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PubMed2002

An ectopic human XIST gene can induce chromosome inactivation in postdifferentiation human HT-1080 cells.

It has been believed that XIST RNA requires a discrete window in early development to initiate the series of chromatin-remodeling events that form the heterochromatic inactive X chromosome. Here we investigate four adult male HT-1080 fibrosarcoma cell lines expressing ectopic human XIST and demonstrate that these postdifferentiation cells can undergo chromosomal inactivation outside of any normal developmental context. All four clonal lines inactivated the transgene-containing autosome to varying degrees and with variable stability. One clone in particular consistently localized the ectopic XIST RNA to a discrete chromosome territory that exhibited striking hallmarks of inactivation, including long-range transcriptional inactivation. Results suggest that some postdifferentiation cell lines are capable of de novo chromosomal inactivation; however, long-term retention of autosomal inactivation was less common, which suggests that autosomal inactivation may confer a selective disadvantage. These results have fundamental significance for understanding genomic programming in early development.

Acetylation↗
PubMed2001

Identification of an autoimmune serum containing antibodies against the Barr body.

Transcriptional inactivation of one X chromosome in mammalian female somatic cells leads to condensation of the inactive X chromosome into the heterochromatic sex chromatin, or Barr body. Little is known about the molecular composition and structure of the Barr body or the mechanisms leading to its formation in female nuclei. Because human sera from patients with autoimmune diseases often contain antibodies against a variety of cellular components, we reasoned that some autoimmune sera may contain antibodies against proteins associated with the Barr body. Therefore, we screened autoimmune sera by immunofluorescence of human fibroblasts and identified one serum that immunostained a distinct nuclear structure with a size and nuclear localization consistent with the Barr body. The number of these structures was consistent with the number of Barr bodies expected in diploid female fibroblasts containing two to five X chromosomes. Immunostaining with the serum followed by fluorescence in situ hybridization with a probe against XIST RNA demonstrated that the major fluorescent signal from the autoantibody colocalized with XIST RNA. Further analysis of the serum showed that it stains human metaphase chromosomes and a nuclear structure consistent with the inactive X in female mouse fibroblasts. However, it does not exhibit localization to a Barr body-like structure in female mouse embryonic stem cells or in cells from female mouse E7.5 embryos. The lack of staining of the inactive X in cells from female E7.5 embryos suggests the antigen(s) may be involved in X inactivation at a stage subsequent to initiation of X inactivation. This demonstration of an autoantibody recognizing an antigen(s) associated with the Barr body presents a strategy for identifying molecular components of the Barr body and examining the molecular basis of X inactivation.

Animals↗
PubMed2001

A functional role for Tsix transcription in blocking Xist RNA accumulation but not in X-chromosome choice.

In female mammals, up-regulation of Xist triggers X-chromosome inactivation in cis. Up-regulation is inhibited by sequences 3' to Xist contained within the antisense locus, Tsix. Inhibition could depend on transcription of Tsix and/or on DNA elements therein. Here we test the role of Tsix transcription by augmenting the duration and strength of Tsix expression. We find that Tsix hypertranscription is sufficient to block Xist RNA accumulation in a cis-limited manner. We propose that Tsix transcription is necessary to restrict Xist activity on the future active X and, conversely, that Tsix repression is required for Xist RNA accumulation on the future inactive X. We also find that Tsix hypertranscription does not affect X-chromosome choice. Thus, choice is mediated by elements within Tsix that are independent of promoter activity.

Animals↗
PubMed2002

Distinct RNA motifs are important for coactivation of steroid hormone receptors by steroid receptor RNA activator (SRA).

Steroid receptor RNA activator (SRA) is an RNA transcript that functions as a eukaryotic transcriptional coactivator for steroid hormone receptors. We report here the isolation and functional characterization of distinct RNA substructures within the SRA molecule that constitute its coactivation function. We used comparative sequence analysis and free energy calculations to systematically study SRA RNA subdomains for identification of structured regions and base pairings, and we used site-directed mutagenesis to assess their functional consequences. Together with genetic deletion analysis, this approach identified six RNA motifs in SRA important for coactivation. Because all nucleotide changes in the mutants that disrupted SRA function were silent mutations presumed not to alter deduced encoded amino acid sequence, our analysis provides strong evidence that SRA-mediated coactivation is executed by distinct RNA motifs and not by an encoded protein.

Animals↗
PubMed1994

Rodent BC1 RNA gene as a master gene for ID element amplification.

