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Huntington F. Willard - One of the best experts on this subject based on the ideXlab platform.
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an ectopic human XIST Gene can induce chromosome inactivation in postdifferentiation human ht 1080 cells
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Lisa L Hall, Huntington F. Willard, Meg Byron, Kosuke Sakai, Laura Carrel, Jeanne B LawrenceAbstract: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.
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a promoter mutation in the XIST Gene in two unrelated families with skewed x chromosome inactivation
Nature Genetics, 1997Co-Authors: Robert M Plenge, Charles E Schwartz, Anna K. Naumova, B D Hendrich, J F Arena, Carmen Sapienza, R M Winter, Huntington F. WillardAbstract:X-chromosome inactivation is the process by which a cell recognizes the presence of two copies of an X chromosome early in the development of XX embryos and chooses one to be active and one to be inactive1. Although it is commonly believed that the initiation of X inactivation is random, with an equal probability (50:50) that either X chromosome will be the inactive X in a given cell, significant variation in the proportion of cells with either X inactive is observed both in mice heterozygous for alleles at the Xce locus2 and among normal human females in the population3–5. Families in which multiple females demonstrate extremely skewed inactivation patterns that are otherwise quite rare in the General population are thought to reflect possible Genetic influences on the X-inactivation process5–7. Here we report a rare cytosine to guanine mutation in the XIST minimal promoter that underlies both epiGenetic and functional differences between the two X chromosomes in nine females from two unrelated families. All females demonstrate preferential inactivation of the X chromosome carrying the mutation, suggesting that there is an association between alterations in the regulation of XIST expression and X-chromosome inactivation.
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identification and characterization of the human XIST Gene promoter implications for models of x chromosome inactivation
Nucleic Acids Research, 1997Co-Authors: Brian Hendrich, Robert M Plenge, Huntington F. WillardAbstract:The XIST Gene in both humans and mice is expressed exclusively from the inactive X chromosome and is required for X chromosome inactivation to occur early in development. In order to understand transcriptional regulation of the XIST Gene, we have identified and characterized the human XIST promoter and two repeated DNA elements that modulate promoter activity. As determined by reporter Gene constructs, the XIST minimal promoter is constitutively active at high levels in human male and female cell lines and in transgenic mice. We demonstrate that this promoter activity is dependent in vitro upon binding of the common transcription factors SP1, YY1 and TBP. We further identify two cis -acting repeated DNA sequences that influence reporter Gene activity. First, DNA fragments containing a set of highly conserved repeats located within the 5'-end of XIST stimulate reporter activity 3-fold in transiently transfected cell lines. Second, a 450 bp alternating purine-pyrimidine repeat located 25 kb upstream of the XIST promoter partially suppresses promoter activity by approximately 70% in transient transfection assays. These results indicate that the XIST promoter is constitutively active and that critical steps in the X inactivation process must involve silencing of XIST on the active X chromosome by factors that interact with and/or recognize sequences located outside the minimal promoter.
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XIST rna paints the inactive x chromosome at interphase evidence for a novel rna involved in nuclear chromosome structure
Journal of Cell Biology, 1996Co-Authors: Christine Moulton Clemson, Huntington F. Willard, John Mcneil, Jeanne B LawrenceAbstract:The XIST Gene is implicated in X chromosome inactivation, yet the RNA contains no apparent open reading frame. An accumulation of XIST RNA is observed near its site of transcription, the inactive X chromosome (Xi). A series of molecular cytoGenetic studies comparing properties of XIST RNA to other protein coding RNAs, support a critical distinction for XIST RNA; XIST does not concentrate at Xi simply because it is transcribed and processed there. Most notably, morphometric and 3-D analysis reveals that XIST RNA and Xi are coincident in 2- and 3-D space; hence, the XIST RNA essentially paints Xi. Several results indicate that the XIST RNA accumulation has two components, a minor one associated with transcription and processing, and a spliced major component, which stably associates with Xi. Upon transcriptional inhibition the major spliced component remains in the nucleus and often encircles the extra-prominent heterochromatic Barr body. The continually transcribed XIST Gene and its polyadenylated RNA consistently localize to a nuclear region devoid of splicing factor/poly A RNA rich domains. XIST RNA remains with the nuclear matrix fraction after removal of chromosomal DNA. XIST RNA is released from its association with Xi during mitosis, but shows a unique highly particulate distribution. Collective results indicate that XIST RNA may be an architectural element of the interphase chromosome territory, possibly a component of nonchromatin nuclear structure that specifically associates with Xi. XIST RNA is a novel nuclear RNA which potentially provides a specific precedent for RNA involvement in nuclear structure and cis-limited Gene regulation via higher-order chromatin packaging.
