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PubMed2005

Regulation of X-chromosome counting by Tsix and Xite sequences.

In mammals, X-inactivation establishes X-chromosome dosage parity between males and females. How X-chromosome counting regulates this process remains elusive, because neither the hypothesized inactivation "blocking factor" nor the required cis-elements have been defined. Here, a mouse knockout and transgenic analysis identified DNA sequences within the noncoding Tsix and Xite genes as numerators. Homozygous deficiency of Tsix resulted in "chaotic choice" and a variable number of inactive X's, whereas overdosage of Tsix/Xite inhibited X-inactivation. Thus, counting was affected by specific Tsix/Xite mutations, suggesting that counting is genetically separable from but molecularly coupled to choice. The mutations affect XX and XY cells differently, demonstrating that counting and choice are regulated not by one "blocking factor," but by both a "blocking" and a "competence" factor.

Animals↗
PubMed2000

Sequence and comparative analysis of the mouse 1-megabase region orthologous to the human 11p15 imprinted domain.

A major barrier to conceptual advances in understanding the mechanisms and regulation of imprinting of a genomic region is our relatively poor understanding of the overall organization of genes and of the potentially important cis-acting regulatory sequences that lie in the nonexonic segments that make up 97% of the genome. Interspecies sequence comparison offers an effective approach to identify sequence from conserved functional elements. In this article we describe the successful use of this approach in comparing a approximately 1-Mb imprinted genomic domain on mouse chromosome 7 to its orthologous region on human 11p15.5. Within the region, we identified 112 exons of known genes as well as a novel gene identified uniquely in the mouse region, termed Msuit, that was found to be imprinted. In addition to these coding elements, we identified 33 CpG islands and 49 orthologous nonexonic, nonisland sequences that met our criteria as being conserved, and making up 4.1% of the total sequence. These conserved noncoding sequence elements were generally clustered near imprinted genes and the majority were between Igf2 and H19 or within Kvlqt1. Finally, the location of CpG islands provided evidence that suggested a two-island rule for imprinted genes. This study provides the first global view of the architecture of an entire imprinted domain and provides candidate sequence elements for subsequent functional analyses.

Amino Acid Sequence↗
PubMed2004

Tsix transcription- versus RNA-based mechanisms in Xist repression and epigenetic choice.

Recent inquiries have revealed a surprisingly large number (>2500) of naturally occurring antisense transcripts, but their function remains largely undiscovered. A better understanding of antisense mechanisms is clearly needed because of their potentially diverse roles in gene regulation and disease. A well-documented case occurs in X inactivation, the mechanism by which X-linked gene expression is equalized between XX females and XY males. The antisense gene Tsix determines X chromosome choice and represses the noncoding silencer, Xist. In principle, Tsix action may involve RNA, the act of transcription, or local chromatin. Here, we create novel Tsix alleles to distinguish transcription- versus RNA-based mechanisms. When Tsix transcription is terminated before Xist (TsixTRAP), Tsix cannot block Xist upregulation, suggesting the importance of overlapping antisense transcription. To separate the act of transcription from RNA, we knocked in Tsix cDNA in the reverse orientation (Tsix(cDNA)) to restore RNA levels in cis without concurrent transcription across Xist. However, Tsix(cDNA) cannot complement TsixTRAP. Surprisingly, both mutations disrupt choice, indicating that this epigenetic step requires transcription. We conclude that the processed antisense RNA does not act alone and that Tsix function specifically requires antiparallel transcription through Xist. A mechanism of transcription-based feedback regulation is proposed.

Cells, Cultured↗
PubMed2006

X chromosomes alternate between two states prior to random X-inactivation.

Early in the development of female mammals, one of the two X chromosomes is silenced in half of cells and the other X chromosome is silenced in the remaining half. The basis of this apparent randomness is not understood. We show that before X-inactivation, the two X chromosomes appear to exist in distinct states that correspond to their fates as the active and inactive X chromosomes. Xist and Tsix, noncoding RNAs that control X chromosome fates upon X-inactivation, also determine the states of the X chromosomes prior to X-inactivation. In wild-type ES cells, X chromosomes switch between states; among the progeny of a single cell, a given X chromosome exhibits each state with equal frequency. We propose a model in which the concerted switching of homologous X chromosomes between mutually exclusive future active and future inactive states provides the basis for the apparently random silencing of one X chromosome in female cells.

