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PubMed1995

Towards unravelling the Igf2/H19 imprinted domain.

Genomic imprinting is an epigenetic marking process that confers parent-of-origin-dependent expression on certain genes. These imprinted genes are sometimes found in clusters, suggesting a possible involvement of higher order regulatory elements controlling expression and imprinting of genes organised in such clusters. In the distal chromosome 7 there are at least four imprinted genes: Mash2, Ins2, Igf2 and H19. Recent evidence suggests that imprinting and expression of at least Igf2 and H19 may be mechanistically linked.

Animals↗

PubMed1995

Epigenetic programming of differential gene expression in development and evolution.

This review covers data on changing patterns of DNA methylation and the regulation of gene expression in mouse embryonic development. Global demethylation occurs from the eight-cell stage to the blastocyst stage in preimplantation embryos, and global de novo methylation begins at implantation. We have used X-chromosome inactivation in female embryos as a model system to study specific CpG sites in the X-linked Pgk-1 and G6pd housekeeping genes and in the imprinted regulatory Xist gene to elucidate the role of methylation in the initiation and maintenance of differential gene activity. Methylation of the X-linked housekeeping genes occurs very close in time to their inactivation, thus raising the question as to whether methylation could be causal to inactivation, as well as being involved in its maintenance. A methylation difference between sperm and eggs in the promoter region of the Xist gene, located at the X-chromosome inactivation centre, is correlated with imprinted preferential inactivation of the paternal X chromosome in extra-embryonic tissues. Based on our data, a picture of the inheritance of methylation imprints and speculation on the significance of the Xist imprint in development is presented. On a more general level, an hypothesis of evolution by "adaptive epigenetic/genetic inheritance" is considered. This proposes modification of germ line DNA in response to a change in environment and mutation at the site of modification (e.g., of methylated cytosine to thymine). Epigenetic inheritance could function to shift patterns of gene expression to buffer the evolving system against changes in environment. If the altered patterns of gene activity and inactivity persist, the modifications may become "fixed" as mutations; alternatively, previously silenced gene networks might be recruited into function, thus appearing as if they are "acquired characteristics." An extension of this hypothesis is "foreign gene acquisition and sorting" (selection or silencing of gene function according to use). "Kidnapping" and sorting of foreign genes in this way could explain the observation that increased complexity in evolution is associated with more "junk" DNA. Adaptive epigenetic/genetic inheritance challenges the "central dogma" that information is unidirectional from the DNA to protein and the idea that Darwinian random mutation and selection are the sole mechanisms of evolution.

Animals↗

PubMed1995

Expression of X-linked genes in androgenetic, gynogenetic, and normal mouse preimplantation embryos.

A quantitative RT-PCR approach has been used to examine the expression of a number of X-linked genes during preimplantation development of normal mouse embryos and in androgenetic and gynogenetic mouse embryos. The data reveal moderately reduced expression of the Prps1, Hprt, and Pdha1 mRNAs in androgenetic eight-cell and morula stage embryos, but not in androgenetic blastocysts. Pgk1 mRNA abundance was severely reduced in androgenones at the eight-cell and morula stages and remained reduced, but to a lesser degree, in androgenetic blastocysts. These data indicate that paternally inherited X chromosomes are at least partially repressed in androgenones, as they are in normal XX embryos, and that the degree of this repression is chromosome position-dependent or gene-dependent. Gynogenetic embryos expressed elevated amounts of some mRNAs at the morula and blastocyst stages, indicative of a delay in dosage compensation that may be chromosome position-dependent. The Xist RNA was expressed at a greater abundance in androgenones than in gynogenones at the eight-cell and morula stages, consistent with previous studies. Xist expression was observed in both androgenones and gynogenones at the blastocyst stage. We conclude that the developmental arrest in early androgenones may be, in part, due to reduced expression of essential X-linked genes, particularly those near the X inactivation center, whereas the developmental defects of gynogenones and parthenogenones, by contrast, may be partially due to overexpression of X-linked genes in extraembryonic tissues, possibly those farthest away from the X inactivation center.

