The 5' flank of mouse H19 in an unusual chromatin conformation unidirectionally blocks enhancer-promoter communication.
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BACKGROUND: During mouse prenatal development, the neighbouring insulin-like growth factor II (Igf2) and H19 loci are expressed monoallelically from the paternal and maternal alleles, respectively. Identical spatiotemporal expression patterns and enhancer deletion experiments show that the Igf2 and H19 genes share a common set of enhancers. Deletion of a differentially methylated region in the 5' flank of the H19 gene partially relieves the repression of the maternal Igf2 and paternal H19 alleles in the soma. The mechanisms underlying the function of the 5' flank of the H19 gene are, however, unknown. RESULTS: Chromatin analysis showed that the 5' flank of the mouse H19 gene contains maternal-specific, multiple nuclease hypersensitive sites that map to linker regions between positioned nucleosomes. These features could be recapitulated in an episomal-based H19 minigene, which was propagated in human somatic cells. Although the 5' flank of the H19 promoter has no intrinsic silencer activity under these conditions, it unidirectionally extinguished promoter-enhancer communications in a position-dependent manner, without directly affecting the enhancer function. CONCLUSIONS: The unmethylated 5' flank of the H19 gene adopts an unusual and maternal-specific chromatin conformation in somatic cells and regulates enhancer-promoter communications, thereby providing an explanation for its role in manifesting the repressed state of the maternally inherited Igf2 allele. (+info)
Comparative genomic sequencing identifies novel tissue-specific enhancers and sequence elements for methylation-sensitive factors implicated in Igf2/H19 imprinting.
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A differentially methylated region (DMR) and endoderm-specific enhancers, located upstream and downstream of the mouse H19 gene, respectively, are known to be essential for the reciprocal imprinting of Igf2 and H19. To explain the same imprinting patterns in non-endodermal tissues, additional enhancers have been hypothesized. We determined and compared the sequences of human and mouse H19 over 40 kb and identified 10 evolutionarily conserved downstream segments, 2 of which were coincident with the known enhancers. Reporter assays in transgenic mice showed that 5 of the other 8 segments functioned as enhancers in specific mesodermal and/or ectodermal tissues. We also identified a conserved 39-bp element that appeared repeatedly within the DMR and formed complexes with specific nuclear factors. Binding of one of the factors was inhibited when the target sequence contained methylated CpGs. These complexes may contribute to the presumed boundary function of the unmethylated DMR, which is proposed to insulate maternal Igf2 from the enhancers. Our results demonstrate that comparative genomic sequencing is highly efficient in identifying regulatory elements. (+info)
Telomerase activity and expression of hTRT and hTR in gastrointestinal stromal tumors in comparison with extragastrointestinal sarcomas.
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Stromal tumors of the gut (GISTs) have rarely been analyzed for genetic alterations. This study aimed at determining telomerase activity and the expression of the telomerase subunits human telomerase reverse transcriptase (hTRT) and human telomerase RNA (hTR) in GISTs and extragastrointestinal neurogenic or myogenic sarcomas. Telomerase activity was investigated using the telomeric repeat amplification protocol assay in 21 GISTs, recurrences and liver metastases from 16 patients, and in 22 leiomyosarcomas and 21 malignant peripheral nerve sheath tumors (MPNSTs), which served as reference tumors. Expression of hTRT and hTR mRNA was investigated using reverse transcription-PCR. Thirteen GISTs were localized in the stomach and three in the small intestine. Two tumors were benign. In one case, the biological behavior was uncertain. In 67% of GISTs, high telomerase activity was found, whereas high activity was noted in only 18% of leiomyosarcomas and in 48% of MPNSTs. There was no activity in two benign and two malignant GISTs. In one malignant tumor of the small intestine, the primary tumor showed no activity at first but a marked activity in its recurrence. In the tumor with uncertain behavior, telomerase activity and hTRT expression were only weak. In all GISTs showing telomerase activity, the catalytic subunit hTRT was expressed. All GISTs and extragastrointestinal sarcomas expressed hTR. In comparison with leiomyosarcomas and MPNSTs, malignant GISTs showed a higher telomerase activity, which, however, was not seen in benign GISTs. It is possible that telomerase activity occurs during the progression of malignant GISTs. There was a correlation between telomerase activity and the expression of hTRT. (+info)
H19 and Igf2 monoallelic expression is regulated in two distinct ways by a shared cis acting regulatory region upstream of H19.
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H19 and Igf2 are expressed in a monoallelic fashion from the maternal and paternal chromosomes, respectively. A region upstream of H19 has been shown to regulate such imprinted expression of both genes in cis. We have taken advantage of a loxP/cre recombinase-based strategy to delete this region in mice in a conditional manner to determine the temporal requirement of the upstream region in initiating and maintaining the imprinted expression of H19 and Igf2. Analysis of allele-specific expression of H19 and Igf2 and DNA methylation at the H19 promoter demonstrates that this region controls the monoallelic expression of the two genes in different ways, suggesting that it harbors two functionally distinct regulatory elements. Continued presence of the region is required to silence maternal Igf2 in accordance with its proposed role as an insulator. However, it does not have a direct role in keeping the paternal H19 promoter silenced. Instead, on the paternal chromosome, the upstream element mediates epigenetic modifications of the H19 promoter region during development, leading to transcriptional silencing of H19. Thereafter, its presence is redundant for preventing transcription. Presently, this temporal requirement of the silencing element appears to be a unique cis activity in the mammalian system. However, it is likely that other cis-acting elements, positive and negative, have the ability to effect stable changes in the chromatin structure and are not constantly required to give signals to the transcriptional machinery. (+info)
Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo.