Animals↗
PubMed2000

PCGEM1, a prostate-specific gene, is overexpressed in prostate cancer.

A prostate-specific gene, PCGEM1, was identified by differential display analysis of paired normal and prostate cancer tissues. Multiple tissue Northern blot analysis revealed that PCGEM1 was expressed exclusively in human prostate tissue. Analysis of PCGEM1 expression in matched normal and primary tumor specimens revealed tumor-associated overexpression in 84% of patients with prostate cancer by in situ hybridization assay and in 56% of patients by reverse transcription-PCR assay. Among various prostate cancer cell lines analyzed, PCGEM1 expression was detected only in the androgen receptor-positive cell line LNCaP. Extensive DNA sequence analysis of the PCGEM1 cDNA and genomic DNA revealed that PCGEM1 lacks protein-coding capacity and suggests that it may belong to an emerging class of noncoding RNAs, also called "riboregulators." The PCGEM1 locus was mapped to chromosome 2q32. Taken together, the remarkable prostate-tissue specificity and androgen-dependent expression of PCGEM1 as well as its elevated expression in a significant percentage of tumor tissues suggest specific functions of PCGEM1 in the biology and tumorigenesis of the prostate gland.

Androgens↗
PubMed2004

The spinocerebellar ataxia 8 noncoding RNA causes neurodegeneration and associates with staufen in Drosophila.

Spinocerebellar Ataxia 8 (SCA8) appears unique among triplet repeat expansion-induced neurodegenerative diseases because the predicted gene product is a noncoding RNA. Little is currently known about the normal function of SCA8 in neuronal survival or how repeat expansion contributes to neurodegeneration. To investigate the molecular context in which SCA8 operates, we have expressed the human SCA8 noncoding RNA in Drosophila. SCA8 induces late-onset, progressive neurodegeneration in the Drosophila retina. Using this neurodegenerative phenotype as a sensitized background for a genetic modifier screen, we have identified mutations in four genes: staufen, muscle-blind, split ends, and CG3249. All four encode neuronally expressed RNA binding proteins conserved in Drosophila and humans. Although expression of both wild-type and repeat-expanded SCA8 induce neurodegeneration, the strength of interaction with certain modifiers differs between the two SCA8 backgrounds, suggesting that CUG expansions alter associations with specific RNA binding proteins. Our demonstration that SCA8 can recruit Staufen and that the interaction domain maps to the portion of the SCA8 RNA that undergoes repeat expansion in the human disease suggests a specific mechanism for SCA8 function and disease. Genetic modifiers identified in our SCA8-based screens may provide candidates for designing therapeutic interventions to treat this disease.

Animals↗
PubMed2001

CTCF, a candidate trans-acting factor for X-inactivation choice.

In mammals, X-inactivation silences one of two female X chromosomes. Silencing depends on the noncoding gene, Xist (inactive X-specific transcript), and is blocked by the antisense gene, Tsix. Deleting the choice/imprinting center in Tsix affects X-chromosome selection. Here, we identify the insulator and transcription factor, CTCF, as a candidate trans-acting factor for X-chromosome selection. The choice/imprinting center contains tandem CTCF binding sites that function in an enhancer-blocking assay. In vitro binding is reduced by CpG methylation and abolished by including non-CpG methylation. We postulate that Tsix and CTCF together establish a regulatable epigenetic switch for X-inactivation.

Animals↗
PubMed2006

A transient heterochromatic state in Xist preempts X inactivation choice without RNA stabilization.

X chromosome inactivation (XCI) depends on a noncoding sense-antisense transcript pair, Xist and Tsix. At the onset of XCI, Xist RNA accumulates on one of two Xs, coating and silencing the chromosome in cis. The molecular basis for monoallelic Xist upregulation is not known, though evidence predominantly supports a posttranscriptional mechanism through RNA stabilization. Here, we test whether Tsix RNA destabilizes Xist RNA. Unexpectedly, we find that Xist upregulation is not based on transcript stabilization at all but is instead controlled by transcription in a sex-specific manner. Tsix directly regulates its transcription. On the future inactive X, Tsix downregulation induces a transient heterochromatic state in Xist, followed paradoxically by high-level Xist expression. A Tsix-deficient X chromosome adopts the heterochromatic state in pre-XCI cells. This state persists through XCI establishment and "reverts" to a euchromatic state during XCI maintenance. We have therefore identified chromatin marks that preempt and predict asymmetric Xist expression.

