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PubMed1999

Expression of the imprinted H19 oncofetal RNA in epithelial ovarian cancer.

STUDY: To examine the expression of the imprinted maternally expressed H19 gene in benign, low malignant potential (borderline) and malignant surface epithelial ovarian tumors. DESIGN: In situ hybridization for H19 RNA using S-labeled and digoxigenin-labeled probes was performed on paraffin sections of ovarian surface epithelial tumors. The serous tumors included nine section cystadenomas, twelve serous tumors of low malignant potential and twenty serous carcinomas, grade I-IIII (FIGO classification). A smaller group included two mucinous cystadenomas, four mucinous tumors of low malignant potential and two mucinous cystadenocarcinomas. RESULTS: H19 expression was found to be positive in 6/9 (67%) serous cystadenomas, 9/12 (75%) of serous tumors of low malignant potential and 13/20 (65%) of invasive serous carcinomas. Expression in mucinous tumors was confined to the stroma beneath the epithelial lining. CONCLUSION: H19 is expressed in the majority of serous epithelial tumors. Taking into consideration the high percentage of H19 expressing serous ovarian neoplasms we suggest that H19 RNA may be used as an adjuvant tumor marker for the diagnosis and mainly for staging and follow-up of patients with serous ovarian carcinoma.

Adenocarcinoma↗

PubMed1997

Relaxation of insulin-like growth factor-2 imprinting in rat cultured cells.

The parental-specific expression of the insulin-like growth factor-2 (Igf-2) and H19 genes was studied in rat fibroblast cells derived from a 3 day-old first-generation hybrid animal obtained by crossing Fisher and Wistar strains (F x W cells). Results showed that the reciprocal imprinting of the Igf-2 and H19 genes was conserved in the rat tissues and in the derived F x W cells when cultured with frequent transfer. Igf-2 and H19 gene expression was coordinately up-regulated upon reaching confluence, but Igf-2 RNA levels were further increased in a time-dependent manner and the repressed state of the maternal Igf-2 allele was progressively relaxed in cultures held in the confluent state and in the presence of low serum for more than 3 days. The active expression and relaxed imprinting status of the Igf-2 gene persisted over cell generations when the growth-constraining conditions were released by trypsinization and dilution. On the contrary, the imprinting of the H19 gene appeared to be unaffected by changes in growth conditions and its expression was down-regulated when the confluent cells were passaged. Methylation of the H19 promoter and Igf-2 coding regions was increased in the F x W cells extensively held under confluence and in the derived 'post-confluent' cultures. The heritable changes in the expression, and imprinting status of the Igf-2 and H19 genes observed in the F x W cells closely resembles events described in human embryonal cancers and cancer-predisposing syndromes. The occurrence of imprinting relaxation under strong growth-inhibitory conditions supports the hypothesis that it is an epigenetic change.

Animals↗

PubMed1998

Xist and X chromosome inactivation.

X inactivation acts in female mammals to equalise X-linked gene dosage between XX females and XY males. X inactivation is controlled by a single X-linked cis-acting locus called the X inactivation centre (Xic). In 1991 the Xist gene was identified as a candidate for the Xic. Xist is expressed in all adult female tissues, but only from the allele on the inactive X. The Xist transcript does not encode a protein but remains sequestered within the nucleus and co-localises with the inactive X chromosome. Transgenic and knockout studies have shown that a genomic region covering only a few kilobases either side of Xist carries all of the functions attributed to the Xic. The major questions currently occupying researchers studying X inactivation are: how do cells count their number of X chromosomes to determine whether X inactivation is necessary, and how does the Xist transcript inactivate all genes on the X chromosome?

Dosage Compensation, Genetic↗

PubMed1997

Biallelic expression of the H19 and IGF2 genes in hepatocellular carcinoma.