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Direct Detection of Non-Random X Chromosome Inactivation by Use of a Transcribed Polymorphism in the XIST Gene
European Journal of Human Genetics, 1995Co-Authors: Jim L. Rupert, Carolyn J. Brown, Huntington F. WillardAbstract:As a result of X chromosome inactivation, females are mosaic for cell lineages in which either the paternal or the maternal X chromosome is active, and, if inactivation were random, each lineage should be present at approximately the same frequency. Detection of instances of non-random X inactivation can be important both clinically and for the study of X chromosome inactivation. Identification of a single-base polymorphism in an expressed region of the human XIST Gene has permitted the development of a direct PCR-based assay for randomness of X inactivation. Oligonucleotide primers were designed, incorporating the variant base, and conditions established that allowed allele-specific PCR amplification. As the XIST Gene is expressed only from the inactive X chromosome, differential amplification of the alleles in cDNA from heterozygotes can be used as an indicator of non-random inactivation. Using this assay, non-random X chromosome inactivation has been demonstrated in chromosomally abnormal cell lines and in lymphocytes from heterozygous, normal females. Virtually complete non-random X inactivation was also shown in a mother and her daughter, suggesting the eXISTence of some familial factor affecting X chromosome inactivation.
Philip Avner - One of the best experts on this subject based on the ideXlab platform.
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evidence for de novo imprinted x chromosome inactivation independent of meiotic inactivation in mice
Nature, 2005Co-Authors: Ikuhiro Okamoto, Danielle Arnaud, Philip Avner, Patricia Le Baccon, Arie P Otte, Christine M Disteche, Edith HeardAbstract:In mammals, one of the two X chromosomes is inactivated in females to enable dosage compensation for X-linked Gene products. In rodents and marsupials, only the X chromosome of paternal origin (Xp) is silenced during early embryoGenesis. This could be due to a carry-over effect of the X chromosome's passage through the male germ line, where it becomes transiently silenced together with the Y chromosome, during meiotic sex chromosome inactivation (MSCI). Here we show that XIST (X inactive specific transcript) transGenes, located on autosomes, do not undergo MSCI in the male germ line of mice and yet can induce imprinted cis-inactivation when paternally inherited, with identical kinetics to the Xp chromosome. This suggests that MSCI is not necessary for imprinted X-chromosome inactivation in mice. We also show that the Xp is transcribed, like autosomes, at zygotic Gene activation rather than being 'pre-inactivated'. We propose that expression of the paternal XIST Gene at zygotic Gene activation is sufficient to trigger cis-inactivation of the X chromosome, or of an autosome carrying a XIST transGene.
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the region 3 to XIST mediates x chromosome counting and h3 lys 4 dimethylation within the XIST Gene
The EMBO Journal, 2004Co-Authors: Celine Morey, Philip Avner, Emmanuel Debrand, Pablo Navarro, Claire Rougeulle, Philippe ClercAbstract:A counting process senses the X chromosome/autosome ratio and ensures that X chromosome inactivation (XCI) initiates in the female (XX) but not in the male (XY) mouse embryo. Counting is regulated by the X-inactivation centre, which contains the XIST Gene. Deleting 65 kb 3′ to XIST in XO embryonic stem (ES) cells affects counting and results in inappropriate XCI upon differentiation. We show here that normal counting can be rescued in these deleted ES cells using cre/loxP re-insertion, and refine the location of elements controlling counting within a 20 kb bipartite domain. Furthermore, we show that the 65 kb deletion also leads to inappropriate XCI in XY differentiated ES cells, which excludes the involvement of sex-specific mechanisms in the initiation of XCI. At the chromatin level, we have found that the XIST Gene corresponds to a peak of H3 Lys-4 dimethylation, which is dramatically and specifically affected by the deletion 3′ to XIST. Our results raise the possibility that H3 Lys-4 dimethylation within XIST may be functionally implicated in the counting process.
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human XIST yeast artificial chromosome transGenes show partial x inactivation center function in mouse embryonic stem cells
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Edith Heard, Fabien Mongelard, Danielle Arnaud, Corinne Chureau, Claire Vourch, Philip AvnerAbstract: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.
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XIST yeast artificial chromosome transGenes function as x inactivation centers only in multicopy arrays and not as single copies
Molecular and Cellular Biology, 1999Co-Authors: Edith Heard, Fabien Mongelard, Danielle Arnaud, Philip AvnerAbstract:X-chromosome inactivation in female mammals is controlled by the X-inactivation center (Xic). This locus is required for inactivation in cis and is thought to be involved in the counting process which ensures that only a single X chromosome remains active per diploid cell. The XIST Gene maps to the Xic region and has been shown to be essential for inactivation in cis. TransGenesis represents a stringent test for defining the minimal region that can carry out the functions attributed to the Xic. Although YAC and cosmid XIST-containing transGenes have previously been reported to be capable of cis inactivation and counting, the transGenes were all present as multicopy arrays and it was unclear to what extent individual copies are functional. Using two different yeast artificial chromosomes (YACs), we have found that single-copy transGenes, unlike multicopy arrays, can induce neither inactivation in cis nor counting. These results demonstrate that despite their large size and the presence of XIST, the YACs that we have tested lack sequences critical for autonomous function with respect to X inactivation.