Animals↗
PubMed2004

Regulation of nuclear receptor activity by a pseudouridine synthase through posttranscriptional modification of steroid receptor RNA activator.

Nuclear receptors (NRs) induce transcription through association with coactivator complexes. We identified a pseudouridine synthase (PUS), mPus1p, as a coactivator for retinoic acid receptor (mRAR)gamma and other NR-dependent transactivation. mPus1p is a member of the truA subfamily of PUSs, a class of enzymes that isomerize uridine to pseudouridine in noncoding RNAs, such as tRNA, to ensure proper folding and function. mPus1p binds the first zinc finger of mRARgamma and also associates with other NRs. Interestingly, mPus1p pseudouridylates coactivator Steroid Receptor RNA Activator (SRA), and when coexpressed, mPus1p and SRA cooperatively enhance mRARgamma-mediated transcription. mPus1p, mRARgamma, and SRA exist in a retinoid-independent, promoter bound complex in the nucleus although mPus1p is also expressed in the nucleolus, where it likely modifies tRNA. Finally, we show that mPus1p-coactivator function required SRA, mPus1p-associated mRARgamma binding, and PUS activities. mPus1p-dependent pseudouridylation of SRA represents an additional type of posttranscriptional modification of a NR-coactivator complex that is important for NR signaling.

Animals↗
PubMed2004

DLEU2 encodes an antisense RNA for the putative bicistronic RFP2/LEU5 gene in humans and mouse.

Our group previously identified two novel genes, RFP2/LEU5 and DLEU2, within a 13q14.3 genomic region of loss seen in various malignancies. However, no specific inactivating mutations were found in these or other genes in the vicinity of the deletion, suggesting that a nonclassical tumor-suppressor mechanism may be involved. Here, we present data showing that the DLEU2 gene encodes a putative noncoding antisense RNA, with one exon directly overlapping the first exon of the RFP2/LEU5 gene in the opposite orientation. In addition, the RFP2/LEU5 transcript can be alternatively spliced to produce either several monocistronic transcripts or a putative bicistronic transcript encoding two separate open-reading frames, adding to the complexity of the locus. The finding that these gene structures are conserved in the mouse, including the putative bicistronic RFP2/LEU5 transcript as well as the antisense relationship with DLEU2, further underlines the significance of this unusual organization and suggests a biological function for DLEU2 in the regulation of RFP2/LEU5.

Alternative Splicing↗
PubMed2004

Interaction between differentially methylated regions partitions the imprinted genes Igf2 and H19 into parent-specific chromatin loops.

Imprinted genes are expressed from only one of the parental alleles and are marked epigenetically by DNA methylation and histone modifications. The paternally expressed gene insulin-like growth-factor 2 (Igf2) is separated by approximately 100 kb from the maternally expressed noncoding gene H19 on mouse distal chromosome 7. Differentially methylated regions in Igf2 and H19 contain chromatin boundaries, silencers and activators and regulate the reciprocal expression of the two genes in a methylation-sensitive manner by allowing them exclusive access to a shared set of enhancers. Various chromatin models have been proposed that separate Igf2 and H19 into active and silent domains. Here we used a GAL4 knock-in approach as well as the chromosome conformation capture technique to show that the differentially methylated regions in the imprinted genes Igf2 and H19 interact in mice. These interactions are epigenetically regulated and partition maternal and paternal chromatin into distinct loops. This generates a simple epigenetic switch for Igf2 through which it moves between an active and a silent chromatin domain.

Animals↗
PubMed2005

A strategy for probing the function of noncoding RNAs finds a repressor of NFAT.

Noncoding RNA molecules (ncRNAs) have been implicated in numerous biological processes including transcriptional regulation and the modulation of protein function. Yet, in spite of the apparent abundance of ncRNA, little is known about the biological role of the projected thousands of ncRNA genes present in the human genome. To facilitate functional analysis of these RNAs, we have created an arrayed library of short hairpin RNAs (shRNAs) directed against 512 evolutionarily conserved putative ncRNAs and, via cell-based assays, we have begun to determine their roles in cellular pathways. Using this system, we have identified an ncRNA repressor of the nuclear factor of activated T cells (NFAT), which interacts with multiple proteins including members of the importin-beta superfamily and likely functions as a specific regulator of NFAT nuclear trafficking.

