Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “long noncoding RNA”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 577 records · Page 32Linked to original sources
PubMed1999

Further examination of the Xist promoter-switch hypothesis in X inactivation: evidence against the existence and function of a P(0) promoter.

The onset of X inactivation coincides with accumulation of Xist RNA along the future inactive X chromosome. A recent hypothesis proposed that accumulation is initiated by a promoter switch within Xist. In this hypothesis, an upstream promoter (P(0)) produces an unstable transcript, while the known downstream promoter (P(1)) produces a stable RNA. To test this hypothesis, we examined expression and half-life of Xist RNA produced from an Xist transgene lacking P(0) but retaining P(1). We confirm the previous finding that P(0) is dispensable for Xist expression in undifferentiated cells and that P(1) can be used in both undifferentiated and differentiated cells. Herein, we show that Xist RNA initiated at P(1) is unstable and does not accumulate. Further analysis indicates that the transcriptional boundary at P(0) does not represent the 5' end of a distinct Xist isoform. Instead, P(0) is an artifact of cross-amplification caused by a pseudogene of the highly expressed ribosomal protein S12 gene Rps12. Using strand-specific techniques, we find that transcription upstream of P(1) originates from the DNA strand opposite Xist and represents the 3' end of the antisense Tsix RNA. Thus, these data do not support the existence of a P(0) promoter and suggest that mechanisms other than switching of functionally distinct promoters control the up-regulation of Xist.

Animals↗
PubMed1999

Genetic analysis of the mouse X inactivation center defines an 80-kb multifunction domain.

Dosage compensation in mammals occurs by X inactivation, a silencing mechanism regulated in cis by the X inactivation center (Xic). In response to developmental cues, the Xic orchestrates events of X inactivation, including chromosome counting and choice, initiation, spread, and establishment of silencing. It remains unclear what elements make up the Xic. We previously showed that the Xic is contained within a 450-kb sequence that includes Xist, an RNA-encoding gene required for X inactivation. To characterize the Xic further, we performed deletional analysis across the 450-kb region by yeast-artificial-chromosome fragmentation and phage P1 cloning. We tested Xic deletions for cis inactivation potential by using a transgene (Tg)-based approach and found that an 80-kb subregion also enacted somatic X inactivation on autosomes. Xist RNA coated the autosome but skipped the Xic Tg, raising the possibility that X chromosome domains escape inactivation by excluding Xist RNA binding. The autosomes became late-replicating and hypoacetylated on histone H4. A deletion of the Xist 5' sequence resulted in the loss of somatic X inactivation without abolishing Xist expression in undifferentiated cells. Thus, Xist expression in undifferentiated cells can be separated genetically from somatic silencing. Analysis of multiple Xic constructs and insertion sites indicated that long-range Xic effects can be generalized to different autosomes, thereby supporting the feasibility of a Tg-based approach for studying X inactivation.

Acetylation↗
PubMed2000

Loss of imprinting of the insulin-like growth factor II gene occurs by biallelic methylation in a core region of H19-associated CTCF-binding sites in colorectal cancer.

We hypothesize that loss of imprinting (LOI) of the insulin-like growth factor II (IGF2) gene is associated with a predisposition to sporadic colorectal cancer. We confirmed a previously known strong correlation between LOI and microsatellite instability and showed that LOI was not a consequence of microsatellite instability or mismatch repair deficiency. LOI of IGF2 correlated strongly with biallelic hypermethylation of a core of five CpG sites in the insulator region of IGF2/H19, which is a known CTCF-binding element. As this methylation-dependent LOI was present in both tumors and normal colonic mucosa, it is possible that hypermethylation creates a field defect predisposing to cancer.

Adaptor Proteins, Signal Transducing↗
PubMed1997

In vivo ultraviolet and dimethyl sulfate footprinting of the 5' region of the expressed and silent Xist alleles.

