodent BC1 RNA and primate BC200 RNA are small cytoplasmic non-messenger RNAs that are phylogenetically unrelated. Nevertheless, the two RNAs exhibit a large degree of parallelism. In addition to some sequence similarities in their 3' domains, they are prevalently expressed in a similar subset of neurons and belong to a small group of transcripts with a somatodendritic location. Both RNAs are complexed with proteins as ribonucleoprotein particles (RNPs). Their similarities may even extend to analogous functional roles, for example, in the regulation of decentralized dendritic translation. To shed further light on the physiological role(s) of the BC1/BC200 RNPs, we began to analyze protein components that specifically bind to these RNAs. Ultraviolet-crosslinking experiments and affinity purification techniques revealed that the human autoantigen La/SS-B is associated with BC1/BC200 RNA in vitro and in vivo. As with other RNA polymerase III transcripts, La protein binds with high affinity to the 3' end of BC200 RNA. Our results suggest that an additional function of La may be control of dendritic translation by providing a link between the 5' Alu domain of BC200 RNP and the ribosome via the La protein dimer. The fact that La binds both BC1 and BC200 RNAs further supports the notion that the RNAs are functional analogs despite the fact that they arose from two separate retroposition events in two different mammalian lineages.
Animals↗
Cell cycle-regulated trafficking of human telomerase to telomeres.
Telomerase synthesizes telomeres at the ends of human chromosomes during S phase. The results presented here suggest that telomerase activity may be regulated by intranuclear trafficking of the key components of the enzyme in human cells. We examined the subcellular localization of endogenous human telomerase RNA (hTR) and telomerase reverse transcriptase (hTERT) in HeLa cervical carcinoma cells. Throughout most of the cell cycle, we found that the two essential components of telomerase accumulate at intranuclear sites separate from telomeres. However, during S phase, both hTR and hTERT are specifically recruited to subsets of telomeres. The localization of telomerase to telomeres is dynamic, peaking at mid-S phase. We also found complex associations of both hTR and hTERT with nucleoli and Cajal bodies during S phase, implicating both structures in the biogenesis and trafficking of telomerase. Our results mark the first observation of human telomerase at telomeres and provide a mechanism for the cell cycle-dependent regulation of telomere synthesis in human cells.
Cell Cycle↗
Cell cycle-dependent recruitment of telomerase RNA and Cajal bodies to human telomeres.
Telomerase is a ribonucleoprotein enzyme that counteracts replicative telomere erosion by adding telomeric sequence repeats onto chromosome ends. Despite its well-established role in telomere synthesis, telomerase has not yet been detected at telomeres. The RNA component of human telomerase (hTR) resides in the nucleoplasmic Cajal bodies (CBs) of interphase cancer cells. Here, in situ hybridization demonstrates that in human HeLa and Hep2 S phase cells, besides accumulating in CBs, hTR specifically concentrates at a few telomeres that also accumulate the TRF1 and TRF2 telomere marker proteins. Surprisingly, telomeres accumulating hTR exhibit a great accessibility for in situ oligonucleotide hybridization without chromatin denaturation, suggesting that they represent a structurally distinct, minor subset of HeLa telomeres. Moreover, we demonstrate that more than 25% of telomeres accumulating hTR colocalize with CBs. Time-lapse fluorescence microscopy demonstrates that CBs moving in the nucleoplasm of S phase cells transiently associate for 10-40 min with telomeres. Our data raise the intriguing possibility that CBs may deliver hTR to telomeres and/or may function in other aspects of telomere maintenance.
Cell Cycle↗
The dynamics of the imprinted H19 gene expression in the mouse model of bladder carcinoma induced by N-butyl-N-(4-hydroxybutyl)nitrosamine.
The imprinted H19 gene product is an oncofetal RNA molecule in humans. It is expressed in fetal bladder, down-regulated postnatally and is re-expressed in human bladder carcinoma. This study was designed to investigate the dynamics of the expression of H19 in the mouse bladder carcinoma induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) and its relation to stages of neoplastic transformation. BBN was administered to mice in the drinking water for 26-28 weeks. The bladders were removed at 5-10 week intervals for histopathological examination and for in situ hybridization for H19 RNA, using a 35S-labeled probe. Following BBN administration expression of H19 first appeared after 5 weeks in the lamina propria adjacent to the basement membrane, concomitant with mucosal hyperplasia. At 11 weeks focal expression was noted in epithelial cells. Invasive carcinomas, of the transitional and squamous sub-types, were seen after 20 weeks and more of BBN administration. At this stage H19 expression was observed in scattered tumor cells, in the connective tissue stroma of the tumor and in the lamina propria underlying the remaining hyperplastic/dysplastic mucosa. Abundant expression of H19 was evident in fetal bladder but was absent in normal adult bladder. We conclude that, similar to humans, the H19 gene product is an oncofetal RNA molecule in the experimental mouse model of bladder carcinoma. In this model H19 is expressed in the connective tissue of the lamina propria prior to its expression in epithelial cells, concurrent with preneoplastic changes in the transitional epithelium of the bladder.