Animals↗
PubMed2006

Expression of a modified H19 RNA does not cause embryonic lethality in mice.

The imprinted H19 gene produces a noncoding RNA of unknown function. Targeted and transgenic mouse mutations have shown that this RNA can be deleted and overexpressed without adverse effect. Yet one mutation of the H19 gene displayed an embryonic lethal phenotype in the mouse--the expression of an RNA modified by a short insertion near the 5' end of the transcript (H19(Xba) allele). Expression of this RNA in transgenic mice conferred lethality at day 14 of development. The potential for this mutant to elucidate the function of the H19 RNA supported further investigation of the H19(Xba) phenotype. Since all H19(Xba) transgenic founders died as embryos, an experiment was designed to generate H19(Xba)-expressing mice that could be maintained as an established line. This strategy took advantage of the maternal-specific expression of H19, passing an H19(Xba) knockin allele silently through males and transferring it to females only to generate animals for study. Surprisingly, H19(Xba) knockin mice are fully viable, whether the H19(Xba) allele is inherited paternally or maternally. Experiments to reproduce the original transgene-based lethality were also performed and yielded live-born transgene-expressing animals. These data demonstrate that, contrary to published reports, expression of the H19(Xba) RNA does not cause embryonic lethality in mice.

Animals↗
PubMed1995

XIST expression is repressed when X inactivation is reversed in human placental cells: a model for study of XIST regulation.

Considerable evidence suggests that the X inactive transcript gene, XIST/Xist, has a role in the initial steps of X chromosome inactivation in the female mammalian embryo. It is transcribed exclusively from inactive X chromosomes, and its noncoding transcript seems to be essential for cis inactivation. Unexpected for a developmental gene, XIST continues to be expressed in adult somatic cells. To determine the effect of reversal of inactivation on the expression of XIST, we studied human X chromosomes that had been induced to reverse X inactivation by hybridization of chorionic villi cells from term placentas with mouse A9 cells. In nine hybrids with a reactivated X chromosome, XIST was either not expressed or expressed much less than the locus on the inactive X chromosome in the chorionic villi cells from which they were derived. The repressibility of XIST by reversal of inactivation in these placental cells mirrors events that occur during the ontogeny of oocytes and indicates that the locus is subject to regulation in somatic cells long after inactivation is established in the embryo. The small residual XIST activity from these active chromosomes suggests that low levels of XIST expression do not interfere with chromosome activity and raises the possibility that the induction of cis inactivation requires a certain level of XIST transcription. The chorionic villi hybrids provide an experimental system to study the developmental regulation of XIST.

Base Sequence↗
PubMed2005

H19 mRNA-like noncoding RNA promotes breast cancer cell proliferation through positive control by E2F1.

The imprinted H19 gene has riboregulatory functions. We show here that H19 transcription is up-regulated during the S-phase of growth-stimulated cells and that the H19 promoter is activated by E2F1 in breast cancer cells. H19 repression by pRb and E2F6 confirms the E2F1-dependent control of the H19 promoter. Consistently, we demonstrate by chromatin immunoprecipitation assays that endogenous E2F1 is recruited to the H19 promoter in vivo. The functionality of E2F promoter sites was further confirmed by gel shift and mutagenesis experiments, revealing that these sites are required for binding and promoter response to E2F1 exogenous expression and serum stimulation. Furthermore, we show that H19 overexpression confers a growth advantage on breast cancer cells released from growth arrest as well as in asynchronously growing cells. The H19 knockdown by small interfering RNA duplexes impedes S-phase entry in both wild-type and stably H19-transfected cells. Based on these findings, we conclude that the H19 RNA is actively linked to E2F1 to promote cell cycle progression of breast cancer cells. This clearly supports the H19 oncogenic function in breast tumor genesis.

Base Sequence↗
PubMed2006

Nuclear mRNA degradation pathway(s) are implicated in Xist regulation and X chromosome inactivation.