ID elements are short interspersed repetitive DNA elements (SINEs) which have amplified in rodent genomes via retroposition, a process involving an RNA intermediate. BC1, an abundant ID-related transcript, is transcribed from a conserved, single-copy gene in rodents. The gene encoding BC1 RNA represents one of the earliest and possibly the first ID-containing sequence. Comparison of consensus sequences of each rodent ID with its corresponding BC1 RNA gene showed that the variations of BC1 RNA within rodents corresponded to specific changes within the ID consensus sequence for each rodent species. This supports the hypothesis that the BC1 gene is a master gene responsible for the amplification and evolution of ID elements. The rat ID family consists of at least four subfamilies, with the oldest subfamily having been derived from the BC1 RNA. The other three subfamilies appear to have been derived from a new master gene(s), which has been responsible for the large increase in ID element copy number within the rat genome. We have found that the guinea pig genome contains two copies of the BC1 gene, apparently the result of a DNA-mediated duplication event. Both of these guinea pig BC1 genes have a conserved TATA-like element in the 5' flanking region and have contributed to guinea pig ID amplifications.

Animals↗
PubMed1995

Characterization of the promoter region of the mouse Xist gene.

The mouse Xist gene is expressed exclusively from the inactive X chromosome and may be implicated in initiating X inactivation. To better understand the mechanisms underlying the control of Xist expression, we investigated the upstream regulatory region of the mouse Xist promoter. A 1.2-kb upstream region of the Xist gene was sequenced and promoter activity was studied by chloramphenicol acetyltransferase (CAT) assays after transfection in murine XX and XY cell lines. The region analyzed (-1157 to +917 showed no in vitro sex-specific promoter activity. However, a minimal constitutional promoter was assigned to a region from -81 to +1, and a cis element from -41 to -15 regulates promoter activity. We showed that a nuclear factor binds to an element located at -30 to -25 (TTAAAG). A second sequence at -41 to -15 does not act as an enhancer and is unable to confer transcriptional activity to the Xist gene on its own. A third region from -82 to -41 is needed for correct expression. Deletion of the segment -441 to -231 is associated with an increase in CAT activity and may represent a silencer element.

3T3 Cells↗
PubMed1995

Xce haplotypes show modified methylation in a region of the active X chromosome lying 3' to Xist.

During early mammalian embryogenesis, one of the two X chromosomes in somatic cells of the female becomes inactivated through a process that is thought to depend on a unique initiator region, the X-chromosome inactivation center (Xic). The recently characterized Xist sequence (X-inactive-specific transcript) is thought to be a possible candidate for Xic. In mice a further genetic element, the X chromosome-controlling element (Xce), is also known to influence the choice of which of the two X chromosomes is inactivated. We report that a region of the mouse X chromosome lying 15 kb distal to Xist contains several sites that show hypermethylation specifically associated with the active X chromosome. Analysis of this region in various Xce strains has revealed a correlation between the strength of the Xce allele carried and the methylation status of this region. We propose that such a region could be involved in the initial stages of the inactivation process and in particular in the choice of which of the two X chromosomes present in a female cell will be inactivated.

Animals↗
PubMed1996

The structural H19 gene is required for transgene imprinting.

The product of the H19 gene is an untranslated RNA that is expressed exclusively from the maternal chromosome during mammalian development. The H19 gene and its 5'-flanking sequence are required for the genomic imprinting of two paternally expressed genes, Ins-2 (encodes insulin-2) and Igf-2 (encodes insulin-like growth factor-2), that lie 90 and 115 kb 5' to the H19 gene, respectively. In this report, the role of the H19 gene in its own imprinting is investigated by introducing a Mus spretus H19 gene into heterologous locations in the mouse genome. Multiple copies of the transgene were sufficient for its paternal silencing and DNA methylation. Replacing the H19 structural gene with a luciferase reporter gene resulted in loss of imprinting of the transgene. That is, high expression and low levels of DNA methylation were observed upon both paternal and maternal inheritance. The removal of 701 bp at the 5' end of the structural gene resulted in a similar loss of paternal-specific DNA methylation, arguing that those sequences are required for both the establishment and maintenance of the sperm-specific gametic mark. The M. spretus H19 transgene could not rescue the loss of Igf-2 imprinting in trans in H19 deletion mice, implying a cis requirement for the H19 gene. In contrast to a previous report in which overexpression of a marked H19 gene was a prenatal lethal, expression of the M. spretus transgene had no deleterious effect, leading to the conclusion that the 20-base insertion in the marked gene created a neomorphic mutation.

Animals↗
PubMed1997

Epigenetic changes at the insulin-like growth factor II/H19 locus in developing kidney is an early event in Wilms tumorigenesis.