The Xist (X inactive specific transcript) gene plays an essential role in X chromosome inactivation. To elucidate the mechanisms controlling Xist expression and X inactivation, we examined in vivo DNA-protein interactions in the Xist promoter region in a female mouse cell line (BMSL2), which has distinguishable Xist alleles. In vivo footprinting was accomplished by treatment of cells with dimethyl sulfate or ultraviolet light, followed by ligation-mediated polymerase chain reaction of purified DNA. The expressed allele on the inactive X chromosome and the silent allele on the active X chromosome were separated by the use of a restriction fragment length polymorphism prior to ligation-mediated polymerase chain reaction. The chromatin structure of the Xist promoter was found to be consistent with the activity state of the Xist gene. The silent allele (on the active X chromosome) showed no footprints, while the expressed allele (on the inactive X chromosome) showed footprints at a consensus sequence for a CCAAT box, two weak Sp1 sites, and a weak TATA box.

Alleles↗
PubMed1997

The RNA molecule CsrB binds to the global regulatory protein CsrA and antagonizes its activity in Escherichia coli.

The RNA-binding protein CsrA (carbon storage regulator) is a new kind of global regulator, which facilitates specific mRNA decay. A recombinant CsrA protein containing a metal-binding affinity tag (CsrA-H6) was purified to homogeneity and authenticated by N-terminal sequencing, matrix-assisted laser desorption/ionization time of flight mass spectrometry, and other studies. This protein was entirely contained within a globular complex of approximately 18 CsrA-H6 subunits and a single approximately 350-nucleotide RNA, CsrB. cDNA cloning and nucleotide sequencing revealed that the csrB gene is located downstream from syd in the 64-min region of the Escherichia coli K-12 genome and contains no open reading frames. The purified CsrA-CsrB ribonucleoprotein complex was active in regulating glg (glycogen biosynthesis) gene expression in vitro, as was the RNA-free form of the CsrA protein. Overexpression of csrB enhanced glycogen accumulation in E. coli, a stationary phase process that is repressed by CsrA. Thus, CsrB RNA is a second component of the Csr system, which binds to CsrA and antagonizes its effects on gene expression. A model for regulatory interactions in Csr is presented, which also explains previous observations on the homologous system in Erwinia carotovora. A highly repeated nucleotide sequence located within predicted stem-loops and other single-stranded regions of CsrB, CAGGA(U/A/C)G, is a plausible CsrA-binding element.

Bacterial Proteins↗
PubMed1997

Genomic deletion of an imprint maintenance element abolishes imprinting of both insulin-like growth factor II and H19.

Insulin-like growth factor II (Igf2) is maternally imprinted in normal tissues with only the paternal copy of the gene being transcribed, whereas the contiguous gene H19 is paternally imprinted. Dysregulation of IGF2 imprinting is commonly observed in Wilms' tumor and other human tumors. Previous work comparing promoter-specific imprinting of human and mouse Igf2 suggested the presence of a cis element upstream of Igf2 that regulates or maintains the imprinting of three downstream promoters. To explore the molecular mechanism of maintenance of genomic imprinting, we targeted the region between insulin 2 and Igf2, where the cis imprint maintenance element (IME) resides in mouse fibroblasts. In those clones in which the targeting vector was randomly integrated into the genome, mouse Igf2 remained imprinted. However, when the targeted region containing the IME was deleted by homologous recombination, whether from the paternal or maternal allele, activation of the imprinted maternal allele of Igf2 was observed. In addition, there was a loss of H19 imprinting when the IME was deleted. The requirement of IME from both parental alleles for the maintenance of genomic imprinting thus suggests the importance of a spatial structure of DNA around Igf2 and H19. Modifications in the IME, like abnormal methylation in Wilms' tumors, may represent a novel mechanism for loss of genomic imprinting.

Animals↗
PubMed1999

Methylation sequencing analysis refines the region of H19 epimutation in Wilms tumor.