Animals↗

PubMed2005

IGF2/H19 imprinting analysis of human germ cell tumors (GCTs) using the methylation-sensitive single-nucleotide primer extension method reflects the origin of GCTs in different stages of primordial germ cell development.

Previous studies have demonstrated biallelic expression of the imprinted genes H19 and IGF2 and loss of DNA methylation of the SNRPN gene, indicating a common precursor cell of human germ cell tumors (GCTs), namely, the primordial germ cell (PGC). In this study, we applied the methylation-sensitive single-nucleotide primer extension (MS-SNuPE) technique to the analysis of the IGF2/H19 imprinting control region (ICR) in 55 GCTs from representative clinical and histologic subgroups. Most GCTs showed low methylation at the IGF2/H19 ICR. All 8 ovarian GCTs, 9 of 10 testicular seminomas, 7 of 10 testicular nonseminomas (all in adolescents/adults), 6 of 9 testicular yolk sac tumors (YSTs), and 12 of 14 nongonadal GCTs (all in infants/children) were hypomethylated. The highest methylation was observed in three childhood YSTs (boys) and 2 of 4 spermatocytic seminomas. The latter are derived from more advanced stages of germ-cell development. The predominantly low methylation of most of the other GCTs correlates with studies that demonstrated erasure of the methylation imprint of the IGF2/H19 ICR during embryonal PGC migration and development. These findings suggest that the IGF2/H19 methylation status in GCTs might reflect preservation of the physiologic imprinting erasure in PGCs rather than a loss of imprinting in a sense that is accepted for somatic tumors. Furthermore, this study indicates that imprinting control mechanisms other than the proposed CTCF (CCCTC binding factor) boundary model regulate IGF2 expression during this stage of PGC development as well as in GCTs derived from PGC. (c) 2005 Wiley-Liss, Inc.

Adolescent↗

PubMed2001

An X-linked GFP transgene reveals unexpected paternal X-chromosome activity in trophoblastic giant cells of the mouse placenta.

A GFP transgene has been integrated on the proximal part of the mouse X chromosome just distal of Timp and Syn1. During development, this X-linked GFP transgene exhibits widespread green fluorescence throughout the embryonic and adult life of male mice but displays mosaic expression in tissues as a result of X-inactivation in females. In living female embryos, inactivation of the transgene is imprinted in extraembryonic regions and random in the embryo proper, demonstrating that this reporter is behaving in a similar fashion to the majority of X-linked loci, and so provides a vital readout of X chromosome activity. This is observation is further supported in T16H/X female mice harboring the GFP transgene on the normal X chromosome where reporter inactivation is observed in somatic cells. The differential expression of GFP activity facilitates fluorescence activated cell sorting for the purification of GFP+ vs. GFP- cells from female embryonic tissues, thereby allowing access to populations of cells that have kept active a particular X chromosome. By tracking the activity of this X-linked GFP transgene, we discovered that the primary and secondary giant cells of the X/X placenta maintain an active paternal copy of this transgene on the presumed silenced paternal X-chromosome. This finding implies that the imprint on the paternal X chromosome may be relaxed in these trophectodermal derivatives.

Animals↗

PubMed2002

X-chromosome inactivation during spermatogenesis is regulated by an Xist/Tsix-independent mechanism in the mouse.

Transcriptional inactivation of the single X chromosome occurs in spermatogenic cells during male meiosis in mammals and has been shown to be coincident with expression of the Xist gene in spermatogonia and spermatocytes in mice. However, male mice carrying an ablated Xist gene show normal fertility. Here we examined expression from the Xist locus during spermatogenesis in wild-type mice and detected sense (Xist), but not antisense (Tsix) transcripts. In addition, we examined expression and chromatin conformation of X-linked structural genes in meiotic and postmeiotic spermatogenic cells from wild-type and Xist(-) mice and found no differences associated with the absence of a functional Xist gene. These results, along with the formation of a morphologically normal XY body in primary spermatocytes in Xist(-) mice, indicate that a functional Xist gene is not required for X-chromosome inactivation during spermatogenesis and that this process is therefore regulated by a different mechanism than that which regulates X-chromosome inactivation in female embryonic cells.