Animals↗
Overexpression of an ectopic H19 gene enhances the tumorigenic properties of breast cancer cells.
The maternally expressed H19 gene is transcribed as an untranslated RNA that serves as a riboregulator. We have previously reported that this transcript accumulates in epithelial cells in approximately 10% of breast cancers. To gain further insight on how the overexpression of the H19 gene affects the phenotype of human breast epithelial cells, we investigated the oncogenic potential of RNA that was abundantly expressed from MDA-MB-231 breast cancer cells stably transfected with the genomic sequence of the human H19 gene. The amount of H19 RNA did not affect cell proliferation capacity, timing of cell cycle phases or anchorage-dependent ability of H19-transfected clones in vitro. But in anchorage-independent growth assays the H19-recombined cells formed more and larger colonies in soft-agar versus control cells. To explore this phenotypic change, we analysed tumour development after subcutaneous injection of H19-recombined cells into scid mice. Results showed that H19 overexpression promotes tumour progression. These data support the hypothesis that an overload of H19 transcript is associated with cells exhibiting higher tumorigenic phenotypes and therefore we conclude that the H19 gene has oncogenic properties in breast epithelial cells.
Adenocarcinoma↗
A novel gene isolated from human placenta located in Down syndrome critical region on chromosome 21.
Down syndrome is the most common birth defect, which is caused by trisomy 21. We identified a novel gene in the so-called Down syndrome critical region by EST mapping to genomic DNA and following cDNA cloning. The gene, designated DCRB (Down syndrome Critical Region gene B), consisted of three exons of 1095 bp in total and encoded a large open reading frame of 118 amino acid residues. The amino acids sequence of DCRB showed no significant homology to any known protein. Northern blot analysis showed that DCRB is mainly expressed in the placenta, in which a major 1.1-kb band and a minor 2.0-kb band were detected. Minor bands of 1.4 kb and 2.2 kb were also detected in adult heart and skeletal muscle.
Adult↗
XIST repression in the absence of DNMT1 and DNMT3B.
X chromosome inactivation (XCI) in human and mice involves XIST/Xist gene expression from the inactive X (Xi) and repression from the active X (Xa). Repression of the XIST/Xist gene on the Xa has been associated with methylation of its 5' region. In mice, Dnmt1 has been shown to be involved in the methylation and transcriptional repression of Xist on Xa. We examined maintenance of XIST gene repression on Xa in HCT116 cell lines knockout for either DNMT1 or DNMT3B and for DNMT1 and DNMT3B simultaneously. Methylation of the XIST promoter and XIST transcriptional repression is sustained in DNMT1-, DNMT3B- and DNMT1/DNMT3B knockout cells. Despite global DNA demethylation, the double knockout cells present only partial demethylation of the XIST promoter, which is not sufficient for gene reactivation. In contrast, global DNA demethylation with 5-aza-2'-deoxycytidine leads to XIST expression. Therefore, in these human cells maintenance of XIST methylation is controlled differently than global genomic methylation and in the absence of both DNMT1 and DNMT3B.
Cell Line↗
Turnover of primary transcripts is a major step in the regulation of mouse H19 gene expression.
In the gene expression pathway, RNA biogenesis is a central multi-step process where both message fidelity and steady-state levels of the mature RNA have to be ascertained. An emerging question is whether RNA levels could be regulated at the precursor stage. Until recently, because it was technically very difficult to determine the level of a pre-mRNA, discrimination between changes in transcriptional activity and in pre-mRNA metabolism was extremely difficult. H19 RNA, the untranslated product of an imprinted gene, undergoes post-transcriptional regulation. Here, using a quantitative real-time RT-PCR approach, we accurately quantify its precursor RNA levels and compare these with the transcriptional activity of the gene, assessed by run-on assays. We find that the levels of H19 precursor RNA are regulated during physiological processes and this regulation appears to be related to RNA polymerase II transcription termination. Our results provide direct evidence that turnover of polymerase II primary transcripts can regulate gene expression in mammals.
Animals↗