A critical step in X-chromosome inactivation (XCI), which results in the dosage compensation of X-linked gene expression in mammals, is the coating of the presumptive inactive X chromosome by the large noncoding Xist RNA, which then leads to the recruitment of other factors essential for the heterochromatinisation of the inactive X and its transcriptional silencing. In an approach aimed at identifying genes implicated in the X-inactivation process by comparative transcriptional profiling of female and male mouse gastrula, we identified the Eif1 gene involved in translation initiation and RNA degradation. We show here that female embryonic stem cell lines, silenced by RNA interference for the Eif1 gene, are unable to form Xist RNA domains upon differentiation and fail to undergo X-inactivation. To probe further an effect involving RNA degradation pathways, the inhibition by RNA interference of Rent1, a factor essential for nonsense-mediated decay and Exosc10, a specific nuclear component of the exosome, was analysed and shown to similarly impair Xist upregulation and XCI. In Eif1-, Rent1-, and Exosc10-interfered clones, Xist spliced form(s) are strongly downregulated, while the levels of unspliced form(s) of Xist and the stability of Xist RNA remain comparable to that of the control cell lines. Our data suggests a role for mRNA nuclear degradation pathways in the critical regulation of spliced Xist mRNA levels and the onset of the X-inactivation process.

Alternative Splicing↗
PubMed2006

Tsix defective in splicing is competent to establish Xist silencing.

Dosage differences of X-linked genes between male and female mammals are compensated for by a mechanism known as X-inactivation, and the noncoding Xist gene plays a crucial role in this process. The expression of Xist is regulated in cis by its noncoding antisense gene, Tsix, whose transcripts (though a fraction of them stay unspliced), are processed like common proteincoding RNAs. It has been suggested that certain classes of sense-antisense pairs of RNA are causally involved in not only gene regulation but also higher order chromatin structure in various organisms. In fact, recent studies demonstrated that Tsix modulates Xist expression through modification of the chromatin structure. It is still unknown, however, whether the RNA product is important for the function of Tsix or whether the antisense transcription is sufficient. To obtain insight into this issue, we eliminated the splicing products of Tsix in the mouse and explored the effects of this elimination on Tsix-mediated Xist silencing. To our surprise, the Xist locus was stably repressed on the X carrying the splicing-defective Tsix allele. Moreover, the repressive chromatin configuration was properly established at the Xist locus. These unexpected results indicate that the splicing products are dispensable for Tsix-mediated Xist silencing.

Animals↗
PubMed2006

Rapid identification of UCA1 as a very sensitive and specific unique marker for human bladder carcinoma.

PURPOSE: The most common genitourinary malignancy in China is bladder transitional cell carcinoma (TCC). Early diagnosis of new and recurrent bladder cancers, followed by timely treatment, will help decrease mortality. There are currently no satisfactory markers for bladder cancer available in clinics. Better diagnostic methods are highly demanded. EXPERIMENTAL DESIGN: In this research, we have used comprehensive expressed sequence tag analysis, serial analysis of gene expression, and microarray analysis and quickly discovered a candidate marker, urothelial carcinoma associated 1 (UCA1). The UCA1 gene was characterized and its performance as a urine marker was analyzed by reverse transcription-PCR with urine sediments. A total of 212 individuals were included in this study, 94 having bladder cancers, 33 ureter/pelvic cancers, and 85 normal and other urinary tract disease controls. RESULTS: UCA1 was identified as a novel noncoding RNA gene dramatically up-regulated in TCC and it is the most TCC-specific gene yet identified. The full-length cDNA was 1,439 bp, and sequence analysis showed that it belonged to the human endogenous retrovirus H family. Clinical tests showed that UCA1 assay was highly specific (91.8%, 78 of 85) and very sensitive (80.9%, 76 of 94) in the diagnosis of bladder cancer and was especially valuable for superficial G2-G3 patients (sensitivity 91.1%, 41 of 45). It showed excellent differential diagnostic performance in various urinary tract diseases without TCC. CONCLUSIONS: UCA1 is a very sensitive and specific unique marker for bladder cancer. It could have important implications in postoperative noninvasive follow-up. This research also highlights a shortcut to new cancer diagnostic assays through integration of in silico isolation methods with translational clinical tests based on RNA detection protocols.

Biomarkers, Tumor↗