Differential DNA methylation of the parental alleles has been implicated in the establishment and maintenance of the monoallelic expression of imprinted genes. H19 and IGF2 are oppositely imprinted with only the maternal and the paternal alleles expressed, respectively. In Wilms tumor, a childhood renal neoplasm, loss of the H19/IGF2 imprinted expression pattern results in silencing of H19 and biallelic expression of IGF2. This was shown to be associated with biallelic methylation of the H19 promoter in the tumor and the adjacent kidney tissue suggesting that epigenetic H19 silencing is an early event in Wilms tumorigenesis. An imprinting mark region characterized by paternal allele-specific methylation has been suggested to reside in a GC-rich region of 400-base pair direct repeats starting at -2 kilobase pairs (kb) relative to the H19 transcription start and extending upstream. The upstream boundary of the potential paternal methylation imprint of the H19 gene has yet to be defined. We sought to define this upstream imprint boundary and investigate whether Wilms tumors with loss of imprinting are biallelically methylated in this imprinting mark region. The analysis of 6.6 kb of new upstream H19 sequence determined in this study identified a series of the direct 400-base pair repeats that extends to approximately -5.3 kb relative to the transcription start. DNA methylation analyses indicated that the upstream boundary of the potential imprint may coincide with the 5' end of the direct repeats. We found that Wilms tumors with loss of imprinting are biallelically methylated in the H19 upstream repeat region, and we suggest that pathological methylation in this region is the epigenetic error that initiates H19 silencing.

Blotting, Southern↗
PubMed2000

XIST RNA associates with specific regions of the inactive X chromatin.

Microscopy studies have shown that XIST RNA colocalizes with the inactive X chromosome (Xi). However, the molecular basis for this colocalization is unknown. Here we provide two lines of evidence from chromatin immunoprecipitation experiments that XIST RNA physically associates with the Xi chromatin. First, XIST RNA can be co-precipitated by antiserum against macroH2A, a histone H2A variant enriched in the Xi. Second, XIST RNA can be co-precipitated by antisera that recognize unacetylated, but not acetylated, isoforms of histones H3 and H4. The specificity of XIST RNA association with hypoacetylated chromatin, together with the previous finding that hypoacetylated histone H4 is enriched at promoters of X-inactivated genes, raises the possibility that XIST RNA may contribute to the hypoacetylation of specific regions of the Xi so as to alter the expression of X-linked genes.

Animals↗
PubMed2002

Multiple cis elements within the Igf2/H19 insulator domain organize a distance-dependent silencer. A cautionary note.

The 5'-flank of the H19 gene harbors a differentially methylated imprinting control region that represses the maternally derived Igf2 and paternally derived H19 alleles. Here we show that the H19 imprinting control region (ICR) is a potent silencer when positioned in a promoter-proximal position. The silencing effect is not alleviated by trichostatin A treatment, suggesting that it does not involve histone deacetylase functions. When the H19 ICR is separated from the promoter by more than 1.2 +/- 0.3 kb, however, trichostatin A stimulates promoter activity 10-fold. Deletion analyses revealed that the silencing feature extended throughout the ICR segment. Finally, chromatin immunopurification analyses revealed that the H19 ICR prevented trichostatin A-dependent reacetylation of histones in the promoter region in a proximal but not in a distal position. We argue that these features are likely to be side effects of the H19 ICR, rather than explaining the mechanism of silencing of the paternal H19 allele. We issue a cautionary note, therefore, that the interpretation of insulator/silencer data could be erroneous should the distance issue not be taken into consideration.

Antifungal Agents↗
PubMed2000

H19 RNA binds four molecules of insulin-like growth factor II mRNA-binding protein.

