Sequence of a 42-kb mouse region containing the imprinted H19 locus: identification of a novel muscle-specific transcription unit showing biallelic expression.
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Alleles↗
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Results for “Long noncoding RNA”
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Genomic imprinting in ruminants: allele-specific gene expression in parthenogenetic sheep.
Studies in the mouse have established that both parental genomes are essential for normal embryonic development. Parthenogenetic mouse embryos (which have two maternal genomes and no paternal genome), for example, are growth-retarded and die at early postimplantation stages. The distinct maternal and paternal contributions are mediated by genomic imprinting, an epigenetic mechanism by which the expression of certain genes is dependent on whether they are inherited from mother or father. Although comparative studies have established that many imprinted mouse (and rat) genes are allele-specifically expressed in humans as well (and vice versa), so far imprinting studies have not been performed in other mammalian species. When considering evolutionary theories of genomic imprinting, it would be important to know how widely it is conserved among placental mammals. We have investigated its conservation in a bovid ruminant, the domestic sheep, by comparing parthenogenetic and normal control embryos. Our study establishes that, like in the mouse, parthenogenetic development in sheep is associated with growth-retardation and does not proceed beyond early fetal stages. These developmental abnormalities are most likely caused by imprinted genes. We demonstrate that, indeed, like in mice and humans, the growth-related PEG1/MEST and Insulin-like Growth Factor 2 (IGF2) genes are expressed from the paternal chromosome in sheep. These observations suggest that genomic imprinting is conserved in a third, evolutionarily rather diverged group of placental mammals, the ruminants.
Alleles↗
Compact chromatin packaging of inactive X chromosome involves the actively transcribed Xist gene.
The Xist gene responsible for X inactivation may take a unique chromatin structure because of exceptional expression from inactive X Chromosome, (Chr). We have examined differential chromatin packaging of the Xist gene region between active and inactive X Chr with a novel method consisting of the chromatin fractionation and allele-specific detection. Analysis of F1 heterozygous female mice from T(X;16)16H x MSM crosses and two cell clones derived from inter-subspecific F1 female mice demonstrated that the packaging level of the transcribed Xist region on inactive X Chr was as tight as that of the repressed Pgk-1 allele on the same chromosome. On the other hand, restriction endonuclease sensitivity assay of chromatin showed that the promoter region, but not transcribed regions, of the transcribed Xist allele retained accessibility to nucleases. These results may suggest a cis-element(s) in a regulatory region of the Xist gene to prevent the transcriptionally inhibitory effect of the chromatin packaging.
Animals↗
Scaffold attachment factor A (SAF-A) is concentrated in inactive X chromosome territories through its RGG domain.
Female mammalian cells inactivate transcription from one of their X chromosomes to equalize gene expression of X-linked genes between males and females. Inactivation is a multistep process that involves a large non-coding RNA termed XIST, a variety of epigenetic modifications of chromatin, and alterations in protein composition such as enrichment of the histone variant macroH2A. We show here that inactive X chromosomes are also enriched in a well-characterized protein component of the nuclear scaffold, SAF-A. This protein has been implicated in chromatin organization, owing to its high specificity for scaffold-associated region (SAR)-DNA, in transcriptional regulation, e.g. of hormone-regulated genes, owing to its functional interaction with steroid receptors, and in RNA processing, owing to its interaction with RNA and heterogeneous nuclear ribonucleoprotein (hnRNP) particles. After near complete removal of DNA and associated chromatin proteins such as macroH2A, SAF-A remains with the "nuclear matrix", still highlighting the former position of inactive X chromosomes. Interestingly, the enrichment of SAF-A in the inactive X chromosome depends on the RNA binding domain of the protein, the RGG box, raising the possibility that interaction of SAF-A with XIST RNA may contribute to the silencing of X-linked genes by local changes in nuclear architecture.
Cells, Cultured↗
Ubiquitinated proteins including uH2A on the human and mouse inactive X chromosome: enrichment in gene rich bands.