The imprinted genes, H19 and insulin-like growth factor II (IGF2), have been demonstrated to be necessary for embryonal development in humans. Both genes are reciprocally imprinted, with expression of the maternal H19 and paternal IGF2 alleles, and are normally characterized by monoallelic expression. Recently, loss of imprinting of these genes producing biallelic expression has been observed in childhood tumors including Wilms' tumors (WT), embryonal rhabdomyosarcoma, and adulthood tumors such as lung cancer. To test the existence of loss of imprinting in hepatocellular carcinoma (HCC), we analyzed the status of imprinting of H19 and IGF2 genes in three independent tumors, three HCC and one hepatoblastoma cell lines using AluI and ApaI polymorphisms of these genes, respectively. In contrast to the previous report, all the cases except one tumor and one HCC cell line showed biallelic expression of both H19 and IGF2 genes. Unlike WT, loss of imprinting (LOI) of IGF2 in HCC was not linked to down-regulation of H19 expression, but rather associated with coexpression for H19 and IGF2. Thus, Hl9 and IGF2 expression can be uncoupled in tumors with LOI. The frequent biallelic expression of H19 and IGF2 in hepatocellular carcinoma might play a causal role in the epigenetic mechanism involved in tumor development and/or process.

Carcinoma, Hepatocellular↗

PubMed1997

Loss of imprinting and overexpression of IGF2 gene in gastric adenocarcinoma.

Both insulin-like growth factor II (IGF2) and H19 gene are located on chromosome 11p15.5 in close vicinity to each other, and are imprinted on different parental alleles. Although the exact mechanism remains unclear, loss of imprinting (LOI) leading to the biallelic expression of IGF2 and H19 genes has recently been reported in a variety of tumors. To study the role of IGF2 and H19 genes in gastric carcinogenesis, the LOI and loss of heterozygosity (LOH) status of these two genes were determined in 70 patients with gastric cancer. Among them, 30 patients were heterozygous for IGF2, 28 patients were heterozygous for H19, and 42 patients were heterozygous for either IGF2 or H19 gene. Among the 30 patients who were heterozygous for IGF2, one exhibited LOH (1/30, 3.3%) and 10 exhibited LOI (10/29, 34.5%). None of the 28 patients heterozygous for H19 gene had either LOH or LOI. LOI of IGF2 was more frequently found in the diffuse type (8/15, 53.3%) than the intestinal type (2/14, 14.3%, P < 0.05) gastric cancer. Five out of the six tumors with LOI of IGF2 exhibited overexpression of mRNA, but no obvious alterations of expression of H19 were noted by Northern hybridization. These data suggest that LOI leading to overexpression of IGF2 plays an important role in carcinogenesis of diffuse type gastric cancer.

Adenocarcinoma↗

PubMed2003

Understanding the dynamics of Spinocerebellar Ataxia 8 (SCA8) locus through a comparative genetic approach in humans and apes.

Spinocerebellar Ataxia 8 (SCA8) is a neurodegenerative disorder caused by expansion of a trinucleotide repeat. We undertake a comparative genetic analysis among human populations and primate species in the normal variation range, where forces that shaped present diversity can be recognised. We determinate number of repeats of the short tandem repeat through allele length sizing and sequencing methods. Human allele distributions are very similar among populations, ruling out ethnicity as a genetic risk for allele expansion. Primate comparison shows human-specific features, with longer human alleles due to a novel variable trinucleotide repeat, not present in non-human primates, which increased the disease-causing expansion likelihood. SCA8 seems to be a human specific disease.

Alanine↗

PubMed2000

Replication timing of the human X-inactivation center (XIC) region: correlation with chromosome bands.

The human genome is composed of long-range G+C% mosaic structures, which are thought to be related to chromosome bands. Replication timing during S phase is associated with chromosomal band zones; thus, band boundaries are thought to correspond to regions where replication timing switches. The proximal limit of the human X-inactivation center (XIC) has been localized cytologically to the junction zone between Xq13.1 and Xq13.2. Using PCR-based quantification of the newly replicated DNA from cell-cycle fractionated THP-1 cells, the replication timing in and around the XIC was determined at the genome sequence level. We found two regions where replication timing changes from the early to late period during S phase. One is located near a large inverted duplication proximal to the XIC, and the other is near the XIST locus. We propose that the 1Mb late-replicated zone (from the large inverted duplication to XIST) corresponds to a G-band Xq13.2. Several common characteristics were observed in the XIST region and the MHC class II-III junction which was previously defined as a band boundary. These characteristics included differential high-density clustering of Alu and LINE repeats, and the presence of polypurine/polypyrimidine tracts, MER41A, MER57 and MER58B.