H19 RNA is a major oncofetal 2.5-kilobase untranslated RNA of unknown function. The maternally expressed H19 gene is located 90 kilobase pairs downstream from the paternally expressed insulin-like growth factor II (IGF-II) gene on human chromosome 11 and mouse chromosome 7; and due to their reciprocal imprinting and identical spatiotemporal expression, it is assumed that the two genes are functionally coupled. Here we show that human H19 RNA contains four attachment sites for the oncofetal IGF-II mRNA-binding protein (IMP) with apparent K(d) values in the 0.4-1.3 nm range. The multiple attachment sites are clustered within a 700-nucleotide segment encoded by exons 4 and 5. This 3'-terminal segment targets H19 RNA to lamellipodia and perinuclear regions in dispersed fibroblasts where IMP is also localized. The results suggest that IMP participates in H19 RNA localization and provides a link between the IGF-II and H19 genes at post-transcriptional events during mammalian development.

Animals↗
PubMed2002

Unregulated expression of the imprinted genes H19 and Igf2r in mouse uniparental fetuses.

The present study shows that the H19 and Igf2r genes, which are imprinted and expressed solely from maternal alleles, are expressed in an unregulatable manner in mouse uniparental, androgenetic, and parthenogenetic fetuses at day 9.5 of gestation. In the androgenetic fetuses, the H19 and Igf2r genes were respectively expressed at 12 and 40% of the levels in biparental fetuses. In addition, the expression of both genes was excessive (1259 and 482%, respectively) in the parthenotes. These expressions of the imprinted genes were not regulated by methylation in the regulatory regions. Moreover, the expression of the antisense Igf2r RNA (Air) was also excessive and was not correlated with Igf2r gene expression in the uniparental fetuses. Taken together, these results indicate that the parental specific expression of imprinted genes is not maintained in particular genes in uniparental embryos, which in turn suggests that both parental genomes are required to establish maternal specific expression of the H19 and Igf2r genes by trans-acting mechanisms.

Alleles↗
PubMed2002

A dyad oct-binding sequence functions as a maintenance sequence for the unmethylated state within the H19/Igf2-imprinted control region.

DNA methylation of an imprinted control region (ICR) directs the allele-specific and reciprocal expression of the mouse H19 and the insulin-like growth factor 2 (Igf2) genes, mediated by controlling enhancer access. The ICR shows enhancer blocking activity through CTCF binding to an unmethylated sequence. The unmethylated state of the maternal ICR is maintained throughout development after establishment in the germ line; however, little is known of the molecular mechanisms that regulate DNA methylation. Hence, in this study we show that a dyad Oct-binding sequence (DOS) in the ICR mediates the demethylation of low-density methylation but not hypermethylation and is required to maintain the unmethylated state against the tendency for de novo methylation within the ICR in the embryonic carcinoma cell line P19. Furthermore, we also reveal that the unmethylated state of at least one CTCF-binding site within the ICR is under the control of DOS. Our results suggest that the ICR, as a CTCF-dependent insulator, requires DOS as well as CTCF-binding sites and that DOS maintains the maternal specific unmethylated state of the ICR at postimplantation stages.

Animals↗
PubMed2002

Loss of Igf2 imprinting in monoclonal mouse hepatic tumor cells is not associated with abnormal methylation patterns for the H19, Igf2, and Kvlqt1 differentially methylated regions.

IGFII, the peptide encoded by the Igf2 gene, is a broad spectrum mitogen with important roles in prenatal growth as well as cancer progression. Igf2 is transcribed from the paternally inherited allele, whereas the linked H19 is transcribed from the maternal allele. Igf2 imprinting is thought to be maintained by differentially methylated regions (DMRs) located at multiple sites such as upstream of H19 and Igf2 and within Kvlqt1 loci. Biallelic expression (loss of imprinting (LOI)) of Igf2 is frequently observed in cancers, and a subset of Wilms' and intestinal tumors have been shown to exhibit abnormal methylation at H19DMR associated with loss of maternal H19 expression, but it is not known whether such changes are common in other neoplasms. Because cancers consist of diverse cell populations with and without Igf2 LOI, we established four independent monoclonal cell lines with Igf2 LOI from mouse hepatic tumors. We here demonstrate retention of normal differential methylation at H19, Igf2, or Kvlqt1 DMR by all of the cell lines. Furthermore, H19 was found to be expressed exclusively from the maternal allele, and levels of CTCF, a multifunctional nuclear factor that has an important role in the Igf2 imprinting, were comparable with those in normal hepatic tissues with no mutational changes detected. These data indicate that Igf2 LOI in tumor cells is not necessarily linked to abnormal methylation at H19, Igf2, or Kvlqt1 loci.