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The H19 gene is imprinted with preferential expression from the maternal allele. The putative imprinting control region for this locus is hypermethylated on the repressed paternal allele. Although maternal-specific expression of H19 is observed in mouse blastocysts that develop in vivo, biallelic expression has been documented in embryos and embryonic stem cells experimentally manipulated by in vitro culture conditions. In this study the effect of culture on imprinted H19 expression and methylation was determined. After culture of 2-cell embryos to the blastocyst stage in Whitten's medium, the normally silent paternal H19 allele was aberrantly expressed, whereas little paternal expression was observed following culture in KSOM containing amino acids (KSOM+AA). Analysis of the methylation status of a CpG dinucleotide located in the upstream imprinting control region revealed a loss in methylation in embryos cultured in Whitten's medium but not in embryos cultured in KSOM+AA. Thus, H19 expression and methylation were adversely affected by culture in Whitten's medium, while the response of H19 to culture in KSOM+AA approximated more closely the in vivo situation. It is unlikely that biallelic expression of H19 following culture in Whitten's medium is a generalized effect of lower methylation levels, since the amount of DNA methyltransferase activity and the spatial distribution of Dnmt1 protein were similar in in vivo-derived and cultured embryos. Moreover, imprinted expression of Snrpn was maintained following culture in either medium, indicating that not all imprinted genes are under the same stringent imprinting controls. The finding that culture conditions can dramatically, but selectively, affect the expression of imprinted genes provides a model system for further study of the linkage between DNA methylation and gene expression. (+info)
Treatment of prostate cancer in vitro and in vivo with 2-5A-anti-telomerase RNA component.
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Prostate cancer is the most common malignancy of elderly men in the United States. Since there is no curative treatment for advanced prostate cancer, exploration of novel modalities of treatment is essential. Telomerase, a ribonucleoprotein, is detected in the vast majority of prostate cancer, but not in normal or benign prostatic hyperplasia tissues. Thus, telomerase is expected to be a very strong candidate for targeted therapy of prostate cancer. In this study, we synthesized a 19-mer antisense oligonucleotide against the RNA component of human telomerase (hTR) linked to a 2-5A molecule (2-5A-anti-hTR) and examined its cytotoxic effect on prostate cancer cells. The 2-5A antisense strategy relies on the recruitment and activation of RNase L at the site of targeted RNA sequence. We here show that treatment with 2-5A-anti-hTR in the presence of a cationic liposome reduced cell viability of tumor cell lines tested to 9-18% within 6 days. In contrast, normal fibroblast cells were resistant to the treatment. Its effect was mainly due to induction of apoptosis by activated caspase family members. Furthermore, treatment of subcutaneous tumors in nude mice with 2-5A-anti-hTR significantly suppressed the tumor growth through induction of apoptosis (P<0.001). The treatment with 2-5A-anti-hTR may be a promising strategy for the treatment modality of prostate cancer with telomerase activity. (+info)
Temporal and spatial order of events during the induction of cortical cell divisions in white clover by Rhizobium leguminosarum bv. trifolii inoculation or localized cytokinin addition.
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We examined the timing and location of several early root responses to Rhizobium leguminosarum bv. trifolii infection, compared with a localized addition of cytokinin in white clover, to study the role of cytokinin in early signaling during nodule initiation. Induction of ENOD40 expression by either rhizobia or cytokinin was similar in timing and location and occurred in nodule progenitor cells in the inner cortex. Inoculation of rhizobia in the mature root failed to induce ENOD40 expression and cortical cell divisions (ccd). Nitrate addition at levels repressing nodule formation inhibited ENOD40 induction by rhizobia but not by cytokinin. ENOD40 expression was not induced by auxin, an auxin transport inhibitor, or an ethylene precursor. In contrast to rhizobia, cytokinin addition was not sufficient to induce a modulation of the auxin flow, the induction of specific chalcone synthase genes, and the accumulation of fluorescent compounds associated with nodule initiation. However, cytokinin addition was sufficient for the localized induction of auxin-induced GH3 gene expression and the initiation of ccd. Our results suggest that rhizobia induce cytokinin-mediated events in parallel to changes in auxin-related responses during nodule initiation and support a role of ENOD40 in regulating ccd. We propose a model for the interactions of cytokinin with auxin, ENOD40, flavonoids, and nitrate during nodulation. (+info)
Expression of hTERT and hTR in cis reconstitutes and active human telomerase ribonucleoprotein.
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Telomeres in eukaryotic cells are generally synthesized and maintained by the ribonucleoprotein (RNP) telomerase. This enzyme is composed of at least two subunits, the telomerase reverse transcriptase (TERT) and the telomerase RNA. Human telomerase activity can be reconstituted in vitro by the expression of the telomerase protein catalytic subunit (hTERT) in the presence of recombinant human telomerase RNA (hTR) in a rabbit reticulocyte lysate (RRL) system. The hTERT and hTR subunits are independently expressed in vivo, and little is known about the mechanism of their assembly. To facilitate recombinant telomerase RNP formation and reconstitution, we engineered a construct, termed hTERT-hTR cis, in which the 3' end of the hTERT coding sequence was extended by the addition of the sequence encoding hTR. Expression of the hTERT-hTR cis construct in vitro (in RRL) and in vivo (in the yeast Saccharomyces cerevisiae) produced hTERT-hTR transcripts of the predicted size. Active human telomerase was reconstituted by hTERT-hTR cis expression in both RRL and S. cerevisiae. Assembly of functional human telomerase by the bicistronic expression of the protein and RNA components may facilitate the overexpression and reconstitution of this enzyme in heterologous systems. (+info)