Relaxation of imprinting at the insulin-like growth factor II (IFG-II)/H19 locus is a major mechanism involved in the onset of sporadic Wilms tumor and several other embryonal tumors. The high prevalence of histologically abnormal foci in kidney adjacent to Wilms tumors suggests that tumor-predisposing genetic/epigenetic lesion might also be found at high frequency in Wilms tumor-bearing kidneys. Focusing on Wilms tumors with relaxation of IFG-II imprinting, we determined the frequency of epigenetic change at the IFG-II/H19 locus in adjacent kidney. In all kidneys adjacent to these Wilms tumors, we detected substantial mosaicism for a population of cells with relaxation of IFG-II imprinting and biallelic H19 methylation, regardless of whether the patient had a tumor-predisposing syndrome or not. The high proportion of epigenetically modified cells among "normal" tissue indicates that the epigenetic error occurred very early in development, before the onset of Wilms tumor. Not only does this suggest that the major Wilms tumor-predisposing event occurs within the first few days of development, but it also suggests that sporadic Wilms tumor may represent one end of a spectrum of overgrowth disorders characterized by mosaic epigenetic change at the IFG-II/H19 locus.

Alleles↗
PubMed1997

Loss of the maternal H19 gene induces changes in Igf2 methylation in both cis and trans.

Recent investigations have shown that the maintenance of genomic imprinting of the murine insulin-like growth factor 2 (Igf2) gene involves at least two factors: the DNA (cytosine-5-)-methyltransferase activity, which is required to preserve the paternal specific expression of Igf2, and the H19 gene (lying 90 kb downstream of Igf2 gene), which upon inactivation leads to relaxation of the Igf2 imprint. It is not yet clear how these two factors are related to each other in the process of maintenance of Igf2 imprinting and, in particular, whether the latter is acting through cis elements or whether the H19 RNA itself is involved. By using Southern blots and the bisulfite genomic-sequencing technique, we have investigated the allelic methylation patterns (epigenotypes) of the Igf2 gene in two strains of mouse with distinct deletions of the H19 gene. The results show that maternal transmission of H19 gene deletions leads the maternal allele of Igf2 to adopt the epigenotype of the paternal allele and indicate that this phenomenon is influenced directly or indirectly by the H19 gene expression. More importantly, the bisulfite genomic-sequencing allowed us to show that the methylation pattern of the paternal allele of the Igf2 gene is affected in trans by deletions of the active maternal allele of the H19 gene. Selection during development for the appropriate expression of Igf2, dosage-dependent factors that bind to the Igf2 gene, or methylation transfer between the parental alleles could be involved in this trans effect.

Alleles↗
PubMed1998

Identification of the Meg1/Grb10 imprinted gene on mouse proximal chromosome 11, a candidate for the Silver-Russell syndrome gene.

In a systematic screen for maternally expressed imprinted genes using subtraction hybridization with androgenetic and normal fertilized mouse embryos, seven candidate maternally expressed genes (Megs) have been isolated, including the H19 and p57(Kip2) genes that are known to be maternally expressed. Herein, we demonstrate that an imprinted gene, Meg1, is apparently identical to Grb10 (growth factor receptor-bound protein 10), which is located on mouse proximal chromosome 11. Grb10 protein was reported to bind to the insulin receptor and/or the insulin-like growth factor (IGF) I receptor via its src homology 2 domain and to inhibit the associated tyrosine kinase activity that is involved in the growth promoting activities of insulin and IGFs (IGF-I and -II). Thus, it is probable that Meg1/Grb10 is responsible for the imprinted effects of prenatal growth retardation or growth promotion caused by maternal or paternal duplication of proximal chromosome 11 with reciprocal deficiencies (MatDp.prox11 or PatDp.prox11), respectively. In the human, it has been reported that the maternal uniparental disomy 7 is responsible for the Silver-Russell syndrome (SRS) whose effects include pre- and postnatal growth retardation and other dysmorphologies. The human homologue GRB10 on chromosome 7q11.2-12 is a candidate gene for Silver-Russell syndrome.

Animals↗
PubMed1998

Reactivation of XIST in normal fibroblasts and a somatic cell hybrid: abnormal localization of XIST RNA in hybrid cells.

The XIST gene, expressed only from the inactive X chromosome, is a critical component of X inactivation. Although apparently unnecessary for maintenance of inactivation, XIST expression is thought to be sufficient for inactivation of genes in cis even when XIST is located abnormally on another chromosome. This repression appears to involve the association of XIST RNA with the chromosome from which it is expressed. Reactivated genes on the inactive X chromosome, however, maintain expression in several somatic cell hybrid lines with stable expression of XIST. We describe here another example of an XIST-expressing human-hamster hybrid that lacks X-linked gene repression in which the human XIST gene present on an active X chromosome was reactivated by treatment with 5-aza-2'-deoxycytidine. These data raise the possibility that human XIST RNA does not function properly in human-rodent somatic cell hybrids. As part of our approach to address this question, we reactivated the XIST gene in normal male fibroblasts and then compared their patterns of XIST RNA localization by subcellular fractionation and in situ hybridization with those of hybrid cells. Although XIST RNA is nuclear in all cell types, we found that the in situ signals are much more diffuse in hybrids than in human cells. These data suggest that hybrids lack components needed for XIST localization and, presumably, XIST-mediated gene repression.