The inactive X chromosome (Xi) forms a heterochromatic structure in the nucleus that is known to have several modifications to specific histones involving acetylation or methylation. Using three different antibodies in four different cell lines, we demonstrate that the Xi in human and mouse cells is highly enriched in ubiquitinated protein(s), much of which is polyubiquitinated. This ubiquitination appears specific for the Xi as it was not observed for centromeres or other regions of heterochromatin. Results using an antibody specific to ubiquitinated H2A provide a clear link between H2A ubiquitination and gene repression, as visualized across an entire inactive chromosome. Interestingly, the ubiquitination of the chromosome persists into mitosis and can be seen in a reproducible banded pattern. This pattern matches that of Xist RNA which forms bands as it detaches from the mitotic X chromosome. Both ubiquitination and Xist RNA appear enriched in gene dense regions and depleted in gene poor bands, but do not correlate with L1 LINE elements which have been suggested as key to X-inactivation. These results provide evidence that ubiquitination along with Xist RNA plays an important role in the formation of facultative heterochromatin during X-inactivation.
Animals↗
Developmental regulation of Suz 12 localization.
Chromatin modifications are among the epigenetic alterations essential for genetic reprogramming during development. The Polycomb group (PcG) gene family mediates chromatin modifications that contribute to developmentally regulated transcriptional silencing. Trimethylation of histone H3 on lysine 27, mediated by a PcG protein complex consisting of Eed, Ezh2, and Suz12, is integral in differentiation, stem cell self-renewal, and tumorigenesis. Eed and Ezh2 are also implicated in the developmentally regulated silencing of the inactive X chromosome, as they are transiently enriched on the inactive X chromosome when X chromosome silencing is initiated. Here we analyze the dynamic localization of Suz12 during cellular differentiation and X-inactivation. Though Suz12 is a requisite member of the Eed/Ezh2 complexes, we found that Suz12 exhibits a notable difference from Ezh2 and Eed: while Ezh2 and Eed levels decrease during stem cell differentiation, Suz12 levels remain constant. Despite the differential regulation in abundance of Suz12 and Eed/Ezh2, Suz12 is also transiently enriched on the Xi during early stages of X-inactivation, and this accumulation is Xist RNA dependent. These results suggest that Suz12 may have a function that is not mediated by its association with Eed and Ezh2, and that this additional function is not involved in the regulation of X-inactivation.
Animals↗
Xist RNA is associated with the transcriptionally inactive XY body in mammalian male meiosis.
In eutherian mammals, X inactive-specific transcripts (Xist) are expressed in somatic cells possessing more than one X chromosome, and in germline cells of males, in which the single X chromosome is transcriptionally inactive. In early meiosis of males the sex chromosomes form an inactive XY nuclear compartment (XY body). We show by in situ reverse-transcribed polymerase chain reaction that Xist RNA is concentrated in the XY body. This fine localization suggests that Xist RNA is involved in inactivation of the male X chromosome, and that it has spreading capability, not only in cis but also in a quasi-cis mode, to juxtaposed non-X chromosomes. A hypothetical scheme links the evolution of heteromorphic sex chromosomes to the development of X condensation/inactivation in the male. The mechanism of X inactivation in somatic cells of mammalian females, resulting in male/female dosage compensation, has been recruited from the Xist-activated chromosome condensation machinery that developed in male meiosis earlier in evolution.
Animals↗
Heterochromatin.
The properties of heterochromatin are reconsidered in the context of our present understanding of gene silencing, telomeric and centromeric properties, position-effect variegation and X-chromosome inactivation. It is proposed that the chromatin in heterochromatic chromosomal regions is generally similar in its molecular composition to that in silenced chromosomal regions. Heterochromatic appearance hence reflects not a particular quality of the respective chromosomal regions but only a specific kind of chromatin packaging comparable to that required for the inactivation of genes. This packaging may be initiated by particular signals in the DNA but can be propagated over more extended chromosomal regions by the formation of multiprotein complexes that interact with histones and possibly cell-specific additional components (RNA or proteins) that determine the status of the chromosome in a particular cell type.
Animals↗
Expression of Xist RNA is sufficient to initiate macrochromatin body formation.