Animals↗

PubMed2000

X-chromosome inactivation: lessons from transgenic mice.

X-chromosome inactivation (XCI) is the process by which mammals perform dosage compensation of X-linked gene products between XY males and XX females, resulting in the transcriptional silencing of all but one X chromosome per diploid cell. XCI involves counting the X chromosomes in a cell, randomly choosing those to be inactivated, spreading the inactivation signal in cis throughout the chromosome, and maintaining the inactive state of those X chromosomes during cell divisions thereafter. How the cell performs all these tasks is a fascinating problem and, together with epigenetic inheritance, a basic cellular mechanism that remains to be fully understood. In this review, we describe recent experiments aimed at understanding the first events of XCI and propose a model for initiation of XCI.

Animals↗

PubMed2001

Murine Xist RNA isoforms are different at their 3' ends: a role for differential polyadenylation.

Murine Xist is an essential transcript for X chromosome inactivation (X inactivation). According to recently revised structure, Xist is at least 17.8 kb long. It consists of seven exons and there are two major transcripts in female somatic cells. In this study we further defined the molecular structures of the two isoforms, namely short (S) and long (L) forms by northern blot and RNAse protection assay (RPA). The following lines of evidences suggest that mouse Xist depends on differential polyadenylation, not alternative splicing, to generate the two RNA isoforms: (1) only one band was detectable with the northern probes spanning the 3' end of Xist. (2) RPA showed the 3' termini of both S and L forms, and there are putative polyadenylation signals and hairpin structures close to these ends. (3) Analyses by splice site prediction program did not show any evidence of splice motifs in the sequence of L form. (4) Alignments between Xist 3' end (ESTs) and genomic sequence support the absence of splicing event in the region. The newly revised structure of Xist isoforms may have different stability and roles in the process of X inactivation.

Animals↗

PubMed2002

The sheep (Ovis aries) H19 gene: genomic structure and expression patterns, from the preimplantation embryo to adulthood.

H19, which is one of the most abundantly expressed imprinted genes during mammalian embryonic and foetal development, has been cloned from a ruminant. The sheep (Ovis aries) gene contains five exons interspersed by four exceptionally small introns; only short stretches of the nucleotide sequence, particularly in exon 1, show good homology with the human gene. The size of the exons and introns and the sequences around the splice junctions however, are well conserved between the species. The gene encodes a approximately 2.6 kb transcript which contains several potential short open reading frames, none of which is conserved between the ovine and human or murine transcripts, supporting a previous hypothesis that the gene product is the untranslated RNA itself. H19 mRNA is highly abundant in most ovine embryonic and foetal tissues of mesodermal and endodermal origins but was not detected in tissues of ectodermal origin such as the trophectoderm and the foetal brain. Expression of H19 in the extraembryonic membranes was detected only after the ovine conceptus began attachment to the endometrium and the embryo itself had undergone early organogenesis. This may be regarded as the first step in implantation; thus, in comparison with the mouse, the initiation of H19 expression appears to be determined by the timing of implantation rather than by the stage of development of the embryo itself. In most tissues, H19 expression is temporally linked to IGF2, a major foetal growth factor. The exceptions were the elongated blastocyst, the trophectoderm and brain, where low levels of IGF2 were observed in the absence of detectable H19. The abundance of H19 mRNA was in general, directly correlated with IGF2 mRNA abundance in mesodermal and endodermal tissues, suggesting that the two ovine genes share common regulatory elements that co-ordinately regulate their expression. Though both are generally regarded as embryonic and foetal genes, their expression was still maintained at a fairly high level in the adult sheep liver, lung, skeletal muscle, adrenal gland and kidney, suggesting that these organs are significant sources of IGF II in the adult.

Animals↗

PubMed1997

Evolutionary conservation of putative functional domains in the human homolog of the murine His-1 gene.