Animals↗

PubMed2001

Imprinting defects in mouse embryos: stochastic errors or polymorphic phenotype?

Defects in expression of imprinted genes are believed to cause developmental abnormalities and play a role in carcinogenesis. To determine whether spontaneous imprinting defects may occur in mouse embryos, we studied the expression of two imprinted genes H19 and Igf2 in individual postimplantation 7.5 d.p.c. and 8.5 d.p.c. embryos. Biallelic expression of H19 was found in 1.6% of the embryos, whereas biallelic expression of Igf2 was found in 0.5% of the embryos. The loss of H19 imprinting (LOI) observed in a small fraction of early postimplantation embryos may be purely stochastic. Alternatively, since we never observed it in an inbred background, it may depend on genetic factors acting in trans. Either mechanism could explain the occurrence of polymorphic imprinting as well as the genesis of sporadic imprinting defects, including cancer. The frequency of LOI of H19 was higher than the incidence of sporadic imprinting disorders in humans (about 1 in 20,000). This contradiction may be explained by different incidence of imprinting errors in different imprinted regions of the genome, in different species, or by loss of the majority of nonmosaic embryos with imprinting defects before birth.

Alleles↗

PubMed2004

X-inactivation is stably maintained in mouse embryos deficient for histone methyl transferase G9a.

One of the two X chromosomes becomes inactivated during early development of female mammals. Recent studies demonstrate that the inactive X chromosome is rich in histone H3 methylated at Lys-9 and Lys-27, suggesting an important role for these modifications in X-inactivation. It has been shown that in the mouse Eed is required for maintenance of X-inactivation in the extraembryonic lineages. Interestingly, Eed associates with Ezh2 to form a complex possessing histone methyltransferase activity predominantly for H3 Lys-27. We previously showed that G9a is one of the histone methyltransferases specific for H3 Lys-9 and is essential for embryonic development. Here we examined X-inactivation in mouse embryos deficient for G9a. Expression of Xist, which is crucial for the initiation of X-inactivation, was properly regulated and the inactivated X chromosome was stably maintained even in the absence of G9a. These results demonstrate that G9a is not essential for X-inactivation.

Animals↗

PubMed2001

Loss of imprinting and loss of heterozygosity on 11p15.5 in head and neck squamous cell carcinomas.

BACKGROUND: IGF2 and H19 are reciprocal imprinted genes with paternal and maternal monoallelic expression, respectively. This is interesting, because IGF2 is known as a growth factor, and H19 encodes a RNA with putative tumor suppressor action. Furthermore, IGF2 and H19 are linked genes located on chromosome 11p15.5, a common site of loss of heterozygosity in human cancers. METHODS: We performed an allelic-typing assay using a PCR-RFLP-based method for identification of heterozygous informative cases in head and neck squamous cell carcinomas. Tumoral total RNA was extracted from each of the heterozygotes and further studied by RT-PCR analysis. RESULTS: We detected the expression of the IGF2 gene in 10 of 10 informative cases. Two cases exhibited LOI of the IGF2 gene as evidenced by biallelic expression, and in another case, LOH was coupled with monoallelic expression of this growth factor. LOI for the H19 gene was observed in 1 of 14 informative samples analyzed. In this case, we also detected parallel monoallelic expression of the IGF2 gene. Down-regulation of the H19 gene was observed in 10 of 14 cases. CONCLUSION: These findings support the hypothesis that H19 may be a tumor suppressor gene involved in head and neck carcinogenesis. Furthermore, our data showed that genetic and epigenetic changes at 11p15.5 could lead to abnormal expression of imprinted genes in HNSCC.

Aged↗

PubMed2002

Use of H19 regulatory sequences for targeted gene therapy in cancer.

