Genetic control of X inactivation and processes leading to X-inactivation skewing.
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Animals↗
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Heritability of X chromosome--inactivation phenotype in a large family.
One of the two X chromosomes in each somatic cell of normal human females becomes inactivated very early in embryonic development. Although the inactivation of an X chromosome in any particular somatic cell of the embryonic lineage is thought to be a stochastic and epigenetic event, a strong genetic influence on this process has been described in the mouse. We have attempted to uncover evidence for genetic control of X-chromosome inactivation in the human by examining X chromosome-inactivation patterns in 255 females from 36 three-generation pedigrees, to determine whether this quantitative character exhibits evidence of heritability. We have found one family in which all seven daughters of one male and the mother of this male have highly skewed patterns of X-chromosome inactivation, suggesting strongly that this quantitative character is controlled by one or more X-linked genes in some families.
Alleles↗
Expression of insulin-like growth factor II (IGF)-II) and H19 in murine teratocarcinomas derived from embryonic stem (ES) cells.
Insulin-like growth factor II (IGF-II) is expressed during embryogenesis in rodents and humans, but is not produced in most adult tissues. This pattern of expression is closely shared by the gene H19, which lies 3' to IGF-II. This, together with the fact that the genes are reciprocally imprinted, has led to the proposal that the genes are under common transcriptional control by the H19 enhancers during development. In the present study, embryonic stem (ES) cells have been used to generate teratocarcinomas in mice. These tumours generate a wide range of differentiated tissues which have been subjected to hybridisation histochemistry with RNA probes to H19 and IGF-II. Coexpression of the two genes was found in a range of tissues, a pattern consistent with the idea of common transcriptional control. However, there were some areas in which H19 was expressed strongly in comparison with IGF-II and vice versa suggesting the existence of further control elements other than the H19 enhancers.
Animals↗
H19 gene overexpression in atypical multidrug-resistant cells associated with expression of a 95-kilodalton membrane glycoprotein.
The multidrug resistance phenotype of human breast carcinoma MCF-7/AdrVp cells is characterized by overexpression of a 95-kilodalton membrane glycoprotein (p95), accompanied by a marked reduction in intracellular anthracycline accumulation, without overexpression of P-glycoprotein or the multidrug resistance protein. We discovered that the mRNA of the H19 gene is overexpressed in MCF-7/AdrVp cells relative to parental MCF-7 cells or drug-sensitive MCF-7/AdrVp revertant cells. H19 is an imprinted gene with an important role in fetal differentiation, as well as a postulated function as a tumor suppressor gene. Another p95-overexpressing multidrug-resistant cell line, human lung carcinoma NCI-H1688, also displays high levels of H19 mRNA. In contrast, several multidrug-resistant cell lines that overexpress P-glycoprotein or the multidrug resistance protein do not have higher levels of H19 mRNA than their drug-sensitive counterparts. This is the first report of H19 gene overexpression accompanying any form of drug resistance. The association of H19 and p95 gene expression in drug resistance warrants further study.
Base Sequence↗
Nonrandom X-inactivation patterns in normal females: lyonization ratios vary with age.
The utility of X-inactivation based clonality assays for evaluation of human neoplasia is well-documented. However, excessive Lyonization is a potential limitation of these assays, because it mimics clonal derivation of cells. The incidence of excessive Lyonization in healthy females is controversial, with reported incidence varying from 4% to 33%. Several explanations have been offered for the observed variation, including different criteria for excessive Lyonization, diversity of X-linked clonality assays, small population sizes and more recently, tissue specificity of X-inactivation patterns. However, it is also possible that stem cell depletion, clonal hematopoiesis, or selection pressures on blood cells results in an increased incidence of excessive skewing. If any of the latter is true, then the incidence of excessive skewing should increase with age in blood cells. To test this hypothesis, we determined X-inactivation ratios at the human androgen receptor locus of 295 normal females from three age groups: (1) neonates, (2) females 28 to 32 years old and, (3) females aged > or = 60 years. The incidence of skewing (allele ratios > or = 3:1) was 8.6% (14 of 162) in neonates (P = .104 v 28 to 32); 16.4% (11/67) in 28 to 32 y.o. (P = .0064 v > or = 60), and 37.9% (25 of 66) in women > or = 60 y.o. (P < .0001 v neonates). When a more stringent criterion for skewing was used (allele ratios > or = 10:1), the incidence was 1.9% (3 of 162) in neonates (P = .362 v 28 to 32), 4.5% (3 of 67) in 28 to 32 y.o. (P = .0022 v > or = 60), and 22.7% (15 of 66) in > or = 60 y.o. group (P < .0001 v neonates). Results have been confirmed at the phosphoglycerate kinase locus for 48 heterozygous females. The incidence of excessive skewing increases with age. In neonates, the incidence is low and may correspond to true excessive Lyonization. Acquired skewing occurs with aging in normal females and is present in 38% of females over the age of 60. Further studies are needed to determine whether acquired skewing is a consequence of stem cell depletion, true clonal hematopoiesis, growth advantage conferred by parental-specific X-chromosomes, or other causes. These data provide an explanation for variation in reported incidence of excessive skewing in normal females. Furthermore, these findings suggest that any study of clonality using X-inactivation based assays should incorporate age-matched controls for Lyonization.