Animals↗
PubMed2002

Imprint control element-mediated secondary methylation imprints at the Igf2/H19 locus.

Understanding the molecular basis of monoallelic expression as observed at imprinted loci is helpful in understanding the mechanisms underlying epigenetic regulation. Genomic imprinting begins during gametogenesis with the establishment of epigenetic marks on the chromosomes such that paternal and maternal chromosomes are rendered distinct. During embryonic development, the primary imprint can lead to generation of secondary epigenetic modifications (secondary imprints) of the chromosomes. Eventually, either the primary imprints or the secondary imprints interfere with transcription, leading to parent-of-origin-dependent silencing of one of the two alleles. Here we investigated several aspects pertaining to the generation and functional necessity of secondary methylation imprints at the Igf2/H19 locus. At the H19 locus, these secondary imprints are, in fact, the signals mediating paternal chromosome-specific silencing of that gene. We first demonstrated that the H19 secondary methylation imprints are entirely stable through multiple cell divisions, even in the absence of the primary imprint. Second, we generated mouse mutations to determine which DNA sequences are important in mediating establishment and maintenance of the silent state of the paternal H19 allele. Finally, we analyzed the dependence of the methylation of Igf2DMR1 region on the primary methylation imprint about 90 kilobases away.

Animals↗
PubMed2004

An RNA-binding domain in the thyroid hormone receptor enhances transcriptional activation.

Thyroid hormone plays important roles in development, differentiation, and metabolic homeostasis by binding to nuclear thyroid hormone receptors, which regulate target gene expression by interacting with DNA response elements and coregulatory proteins. We show that thyroid hormone receptors also are single-stranded RNA binding proteins and that this binding is functionally significant. By using a series of deletion mutants, a novel RNA-binding domain was localized to a 41-amino acid segment of thyroid hormone receptor alpha1 between the second zinc finger and the ligand-binding domain. This RNA-binding domain was necessary and sufficient for thyroid hormone receptor binding to the steroid receptor RNA activator (SRA). Although SRA does not bind directly to steroid receptors, it has been identified as a steroid receptor coactivator, and was thought not to be a coactivator for thyroid hormone receptors. However, transfection studies revealed that SRA enhances thyroid hormone induction of appropriate reporter genes and that the thyroid hormone receptor RNA-binding domain is important for this enhancement. We conclude that thyroid hormone receptors bind RNA through a novel domain and that the interaction of this domain with SRA, and perhaps other RNAs, enhances thyroid hormone receptor function.

Amino Acid Sequence↗
PubMed2004

A loss of insulin-like growth factor-2 imprinting is modulated by CCCTC-binding factor down-regulation at senescence in human epithelial cells.