MacroH2A1 is a histone variant that is found as a component of the inactive X chromosome where it is detected as a dense accumulation called a macrochromatin body (MCB). Macrochromatin bodies co-localize with Xist RNA, which is an untranslated RNA that is expressed exclusively from the inactive X chromosome of placental mammals. However, no studies to date have investigated whether Xist RNA expression is necessary or sufficient to cause the formation of MCBs. Here we show that expression of Xist RNA is sufficient to cause the formation of MCBs even when Xist is expressed from an inducible transgene at ectopic autosomal sites. Macrochromatin bodies form at sites of transgenic Xist expression in differentiating mouse ES cell lines and transgenic fibroblasts, but MCBs cannot form in undifferentiated ES cells even after prolonged Xist expression. The kinetics of MCB formation revealed that Xist expression precedes MCB formation and that differentiating ES cells undergo a rapid and synchronous transition that renders them competent to form MCBs. Once MCBs have formed, continued expression of Xist is required for their maintenance. These results show that Xist RNA and macroH2A1 function in a common pathway. Expression of Xist in a permissive nuclear environment is sufficient to initiate a chromatin-remodeling event culminating in the incorporation of macroH2A1. The results also strongly suggest the existence of additional regulatory factors for X inactivation that are regulated developmentally. In addition, we present evidence that macroH2A1 density is not simply a measure of the general degree of DNA compaction.
Animals↗
A parent-of-origin detectable polymorphism in the hypermethylated region upstream of the human H19 gene.
The H19 gene is a paternally imprinted gene located on chromosome 11p15.5. In this study the H19FR haplotype polymorphism including three SNPs upstream of the H19 gene was investigated. Six genotypes derived from three alleles were detected in the Japanese population by means of PCR and subsequent constant denaturing gel electrophoresis. Based on the methylation status of the genomic DNA from blood samples, selective detection of the parental allele for H19FR was examined by using two types of enzyme, the methylation-sensitive restriction enzymes HpaII or HhaI and McrBC. Genomic DNA digested by either HpaII or HhaI, revealed a single band derived from the paternal allele, as a result of cleavage of unmethylated recognition sites on the maternal allele. On the contrary, the use of McrBC, which can digest a methylated paternal sequence, resulted in exclusively amplifying the maternal allele. This method could be one of the useful techniques for discriminating the parental origin of alleles.
Alleles↗
Analysis of SCA8 and SCA12 loci in 134 Italian ataxic patients negative for SCA1-3, 6 and 7 CAG expansions.
Spinocerebellar ataxias (SCA) are a heterogeneous group of neurodegenerative disorders, six of which are caused by expansion of a polyglutamine-coding CAG repeats ( SCA1- 3, 6, 7 and 17). In addition, expansions of a CAG triplet in the 5' region of a gene and a CTG triplet in an antisense RNA have been demonstrated in the SCA12 and SCA8 genes respectively. Our series of 134 ataxic patients (22 familial and 112 sporadic, tested negative for SCAI-3, 6, 7) was investigated for the presence of triplet expansions in the SCA8 and SCA12 genes. No SCA12 expansion was identified. A moderate SCA8 expansion (85-97 repeats) was found in two unrelated families with slowly progressive cerebellar ataxia. The frequency of SCA8 expansion accounts for approximately 4.3 % of the whole pool of our ataxia families (2 out of 46), while none of the 127 controls screened carried > 35 CTG+CTA repeats. Our data suggest a possible pathogenetic role of this mutation, which at present is still controversial, and confirm the rarity of the SCA12 expansion in Italian patients.
Adolescent↗
Correlation of the expression of telomerase RNA with risk factors for recurrence of sebaceous gland carcinoma.