Adult↗
IGF2 but not H19 shows loss of imprinting in human glioma.
Genomic imprinting is a gamete-specific modification resulting in the allele-specific expression of genes in somatic cells. A loss of imprinting (LOI) has been found in many embryonal and adult tumors, suggesting that it plays a role in tumor development. The incidence of LOI, however, does not seem to be ubiquitous among tumors because neuroblastoma and colorectal cancer revealed no LOI. We examined the involvement of LOI of IGF2 and H19 genes in human gliomas. The two genes were imprinted in normal brain subcortex tissues. In glioma, 8 of 14 informative cases (57%) revealed LOI in IGF2. The frequency did not depend on the tumor grade. For H19, in contrast, all 13 informative cases maintained imprinting. These results suggest that LOI of IGF2 but not H19 plays a role in the development of human glioma.
Adult↗
Reversal of loss of imprinting in tumor cells by 5-aza-2'-deoxycytidine.
To determine whether loss of imprinting in cancer might be reversed by altering DNA methylation, we treated tumor cells with 5-aza-2'-deoxycytidine, a specific inhibitor of cytosine DNA methyltransferase. Treated cells showed several significant and reproducible changes. (a) Equal expression of maternal and paternal alleles of insulin-like growth factor 2 switched to predominant expression of a single parental allele. (b) H19 expression was reactivated. (c) Biallelic H19 expression switched to monoallelic expression. (d) Biallelic methylation of H19 switched to preferential allelic methylation. These results imply that abnormally imprinted cells are susceptible to epigenetic modification and that the effect of 5-aza-2'-deoxycytidine on tumor cells with loss of imprinting is not random but specific to one allele.
Azacitidine↗
The 5' part of the human H19 RNA contains cis-acting elements hampering its translatability.
H19 is an imprinted gene developmentally regulated in man and mouse and implicated in various neoplasms. No corresponding protein product has yet been detected, although several open reading frames (ORFs) could be identified along its RNA. The largest ORF found in the human gene could encode a putative 26 kDa protein. We have isolated two H19 cDNAs (AP and ES) that contain this ORF4 and correspond to incomplete copies of the unique 2.3 kb H19 RNA. In transient expression assays, AP was able to synthesize a 26 kDa protein whereas ES was not. With respect to ORF4, ES exhibits a 536 bp long GC-rich 5' untranslated region, whereas AP contains the last 22 nucleotides of this 5'UTR. Using deletions and point mutations, we have found that the length and probably the secondary structure of the 5'UTR strongly hampers the translatability of the RNA. In addition, a potential role of upstream ORFs (uORFs) was detected as stressed by the enhances translation of a construct mutated in uORF3 overlapping ORF4. Interactions between H19 and proteins are indicated by a specific binding between 5'UTR derived RNA segments and two nuclear proteins of about 27 kDa. Our results favor a potential role of these particular structures and binding properties in general trans-regulation of RNA post-transcriptional processes rather than in normal control of H19 mRNA translation.
Animals↗
X-chromosome inactivation during the development of the male urogenital ridge of the mouse.
In the mouse, the activity of Sry (sex-determining gene on the Y chromosome) initiates the transformation of the indifferent gonad into a testis. In humans, a partial Xp21 duplication leads to the development of ovaries instead of testes in XY individuals. This observation indicates that sex determination might also be influenced by a gene-dosage compensation mechanism, in addition to a dominant action of the Sry gene. In female mammals, the regulation of X-linked gene dosage at early embryogenesis is achieved through the inactivation of one of the two X chromosomes. Here we have investigated the possibility that inactivation of the X chromosome may play a role in male sex determination. We have shown, using an X-linked lacZ transgenic mouse line, that loss of beta-galactosidase activity occurs in certain somatic cells of the developing male urogenital ridge. When changes associated with apoptosis of mesonephric tubules in the developing urogenital ridges are taken into account, expression of the Xist (X inactive specific transcript) gene correlates with X inactivation revealed by loss of beta-galactosidase activity in very early mesonephric tubule epithelial cells, gonadal interstitial mesenchymal cells and coelomic epithelial cells.