The mouse His-1 gene encodes a spliced and polyadenylated RNA with no long open reading frame (ORF), making it difficult to distinguish a functional protein coding domain. To identify candidate protein coding ORFs, and other functionally significant regions, we have isolated and sequenced 8.5 kb of a human genomic DNA that is homologous to the mouse His-1 gene. Alignment of the mouse and human sequences required no extensive gapping, indicating that evolutionary constraints have maintained a requirement for colinearity in genomic organization. We have identified the mouse transcriptional start point (tsp) and shown that the sequence of the 5'-flanking region is highly conserved in the human homolog. Sequence comparisons between the mouse and human genes identified conservation of other putative functional domains in exon 3 and in each of the two introns. Southern blot analysis with probes from each of these regions detected homologs in multiple other vertebrate species. However, none of the multiple candidate ORFs in the mouse RNA were conserved in the human sequence, suggesting that the RNA is unlikely to encode a protein. These data suggest that the RNA may be the final and functional product from the mouse His-1 gene.

Animals↗

PubMed1997

Regulatory elements in the minimal promoter region of the mouse Xist gene.

The Xist gene plays a central role in regulating X chromosome inactivation and Xist transcription has recently been shown to be necessary for X inactivation in mouse. We are currently analysing regulation of the Xist gene in order to determine the mechanisms underlying initiation of Xist expression and X inactivation. Sequence comparisons indicate that a region of approximately 0.4 kb upstream of the the major transcriptional start site comprises the Xist minimal promoter. Analysis of reporter constructs demonstrates that the minimal promoter region is active both in embryonic stem (ES) cells and in differentiated derivatives, indicating that sequences either further upstream or downstream are required for appropriate developmental control of Xist transcription. We have examined the minimal promoter region in detail, and in addition to common promoter elements have identified two previously uncharacterised transcription-factor binding sites. Mutation of these sites in reporter constructs indicates that they are functionally important.

Animals↗

PubMed2001

Environmental effects on genomic imprinting in mammals.

Genomic imprinting is an epigenetic marking mechanism by which certain genes become repressed on one of the two parental alleles. Imprinting plays important roles in mammalian development, and in humans its deregulation may result in disease and carcinogenesis. During different medical, technological and scientific interventions, pre-implantation embryos and cells are taken from their natural environment and subjected to culture in artificial media. Studies in the mouse demonstrate that environmental stress, such as in vitro culture, can affect the somatic maintenance of epigenetic marks at imprinted loci. These effects are associated with aberrant growth and morphology at fetal and perinatal stages of development.

Animals↗

PubMed2003

Characterization of expression at the human XIST locus in somatic, embryonal carcinoma, and transgenic cell lines.

X inactivation requires XIST, a functional RNA that is expressed exclusively from, and localizes to, the inactive X in female somatic cells. In mouse, low-level unstable transcription of Xist is observed prior to the time of inactivation, and an antisense transcript, Tsix, is a critical regulator of early Xist expression. To examine the presence and impact of an antisense transcript in humans we have characterized the extent of sense and antisense transcription in human somatic, transgenic, and embryonal carcinoma (EC) cell lines. Downstream antisense expression at the human XIST locus was not detected in somatic cells, but was detected in the EC line N-Tera2D1 and in somatic cells with an ectopic XIST locus. Presence of the antisense did not disrupt the stability or localization of the sense transcript. We have also identified additional sense transcripts in EC and female somatic cells and demonstrate that the 5' flanking JPX/ENOX gene is expressed from both the active and the inactive X chromosome in somatic cell hybrids, delimiting the extent of inactive X-specific transcriptional control in somatic cells. These analyses reveal similarities to and differences from the murine Xist and Tsix transcripts and generate a complex picture of developmentally regulated transcription through the region.

Animals↗

PubMed2004

A gene expressed exclusively in acute B lymphoblastic leukemias.