We present a tumor gene therapy approach based on the use of regulatory sequences of the H19 gene that are differentially expressed between normal and cancer cells. We constructed expression vectors carrying the gene for the A fragment of diphtheria toxin (DT-A) or herpes simplex virus thymidine kinase (HSV-tk), under the control of a 814 bp 5'-flanking region of the H19 gene. The cell killing activity of these constructs was in accordance with the relative activity of the H19 regulatory sequences in the transfected cells. We evaluated the therapeutic potential of the gene expression constructs driven by H19 regulatory sequences in an animal model of bladder cancer induced by subcutaneous injection of syngeneic bladder tumor cell lines. Intratumoral injection of these constructs caused a significant suppression of subcutaneous tumor growth, with no obvious toxicity toward the host.

Animals↗

PubMed2003

Regulation of the H19 imprinting gene expression in human nasopharyngeal carcinoma by methylation.

In East Asia and Singapore, the human nasopharyngeal carcinoma (NPC) presented clinically is mainly of the undifferentiated type. In contrast, the well-differentiated squamous NPC is more commonly detected in the West. To study the potential differences in carcinogenesis between undifferentiated and differentiated human NPC, we employed cDNA microarrays to isolate genes that might be specific for human undifferentiated NPC. One of the genes identified to be specifically upregulated in the undifferentiated human NPC cell line CNE-2 is the human imprinting gene H19. Interestingly, H19 is not expressed in the well-differentiated human HK1 NPC cells. Northern blot and in situ hybridization analyses also confirmed that the H19 gene is strongly expressed in the undifferentiated CNE-2 human NPC cell line but not in the well-differentiated HK1 human NPC cell line. In situ hybridization and reverse transcriptase-polymerase chain reaction also demonstrated that H19 is specifically expressed in NPC biopsies and not in non-NPC human tissue biopsies. Furthermore, we demonstrated that deregulation of H19 gene expression in the well-differentiated human HK1 NPC cells could be induced by the hypomethylation of CpG sites of the H19 promoter region. Hypermethylation of gene promoter regions might therefore be an important epigenetic event that plays a role in the differentiation of human NPC cells and the transcriptional silencing of imprinted genes.

Base Sequence↗

PubMed1994

enod40, a gene expressed during nodule organogenesis, codes for a non-translatable RNA involved in plant growth.

Rhizobium meliloti can interact symbiotically with Medicago plants, thereby inducing root nodules. However, certain Medicago plants can form nodules spontaneously, in the absence of rhizobia. A differential screening was performed using spontaneous nodule versus root cDNAs from Medicago sativa ssp. varia. Transcripts of a differentially expressed clone, Msenod40, were detected in all differentiating cells of nodule primordia and spontaneous nodules, but were absent in fully differentiated cells. Msenod40 showed homology to a soybean early nodulin gene, Gmenod40, although no significant open reading frame (ORF) or coding capacity was found in the Medicago sequence. Furthermore, in the sequences of cDNAs and a genomic clone (Mtenod40) isolated from Medicago truncatula, a species containing a unique copy of this gene, no ORFs were found either. In vitro translation of purified Mtenod40 transcripts did not reveal any protein product. Evaluation of the RNA secondary structure indicated that both msenod40 and Gmenod40 transcripts showed a high degree of stability, a property shared with known non-coding RNAs. The Mtenod40 RNA was localized in the cytoplasm of cells in the nodule primordium. Infection with Agrobacterium tumefaciens strains bearing antisense constructs of Mtenod40 arrested callus growth of Medicago explants, while overexpressing Mtenod40 embryos developed into teratomas. These data suggest that the enod40 genes might have a role in plant development, acting as 'riboregulators', a novel class of untranslated RNAs associated with growth control and differentiation.

Amino Acid Sequence↗

PubMed2003

Epigenetic regulation of Igf2/H19 imprinting at CTCF insulator binding sites.