Animals↗
Regulation of Igf2 imprinting in development and disease.
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Animals↗
Expression of the putative proto-oncogene His-1 in normal and neoplastic tissues.
The His-1 gene is expressed as a 3-kb spliced and polyadenylated RNA that is believed to function in the absence of an encoded protein. The precise function of the His-1 gene is unknown, but its transcriptional activation in a series of mouse leukemias has implicated the His-1 RNA in leukemogenesis when it is abnormally expressed. To study the oncogenic potential of this gene in more detail, we have examined the normal tissue distribution of His-1 RNA during mouse embryogenesis and in various adult tissues. His-1 expression was detected at low levels in the epithelia of the adult mouse stomach, prostate, seminal vesicle, and the developing choroid plexus by in situ hybridization. All other tissues examined lacked detectable levels of hybridizing RNA, suggesting that normal His-1 gene expression is highly restricted to these epithelial sites. These transcripts were not detectable by Northern blot analysis of normal tissues but were readily identified in five mouse leukemias and in five carcinomas of the choroid plexus. These data indicate that the His-1 gene expression is highly restricted and suggest that inappropriate activation of this gene may contribute to carcinogenesis.
Alternative Splicing↗
Altered imprinting of the H19 and insulin-like growth factor II genes in testicular tumors.
PURPOSE: H19 and insulin-like growth factor II (IGF2) genes are imprinted in normal human tissues. Recent reports have described loss of imprinting (LOI) for these genes in some malignancies. We therefore examined the allele-specific expression of the H19 and IGF2 genes in testicular tumors. MATERIALS AND METHODS: Restriction fragment length polymorphisms were examined in 17 testicular germ cell tumors and 2 testicular malignant lymphomas. RESULTS: All 4 informative testicular germ cell tumors showed LOI for the H19 gene. Five of 9 informative germ cell tumors showed LOI for the IGF2 gene, while testicular lymphomas did not show biallelic expression of the H19 or IGF2 gene. CONCLUSION: Loss of imprinting for the H19 and IGF2 genes in testicular seminomatous germ cell tumors may be involved in oncogenesis.
Genes, Tumor Suppressor↗
Disrupted IGF2 promoter control by silencing of promoter P1 in human hepatocellular carcinoma.
Previous investigations have supported or indicated a stimulatory role of the insulin-like growth factor II gene (IGF2) in hepatocarcinogenesis. We have studied the transcript levels, promoter usage, and imprinting status of the ICF2 gene and its relationship to H19 in human hepatocellular carcinomas (HCCs) and liver tumor cell lines. The activity of the IGF2 promoter P1 was lost in about 70% of the cases (18 of 25). This is the most prominent abnormality regarding the IGF2 regulation in this study. Total IGF2 as well as promoter P3 transcription were up-regulated in a small group of the tumors. Twenty genetically informative cases were obtained from 26 cases, thus excluding the probability of loss of heterozygosity of the IGF2 gene. Among these, nine showed abnormal monoallelic expression of IGF2. One HCC and one HCC cell line proved loss of functional imprinting of IGF2. H19 and IGF2 were regulated in parallel, and expression levels were variable. Taken together, the disruption of the IGF2 promoter regulation, particularly the loss of P1 activity, is a common feature of human HCCs. The loss of P1 activity explains the frequent loss of biallelic IGF2 expression and may potentially be used as a diagnostic or monitoring marker for human HCC.
Alleles↗
RNA transport in dendrites: a cis-acting targeting element is contained within neuronal BC1 RNA.
In nerve cells, a select group of RNAs has been localized to dendritic domains. Here we have examined dendritic RNA transport in sympathetic neurons in primary culture, using a microinjection protocol with neuronal BC1 RNA and with BC1-derived sequence segments. After cytoplasmic microinjection, full-length BC1 RNA was selectively transported to dendrites; in contrast, control RNAs such as nuclear RNAs and random-sequence irrelevant RNAs remained restricted to cytoplasmic areas proximal to the injection sites. Chimeric RNAs were constructed that contained the full-length BC1 sequence inserted upstream or downstream of the coding regions of nondendritic mRNAs. After microinjection, such chimeric RNAs were specifically targeted to dendrites; microinjected corresponding nonchimeric mRNAs were not. Dendritic transport of BC1 RNA was rapid: the average dendritic delivery rate within the first hour after microinjection was 242 +/- 25 microm/hr. Whereas a 5'-BC1 segment of 62 nucleotides was transported to dendrites to extents and at levels similar to full-length BC1 RNA, a 3'-BC1 segment of 60 nucleotides did not exit injected somata to any significant degree. A cis-acting dendritic targeting element is thus contained in the 5' part of neuronal BC1 RNA. These results demonstrate that mechanisms exist in neurons for fast and specific transport of selected RNAs to dendrites.