AIMS: To investigate the expression of human telomerase gene RNA (hTR) and human telomerase reverse transcriptase (hTRT) in eyelid sebaceous gland carcinoma and to evaluate risk factors for sebaceous carcinoma recurrence. METHODS: The expression patterns of hTR and hTRT were detected by in situ hybridization (ISH) in paraffin-embedded samples of 55 eyelid sebaceous gland carcinoma, 12 chalazia, and four sebaceous adenoma. The proliferation index (PI) of sebaceous gland carcinoma was determined by Ki-67 immunolabeling and the results were compared with the expression of hTR and hTRT. Several factors for recurrence of sebaceous gland carcinoma were evaluated by statistical analysis. RESULTS: Expression of hTR and hTRT was present mainly in the sebaceous gland carcinoma tissues and not in adjacent tissues of carcinoma, chalazia, and sebaceous adenoma. The prevalence of hTR, hTRT, and Ki-67 expression in 55 sebaceous carcinoma samples were 84.45%, 58.1%, and 78.18%, respectively. hTR expression was highly associated with the degree of carcinoma differentiation (P<0.001) and hTRT expression was correlated with the proliferation index as determined by Ki-67 staining (P<0.005). There was a correlation between the recurrence of sebaceous gland carcinoma and the patient's onset age (OR=0.914, P<0.05), as well as the time to first treatment (OR=1.163, P<0.01). CONCLUSIONS: Telomerase may play an important role in the carcinogenesis of sebaceous gland carcinoma, and expression of hTR and hTRT combined with other features of sebaceous gland carcinoma may be helpful for the diagnosis and evaluation of clinical prognosis. Risk factors for recurrence of sebaceous gland carcinoma are patients' onset age and the time to first treatment.
Adenocarcinoma, Sebaceous↗
Polycomb-group proteins are involved in silencing processes caused by a transgenic element from the murine imprinted H19/Igf2 region in Drosophila.
A subset of autosomal genes undergo genomic imprinting which results in expression from only the paternal or maternal chromosome. While this phenomenon is restricted to mammals and angiosperms, the underlying silencing mechanisms appear to be evolutionarily conserved. A biallelically unmethylated DNaseI hypersensitive region (A6-A4) between the imprinted Igf2 and H19 genes is conserved in humans and mice and functions as a tissue-specific maintenance element for the imprinted growth factor IGF2. In order to analyse A6-A4 for potentially conserved transcriptional maintenance properties, we have generated transgenic Drosophila harbouring the element in a reporter construct. These flies depicted silencing of the reporter genes lacZ and mini -white. The silenced state of the mini -white gene showed variegation and sensitivity to temperature changes. In addition, two members of the conserved Polycomb group, Enhancer of zeste and Posterior sex combs, were needed for repression. Polycomb group proteins are essential for gene silencing during development. Our results indicate that Polycomb group proteins may also be involved in the regulation of mammalian imprinted genes.
Animals↗
The human and mouse H19 imprinting control regions harbor an evolutionarily conserved silencer element that functions on transgenes in Drosophila.
Differentially methylated regions have been characterized at a number of imprinted gene complexes with important roles in the regulation of monoallelic expression of one or more genes. The differentially methylated imprinting control region (ICR) located upstream of the murine H19 gene has been shown to control the imprinted expression of H19 and the coordinately regulated Igf2 gene by acting as a transcriptional silencer. In this study, we show that the murine ICR maintains this function when tested in an in vivo transgenic Drosophila assay in the absence of DNA methylation. Furthermore, the H19 ICR interacts distinctively with Drosophila promoters of different regulatory strengths. We also demonstrate that the comparable region upstream of the human H19 gene is a multipartite cis-regulatory element, demonstrating silencing function when tested in mammalian and Drosophila systems. These results indicate a conservation of the H19/Igf2 imprinting mechanism between humans and mice and further elucidate the functional activities of the H19 ICR. They demonstrate the value of Drosophila as an in vivo system for testing function and interaction of eukaryotic regulatory elements and that mechanisms of transcriptional cis-regulation in mammals and Drosophila are conserved.
Alleles↗
H19 and Igf2 are expressed and differentially imprinted in neuroectoderm-derived cells in the mouse brain.
Igf2 and H19 are reciprocally imprinted genes that are closely linked and coexpressed in tissues of mesodermal and endodermal origin. Here we report that coexpression of these genes is also found in specific fetal tissues of neuroectodermal origin, that is in the ventral midline region of both the hindbrain and spinal cord. For cells of neuroectodermal origin, complete absence of Igf2 and H19 transcription was previously described. Analysis of allele-specific expression of both Igf2 and H19 in the ventral midline region of the hindbrain shows that H19 is expressed monoallelically, with the paternal allele being silent, whereas Igf2 is expressed biallelically. Furthermore, we observed a strong influence of the parental species background, in that the Mus musculus allele was always expressed at higher levels than the M. spretus allele. This was observed when the M. spretus allele was contributed by the mother or by the father. An analysis of Igf2 methylation by bisulphite genomic sequencing provided no clear answer as to whether Igf2 expression and methylation are linked in a tissue of neuroectodermal origin. Taken together, our results provide novel information on H19 and Igf2 expression and imprinting patterns in the fetal mouse brain. In addition, they indicate that some aspects of Igf2 regulation in cells of neuroectodermal origin do not follow the pattern that exists in mesoderm- and endoderm-derived tissues. Apart from the ventral midline region, H19 and Igf2 were found to be coexpressed in the ectodermally derived Rathke's pouch and in some circumventricular organs of the brain, such as the organum vasculosum of the lamina terminalis (OVLT) and the pineal gland.