12E7 Antigen↗
PubMed1999

A new structure for the murine Xist gene and its relationship to chromosome choice/counting during X-chromosome inactivation.

In this report, we present structural data for the murine Xist gene. The data presented in this paper demonstrate that the murine Xist transcript is at least 17.4 kb, not 14.3 kb as previously reported. The new structure of the murine Xist gene described herein has seven exons, not six. Exon VII encodes an additional 3.1 kb of information at the 3' end. Exon VII contains seven possible sites for polyadenylation; four of these sites are located in the newly discovered 3' end. Consequently, it is possible that several distinct transcripts may be produced through differential polyadenylation of a primary transcript. Alternative use of polyadenylation signals could result in size changes for exon VII. Two major species of Xist are detectable by Northern analysis, consistent with differential polyadenylation. In this paper, we propose a model for the role of the Xist 3' end in the process of X-chromosome counting and choice during embryonic development.

3' Untranslated Regions↗
PubMed1999

Human XIST yeast artificial chromosome transgenes show partial X inactivation center function in mouse embryonic stem cells.

Initiation of X chromosome inactivation requires the presence, in cis, of the X inactivation center (XIC). The Xist gene, which lies within the XIC region in both human and mouse and has the unique property of being expressed only from the inactive X chromosome in female somatic cells, is known to be essential for X inactivation based on targeted deletions in the mouse. Although our understanding of the developmental regulation and function of the mouse Xist gene has progressed rapidly, less is known about its human homolog. To address this and to assess the cross-species conservation of X inactivation, a 480-kb yeast artificial chromosome containing the human XIST gene was introduced into mouse embryonic stem (ES) cells. The human XIST transcript was expressed and could coat the mouse autosome from which it was transcribed, indicating that the factors required for cis association are conserved in mouse ES cells. Cis inactivation as a result of human XIST expression was found in only a proportion of differentiated cells, suggesting that the events downstream of XIST RNA coating that culminate in stable inactivation may require species-specific factors. Human XIST RNA appears to coat mouse autosomes in ES cells before in vitro differentiation, in contrast to the behavior of the mouse Xist gene in undifferentiated ES cells, where an unstable transcript and no chromosome coating are found. This may not only reflect important species differences in Xist regulation but also provides evidence that factors implicated in Xist RNA chromosome coating may already be present in undifferentiated ES cells.

Animals↗
PubMed1999

Chromatin structure analysis of the mouse Xist locus.

The Xist gene is expressed exclusively from the inactive X chromosome and plays a central role in regulating X chromosome inactivation. Here we describe experiments aimed at defining the extent of the active chromatin domain of the expressed Xist allele. By using an allele-specific general DNaseI sensitivity assay we show that there is preferential digestion of the expressed allele at sites within the transcribed locus but not in flanking sites located up to 70 kb 5'. A putative proximal boundary for the Xist domain is located within 10 kb upstream of promoter P1. Chromatin in the expressed domain was found to be acetylated at H4 in XX somatic cells but also in XY cells, where Xist is never expressed. A single clear exception to this was the Xist promoter, which is acetylated only in XX cells. These observations concur with the view that H4 acetylation may not be a general marker of active chromatin domains and further support data implicating local promoter acetylation as being of primary functional significance in vivo.

Acetylation↗
PubMed1999

A silencer element identified in Drosophila is required for imprinting of H19 reporter transgenes in mice.

The H19 gene is subject to genomic imprinting because it is methylated and repressed after paternal inheritance and is unmethylated and expressed after maternal inheritance. We recently identified a 1.1-kb control element in the upstream region of the H19 gene that functions as a cis-acting silencer element in Drosophila. Here we investigate the function of this element in mice. We demonstrate that both H19-lacZ and H19-PLAP reporter transgenes can undergo imprinting with repression and hypermethylation after paternal transmission at many integration sites. However, transgenes that were deleted for the 1.1-kb silencer element showed loss of paternal repression, but they did not show marked changes in the paternal methylation of the remaining upstream region. This study demonstrates that the 1.1-kb control element identified in Drosophila is required to silence paternally transmitted H19 minitransgenes in mice.

Alkaline Phosphatase↗
PubMed1999

Enhancer competition between H19 and Igf2 does not mediate their imprinting.

The linked H19 and Igf2 genes on mouse distal chromosome 7 are subject to genomic imprinting. Competition between the promoters of the genes for transcription from shared enhancers has been proposed as an explanation for the coordinate expression and reciprocal imprinting of these two genes. To test this model, we have used Cre-loxP technology to generate in mice a conditional deletion of the H19 promoter and structural gene that leaves no transcription unit in the locus. Contrary to the prediction of enhancer competition we find that transcriptional activity from the H19 promoter is not required for the imprinted silencing of the Igf2 gene.

Alleles↗