The imprinted insulin-like growth factor-2 (IGF2) gene is an auto/paracrine growth factor expressed only from the paternal allele in adult tissues. In tissues susceptible to aging-related cancers, including the prostate, a relaxation of IGF2 imprinting is found, suggesting a permissive role for epigenetic alterations in cancer development. To determine whether IGF2 imprinting is altered in cellular aging and senescence, human prostate epithelial and urothelial cells were passaged serially in culture to senescence. Allelic analyses using an IGF2 polymorphism demonstrated a complete conversion of the IGF2 imprint status from monoallelic to biallelic, in which the development of senescence was associated with a 10-fold increase in IGF2 expression. As a mechanism, a 2-fold decrease in the binding of the enhancer-blocking element CCCTC-binding factor (CTCF) within the intergenic IGF2-H19 region was found to underlie this switch to biallelic IGF2 expression in senescent cells. This decrease in CTCF binding was associated with reduced CTCF expression in senescent cells. No de novo increases in methylation at the IGF2 CTCF binding site were seen. The forced down-regulation of CTCF expression using small interfering RNA in imprinted prostate cell lines resulted in an increase in IGF2 expression and a relaxation of imprinting. Our data suggest a novel mechanism for IGF2 imprinting regulation, that is, the reduction of CTCF expression in the control of IGF2 imprinting. We also demonstrate that altered imprinting patterns contribute to changes in gene expression in aging cells.

Base Sequence↗
PubMed2004

Characterization of interactions between PinX1 and human telomerase subunits hTERT and hTR.

The addition of telomeric repeats to chromosome ends by the enzyme telomerase is a highly orchestrated process. Although much is known regarding telomerase catalytic activity in vitro, less is known about how this activity is regulated in vivo to ensure proper telomere elongation. One protein that appears to be involved in negatively regulating telomerase function in vivo is PinX1 because overexpression of PinX1 inhibits telomerase activity and causes telomere shortening. To understand the nature of this repression, we characterized the interactions among PinX1 and the core components of telomerase, the human telomerase reverse transcriptase (hTERT) and associated human telomerase RNA (hTR). We now show that in vitro PinX1 binds directly to the hTERT protein subunit, primarily to the hTR-binding domain, as well as to the hTR subunit. However, in a cellular context, the association of PinX1 with hTR is dependent on the presence of hTERT. Taken together, we suggest that PinX1 represses telomerase activity in vivo by binding to the assembled hTERT.hTR complex.

Cell Cycle Proteins↗
PubMed2005

Tissue-specific changes in H19 methylation and expression in mice with hyperhomocysteinemia.

Expression of the imprinted genes H19 and insulin-like growth factor 2 (Igf2), which lie in close proximity on mouse chromosome 7, is regulated by methylation of a differentially methylated domain (DMD) located 5' to H19. Biallelic expression of H19 has been observed in renal disease patients with hyperhomocysteinemia, a cardiovascular disease risk factor. The present study determined whether hyperhomocysteinemia produces decreased tissue methylation capacity, hypomethylation of the H19 DMD, and altered expression of H19 and Igf2 in adult mice. Mice heterozygous for disruption of the gene for cystathionine-beta-synthase (Cbs+/-) and C57BL/6 (Cbs+/+) mice were fed a hyperhomocysteinemic or control diet, respectively, from weaning until 9-12 months of age. Higher plasma total homocysteine (p < 0.001) was found in hyperhomocysteinemic mice than in control mice (95 +/- 12 versus 5.0 +/- 0.3 micromol/liter). Hyperhomocysteinemia was accompanied by higher levels of S-adenosylhomocysteine (p < 0.05) and lower S-adenosylmethionine/S-adenosylhomocysteine ratios (p < 0.001) in liver and brain. The effect of hyperhomocysteinemia on H19 DMD methylation was tissue-specific. In liver, hyperhomocysteinemic mice had decreased H19 DMD methylation (p < 0.001). In brain, hyperhomocysteinemia was accompanied by increased H19 DMD methylation (p < 0.001) and a decrease in the ratio of H19/Igf2 transcripts (p < 0.05). In aorta, hyperhomocysteinemia produced an increase in H19 DMD methylation (p < 0.001) and a 2.5-fold increase in expression of H19 transcripts (p < 0.05). Levels of H19 transcripts in aorta correlated positively with plasma total homocysteine concentration (p < 0.05, r = 0.620). We conclude that hyperhomocysteinemia produces tissue-specific changes in H19 DMD methylation and increased vascular expression of H19 in adult mice.

Animals↗