Animals↗
Random monoallelic expression of the imprinted IGF2 and H19 genes in the absence of discriminative parental marks.
The IGF2 and H19 genes are genomically imprinted and expressed preferentially from the paternal and maternal alleles, respectively, during human prenatal development. The exact role of the parental imprint(s), however, is not known. To explore this issue in some detail, we have examined human androgenetic cells which by definition should be incapable of allelic discrimination given the paternal origin of both genomes. Allele-specific in situ hybridisation analysis of dispermic complete hydatidiform moles shows that IGF2 and H19 can be found to be transcriptionally active in a variegated manner, which results in the generation of random monoallelic expression patterns. This data shows that imprinted genes can be expressed monoallelically in the absence of discriminating parental marks and raises the question whether or not mechanisms underlying monoallelic expression preceded the acquisition of parental imprints during evolution.
Alleles↗
Role of a 461-bp G-rich repetitive element in H19 transgene imprinting.
The molecular mechanism leading to the imprinted expression of genes is poorly understood. While no conserved cis-acting elements have been identified within the known loci, many imprinted genes are located near directly repetitive sequence elements, suggesting that such repeats might play a role in imprinted gene expression. The maternally expressed mouse H19 gene is located approximately 1.5 kb downstream from a 461-bp G-rich repetitive element. We have used a transgenic model to investigate whether this element is essential for H19 imprinting. Previous results demonstrated that a transgene, which contains 14 kb of H19 sequence, exhibits parent-of-origin specific expression and methylation analogous to the endogenous H19 imprinting pattern. Here, we have generated transgenes lacking the G-rich repeat. One transgene, containing a deletion of the G-rich repetitive element but which includes an additional 1.7 kb of 5' H19 sequence, is imprinted similarly to the endogenous H19 gene. To determine whether the G-rich repeat is conserved in other imprinted mammalian H19 homologues, additional 5' flanking sequences were cloned from the rat and human. This element is conserved in the rat but not in human DNA. These results suggest that the 461-bp G-rich repetitive element is not essential for H19 imprinting.
Animals↗
Analysis of ENOD40 expression in alb1, a symbiotic mutant of Lotus japonicus that forms empty nodules with incompletely developed nodule vascular bundles.
The alb1 mutant of Lotus japonicus (Ljsym74) forms empty nodules in which most of the bacteria remain in abnormally enlarged infection threads and fail to enter the host plant cells. The alb1 mutant was also found to be defective in differentiation of ramified nodule vascular bundles; only a single vascular bundle differentiates at the proximal end of the alb1 nodules and it fails to differentiate further. Histochemical analysis using fluorescein-conjugated wheat-germ agglutinin (F-WGA) indicated that the mutation in the ALB1 gene specifically affects the differentiation of vascular bundles in nodules. Analysis of nodulin gene expression revealed that the expression of an early nodulin gene, ENOD40, was very low in alb1 nodules. At early developmental stages of alb1 nodules, the pattern of ENOD40 transcription was essentially the same as that in wild-type nodules; transcripts were localized in dividing cortical cells and in the pericycle of the root stele opposite nodule primordia, as in wild-type nodules. However, mature alb1 nodules exhibited very weak or no expression of ENOD40 in the peripheral cells of the undeveloped nodule vascular bundle. The ENOD40 expression pattern in alb1 nodules is distinct from that in another ineffective mutant, fen1 (Ljsym76), in which ENOD40 expression persists prior to premature senescence. These findings lead us to speculate that ENOD40 may play a role in the differentiation of nodule vascular bundles.
Fabaceae↗