Representational difference analysis, a cDNA subtraction approach, was used to identify genes that are expressed in acute leukemia but not in normal hematological tissues. We isolated a cDNA fragment from a cell line derived from a B cell acute lymphoblastic leukemia bearing two Philadelphia chromosomes. The cDNA derives from an orphan gene that was named BLACE. BLACE is located in region 7q36 and encodes a major 5.3-kb transcript and several alternatively spliced minor transcripts. Significant expression of BLACE was detected by RT-PCR and quantitative RT-PCR in bone marrow samples from B cell acute lymphoblastic leukemia patients. BLACE was not or was scarcely expressed in other types of hematological malignancies, in normal tissues, and in solid tumors.

Alternative Splicing↗

PubMed2002

Alterations in promoter usage and expression levels of insulin-like growth factor-II and H19 genes in cervical carcinoma exhibiting biallelic expression of IGF-II.

Biallelic expression of insulin-like growth factor-II (IGF2) has been reported to be associated with progression of several tumors. Here, we report that the promoter usage and expression levels of IGF2 and H19 were altered in cervical carcinoma showing loss of imprinting. The imprinting status was examined in 32 cervical carcinomas and their matched normal tissues. Loss of imprinting (LOI) of IGF2 was observed in seven of 18 (39%) informative cases. LOI of H19 gene was detected in five of 14 informative cases. The usage of promoter P1 was observed in LOI tumors of IGF2, but not in the tumors showing maintenance of IGF2 imprinting (MOI) and in the normal cervical tissues. Unlike MOI tumors, some LOI tumors revealed the lack of IGF2 transcription from promoter P3. LOI tumors of IGF2 showed an increased expression level of IGF2 but a down-regulation of H19 relative to the normal tissues whereas MOI tumors did not reveal significant alterations. These results suggest that promoter P1 could be involved in the biallelic expression of IGF2 and that the altered expression levels of IGF2 and H19 gene might be associated with the progression of cervical carcinoma showing biallelic expression of IGF2.

Alleles↗

PubMed2001

Extensive tissue-specific variation of allelic methylation in the Igf2 gene during mouse fetal development: relation to expression and imprinting.

The imprinted Igf2 gene is active only on the paternal allele in most tissues. Its imprinting involves a cis-acting imprinting-control region (ICR) located upstream of the neighboring and maternally expressed H19 gene. It is thought that differential methylation of the parental alleles at the ICR is crucial for parental imprinting of both genes. Differentially methylated regions (DMRs) have also been identified within the Igf2 gene and their differential methylation is thought to be established during early development. To gain further insight into the function of these DMRs, we performed a quantitative analysis of their allelic methylation levels in different tissues during fetal development and the postnatal period in the mouse. Surprisingly, we found that the methylation levels of Igf2 DMRs vary extensively during fetal development, mostly on the expressed paternal allele. In particular, in skeletal muscle, differential allelic methylation in both DMR 1 and DMR 2 occurs only after birth, whereas correct paternal monoallelic expression is always observed, including in the embryonic stages. This suggests that differential methylation in the DMR 1 and DMR 2 of the Igf2 gene is dispensable for its imprinting in skeletal muscle. Furthermore, progressive methylation of the Igf2 paternal allele appears to be correlated with concomitant postnatal down-regulation and silencing of the gene. We discuss possible relations between Igf2 allelic methylation and expression during fetal development.

Age Factors↗

PubMed2001

Characterization of mouse Dach2, a homologue of Drosophila dachshund.

The Drosophila genes eyeless, eyes absent, sine oculis and dachshund cooperate as components of a network to control retinal determination. Vertebrate homologues of these genes have been identified and implicated in the control of cell fate. We present the cloning and characterization of mouse Dach2, a homologue of dachshund. In situ hybridization studies demonstrate Dach2 expression in embryonic nervous tissues, sensory organs and limbs. This pattern is similar to mouse Dach1, suggesting a partially redundant role for these genes during development. In addition, we determine that Dach2 expression in the forebrain of Pax6 mutants and dermamyotome of Pax3 mutants is not detectably altered. Finally, genetic mapping experiments place mouse Dach2 on the X chromosome between Xist and Esx1. The identification of human DACH2 sequences at Xq21 suggests a possible role for this gene in Allan-Herndon syndrome, Miles-Carpenter syndrome, X-linked cleft palate and/or Megalocornea.

Amino Acid Sequence↗