The mouse insulin-like growth factor II (Igf2) and H19 genes are located adjacent to each other on chromosome 7q11-13 and are reciprocally imprinted. It is believed that the allelic expression of these two genes is regulated by the binding of CTCF insulators to four parent-specific DNA methylation sites in an imprinting control center (ICR) located between these two genes. Although monoallelically expressed in peripheral tissues, Igf2 is biallelically transcribed in the CNS. In this study, we examined the allelic DNA methylation and CTCF binding in the Igf2/H19 imprinting center in CNS, hypothesizing that the aberrant CTCF binding as one of the mechanisms leads to biallelic expression of Igf2 in CNS. Using hybrid F1 mice (M. spretus males x C57BL/6 females), we showed that in CNS, CTCF binding sites in the ICR were methylated exclusively on the paternal allele, and CTCF bound only to the unmethylated maternal allele, showing no differences from the imprinted peripheral tissues. Among three other epigenetic modifications examined, histone H3 lysine 9 methylation correlated well with Igf2 allelic expression in CNS. These results suggest that CTCF binding to the ICR alone is not sufficient to insulate the Igf2 maternal promoter and to regulate the allelic expression of the gene in the CNS, thus challenging the aberrant CTCF binding as a common mechanism for lack of Igf2 imprinting in CNS. Further studies should be focused on the identification of factors that are involved in histone methylation and CTCF-associated factors that may be needed to coordinate Igf2 imprinting.

Acetylation↗

PubMed2005

Deregulation of DNA methyltransferases and loss of parental methylation at the insulin-like growth factor II (Igf2)/H19 loci in p53 knockout mice prior to tumor development.

To ascertain whether p53 deficiency in vivo leads to the deregulation of DNA methylation machinery prior to tumor development, we investigated the expression profile of DNA methyltransferases in the thymus and the liver of p53(+/+), p53(+/-), and p53(-/-) mice at 7 weeks of age before tumor development. The expression of DNA methyltransferases was examined in the thymus at 7 weeks of age, since the malignant T-cell lymphoma develops most frequently in p53(-/-) mice around 20 weeks of age. Both mRNA and protein levels of Dnmt1 and Dnmt3b were increased in the thymus and the liver of p53-deficient mice. The expression of Dnmt3a was also increased in the liver but not in the thymus of p53-deficient mice. Dnmt3L expression was reduced in the thymus of p53(+/-) and p53(-/-) mice. The total 5-methylcytosine (5-MeC) in the genomic DNA of p53(+/+), p53(+/-), and p53(-/-) mice was quantitated by dot-blot using antibody against 5-MeC. Global methylation was increased in the thymus and the liver of p53-deficient mice. To correlate the deregulated expression of DNA methyltransferases with the disturbance of the epigenetic integrity, we examined the DNA methylation of the imprinting control region (ICR) at the insulin-like growth factor II (Igf2)/H19 loci in the thymus and the liver of p53(+/+), p53(+/-), and p53(-/-) mice. The region containing two CCCTC binding factor (CTCF) binding sites in the 5'-ICR tended to be hypomethylated in the thymus of p53(-/-) mice, but not in the liver. The expression profile of Igf2 and H19 indicated that the thymus-specific changes of Igf2 and H19 expression were coherent to the hypomethylation of the ICR in the thymus. Our results suggest that p53 is required for the maintenance of DNA methylation patterns in vivo.

Animals↗

PubMed2007

BRCA1 does not paint the inactive X to localize XIST RNA but may contribute to broad changes in cancer that impact XIST and Xi heterochromatin.

The BRCA1 tumor suppressor involved in breast and ovarian cancer is linked to several fundamental cell regulatory processes. Recently, it was reported that BRCA1 supports localization of XIST RNA to the inactive X chromosome (Xi) in women. The apparent cytological overlap between BRCA1 and XIST RNA across the Xi raised the possibility a direct role of BRCA1 in localizing XIST. We report here that BRCA1 does not paint the Xi or XIST territory, as do markers of Xi facultative heterochromatin. A smaller BRCA1 accumulation abuts Xi, although this is not exclusive to Xi. In BRCA1 depleted normal and tumor cells, or BRCA1 reconstituted cells, BRCA1 status does not closely correlate with XIST localization, however in a BRCA1 inducible system over-expression correlated strongly with enhanced XIST expression. We confirm frequent loss of an Xi in tumor cells. In addition to mitotic loss of Xi, we find XIST RNA expression or localization frequently become compromised in cultured breast cancer cells, suggesting Xi heterochromatin may not be fully maintained. We demonstrate that complex epigenetic differences between tumor cell subpopulations can have striking effects on XIST transcription, accumulation, and localization, but this does not strictly correlate with BRCA1. Although BRCA1 can have indirect effects that impact XIST, our results do not indicate a direct and specific role in XIST RNA regulation. Rather, regulatory factors such as BRCA1 that have broad effects on chromatin or gene regulation can impact XIST RNA and the Xi. We provide preliminary evidence that this may occur as part of a wider failure of heterochromatin maintenance in some cancers.