Animals↗
X inactivation in human testicular tumors. XIST expression and androgen receptor methylation status.
In female mammalian cells, inactivation of one of the X chromosomes compensates the increased dosage of X-linked genes as compared with their male counterparts. This process is initiated by the X-inactive specific transcripts of the xist/XIST gene in cis, resulting in methylation of specific sites of genes to be silenced. However, in male germ cells, X inactivation is established by xist/XIST expression only. We investigated the X inactivation pattern in human testicular tumors of different histogenesis by analysis of XIST expression and methylation of the androgen receptor gene. XIST was expressed only in tumors derived from the germ cell lineage with supernumerical X chromosomes: seminomas, nonseminomas, and spermatocytic seminomas. Although low expression was present in testicular parenchyma with spermatogenesis, XIST was expressed at a higher level in parenchyma with carcinoma in situ, the precursor lesion of seminomas and nonseminomas. Despite the consistent expression of XIST in germ-cell-derived tumors with gain of X chromosomes, methylation of the androgen receptor gene was present in all differentiated but only in a proportion of the undifferentiated nonseminomas. This differential pattern of methylation was also found in a number of representative cell lines. Our data indicate that the counting mechanism resulting in X inactivation is functional in testicular cancers of different histogenesis. Moreover, the differentiation-dependent pattern of X inactivation as reported during normal development in the case of multiple X chromosomes by methylation is retained in these tumors. We conclude therefore that X inactivation allows the excessive gain of X chromosomes found in germ-cell-derived tumors of the adult testis. In addition, this offers an interesting model to study the fundamental mechanisms of these processes.
Adult↗
Role of the H19 gene in Syrian hamster embryo cell tumorigenicity.
Carcinogen-induced transformation in Syrian hamster embryo (SHE) cells is a multistage process characterized by specific genetic alterations at each stage in the transformation process. Loss of H19 gene expression is one of the earliest events observed, occurring in approximately 75% of the morphologically transformed cells and the subsequently derived tumorigenic cells. To investigate the effect the loss of H19 expression has on SHE cell tumorigenicity, H19 expression was reestablished in a tumorigenic SHE cell lineage that lacked H19 expression. H19 reexpression had little effect on cellular growth in vitro but did retard tumor growth in nude mice. Analysis of the tumors that did develop from cells containing the H19 gene indicated that loss of exogenous H19 gene expression was probably due to changes in DNA methylation. These results demonstrate that alterations in H19 gene expression play an important role in SHE cell tumorigenicity.
Animals↗
Inactivation of H19, an imprinted and putative tumor repressor gene, is a preneoplastic event during Wilms' tumorigenesis.
Genetic evidence shows that the parent of origin-dependent expression patterns of the Igf2 and H19 genes is coordinated in mouse, such that H19 controls the activity of Igf2 in cis. Equally compelling evidence for a similar situation in humans is absent, although the frequently observed activation of the maternal IGF2 allele (ie., loss of imprinting) in Wilms' tumors has been attributed to the silencing of the maternal H19 locus. We show here that loss of H19 activity is generally a preneoplastic event, which may be linked with an overgrowth lesion that has been proposed to be permissive for tumor formation. Although our results document one instance in which a postneoplastic loss of H19 activity correlates with loss of IGF2 imprinting at the cellular level, it appears that inactivation of H19 is more generally independent of loss of imprinting of IGF2, at least in our specimens. Our results imply that inactivation of H19 correlates with blastema overgrowth and can be independent of a regulatory role with respect to IGF2 imprinting status in cis.
Alleles↗
Metastasizing mammary carcinomas in H19 enhancers-Igf2 transgenic mice.
The insulin-like growth factor II is mitogenic for a number of cell types and can inhibit apoptosis. The frequent expression of this gene in human and experimental animal tumors indicates that insulin-like growth factor-2 may play an important role in tumor development. It has also been hypothesized that overexpression of this growth factor may be responsible for the increased incidence of childhood tumors in patients with Beckwith-Wiedemann syndrome. To assess the effects of Igf2 on tumor development we produced six transgenic mouse lines that express the gene under the control of the H19 enhancers. Transgenic expression was initiated in the embryonic period but remained high in several adult tissues, including the mammary gland, lung, and liver. Adult transgenic females from five of the transgenic lines developed often multiple mammary tumors that had the ability to metastasize. Increased incidence of other solid tumors was also noted in older mice. These findings indicate that Igf2 expression increases the probability of malignant transformation and that the mammary gland is at a particularly high risk of tumor development in response to chronic increase in Igf2 gene dosage.
Age Factors↗