BRCA1 Protein↗

PubMed2005

Regulatory sequences of H19 and IGF2 genes in DNA-based therapy of colorectal rat liver metastases.

BACKGROUND: Malignant tumors of the liver are among the most common causes of cancer-related death throughout the world. Current therapeutic approaches fail to control the disease in most cases. This study seeks to explore the potential utility of transcriptional regulatory sequences of the H19 and insulin growth factor 2 (IGF2) genes for directing tumor-selective expression of a toxin gene (A fragment of diphtheria toxin), delivered by non-viral vectors. METHODS: The therapeutic potential of the toxin vectors driven by the H19 and the IGF2-P3 regulatory sequences was tested in a metastatic model of rat CC531 colon carcinoma in liver. RESULTS: Intratumoral injection of these vectors into colon tumors implanted in the liver of rats induced an 88% and a 50% decrease respectively in the median tumor volume as compared with the control groups. This therapeutic action was accompanied by increased necrosis of the tumor. Importantly, no signs of toxicity were detected in healthy animals after their treatment by the toxin expression vectors. CONCLUSIONS: DT-A was preferentially expressed in liver metastases after being transfected with H19 or IGF2-P3 promoter-driven DT-A expression plasmids, causing a very significant inhibition of tumor growth as a result of its cytotoxic effect. Our findings strongly support the feasibility of our proposed therapeutic strategy, which may contribute to open new gene therapeutic options for human liver metastases.

Animals↗

PubMed2006

Role of Pur alpha in targeting mRNA to sites of translation in hippocampal neuronal dendrites.

Using genetic inactivation in the mouse, PURA, encoding Pur alpha, is demonstrated to be essential for developmentally-timed dendrite formation in the cerebellum and hippocampus. Comparison of RNA species bound by Pur alpha prompts the hypothesis that Pur alpha functions with non-coding RNA in transport of certain mRNA molecules to sites of translation in dendrites. Pur alpha binds to human BC200 RNA, implicated in dendritic targeting, and this has homologies to 7SL RNA, implicated in compartmentalized translation. Results using hippocampal rat neurons in situ show that Pur alpha binds to BC1 RNA, implicated in dendritic targeting as a mouse counterpart of BC200, and to mRNA molecules translated in dendrites; Pur alpha is specifically located in dendrites, where it is colocalized with Map2, but not in axons, where it fails to colocalize with Ankyrin G. Pur alpha and Staufen are colocalized at dendritic sites of mRNA translation. Microtubule disruptors inhibit Pur alpha dendritic targeting and allow its mislocalization to axons. Using mouse brain, double-RNA immunoprecipitation places Pur alpha together with Staufen or FMRP on BC1 RNA and specific mRNA species in vivo. These results help define a mechanism by which Pur alpha targets specific mRNA molecules to sites of dendritic translation.

Animals↗

PubMed2002

Possible physiological role of H19 RNA.

The product of the imprinted oncofetal H19 gene is an untranslated RNA of unknown function. With the human cDNA Atlas microarray, we detected differentially expressed genes modulated by the presence of H19 RNA. Many of the genes that are upregulated by H19 RNA are known to contribute to the invasive, migratory, and angiogenic capacities of cells. Moreover, we provided experimental data indicating that whereas H19 RNA did not have any growth advantage for the cells when cultured in 10% fetal calf serum, it did confer an advantage when cells were cultured in serum-poor medium. This observation can be explained in part by the inability of the H19-expressing cells to induce the cyclin-dependent kinase inhibitor p57(kip2) in response to serum stress. Our results favor the possible role of the H19 gene in promoting cancer progression, angiogenesis, and metastasis.

Bromodeoxyuridine↗