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role of the region 3 to XIST exon 6 in the counting process of x chromosome inactivation
Nature Genetics, 1998Co-Authors: Philippe Clerc, Philip AvnerAbstract:During early embryoGenesis of female mammals, one of the two X chromosomes is randomly chosen to be inactivated in each cell1, leading to the transcriptional silencing of thousands of Genes on this chromosome. This random X-inactivation process also occurs during in vitro differentiation of female embryonic stem (ES) cells2,3,4. A locus on the X chromosome, the X inactivation centre (Xic) is initially 'counted', given that at least two copies of Xic must be present per diploid genome in order for inactivation to occur2. The counting process ensures that one X chromosome remains active in diploid cells. In the mouse, the essential functions of Xic can be assured by a 450-kb region5,6 containing the XIST Gene. XIST maps within Xic (refs 7, 8, 9, 10) and is necessary in cis for inactivation11,12. The XIST transcript is a 15-kb RNA which is confined within the nucleus and coats the inactive X chromosome13. In order to characterize functional elements within Xic and the XIST Gene, we created a 65-kb cre/loxP deletion extending 3′ to XIST exon 6. In undifferentiated ES cells, XIST expression from the deleted X chromosome was markedly reduced. In differentiated XX ES cells containing one deleted X chromosome, the X inactivation process still occurred but was never initiated from the unmutated X chromosome. In differentiated ES cells that were essentially XO, the mutated Xic was capable of initiating X inactivation, even in the absence of another Xic. These results demonstrate a role for the region 3′ to XIST exon 6 in the counting process and suggest that counting is mediated by a repressive mechanism which prevents inactivation of a single X chromosome in diploid cells.
Neil Brockdorff - One of the best experts on this subject based on the ideXlab platform.
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variability of sequence surrounding the XIST Gene in rodents suggests taxon specific regulation of x chromosome inactivation
PLOS ONE, 2011Co-Authors: Alexander I Shevchenko, Neil Brockdorff, Eugeny A Elisaphenko, Tatyana B Nesterova, S M Zakian, A A Malakhova, N A MazurokAbstract:One of the two X chromosomes in female mammalian cells is subject to inactivation (XCI) initiated by the XIST Gene. In this study, we examined in rodents (voles and rat) the conservation of the microsatellite region DXPas34, the Tsix Gene (antisense counterpart of XIST), and enhancer Xite that have been shown to flank XIST and regulate XCI in mouse. We have found that mouse regions of the Tsix Gene major promoter and minisatellite repeat DXPas34 are conserved among rodents. We have also shown that in voles and rat the region homologous to the mouse Tsix major promoter, initiates antisense to XIST transcription and terminates around the XIST Gene start site as is observed with mouse Tsix. A conservation of Tsix expression pattern in voles, rat and mice suggests a crucial role of the antisense transcription in regulation of XIST and XIC in rodents. Most surprisingly, we have found that voles lack the regions homologous to the regulatory element Xite, which is instead replaced with the Slc7a3 Gene that is unassociated with the X-inactivation centre in any other eutherians studied. Furthermore, we have not identified any transcription that could have the same functions as murine Xite in voles. Overall, our data show that not all the functional elements surrounding XIST in mice are well conserved even within rodents, thereby suggesting that the regulation of XCI may be at least partially taxon-specific.
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XIST Gene regulation at the onset of X inactivation.
Current opinion in genetics & development, 2009Co-Authors: Claire E Senner, Neil BrockdorffAbstract:The large non-coding RNA XIST is the master regulator of X inactivation. XIST is negatively regulated by its antisense transcript Tsix. This repressive antisense transcription across XIST operates at least in part through the modification of the chromatin environment of the locus. However Tsix is not sufficient to repress XIST in pluripotent cells and there is emerging evidence that transcription factors associated with pluripotency are involved in Tsix-independent repression. This review focuses on recent advances in this area and discusses the implications for our understanding of XIST Gene regulation at the onset of X inactivation.
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a dual origin of the XIST Gene from a protein coding Gene and a set of transposable elements
PLOS ONE, 2008Co-Authors: Eugeny A Elisaphenko, Neil Brockdorff, Nikolay N Kolesnikov, Alexander I Shevchenko, Igor B Rogozin, Tatyana B Nesterova, S M ZakianAbstract:X-chromosome inactivation, which occurs in female eutherian mammals is controlled by a complex X-linked locus termed the X-inactivation center (XIC). Previously it was proposed that Genes of the XIC evolved, at least in part, as a result of pseudogenization of protein-coding Genes. In this study we show that the key XIC Gene XIST, which displays fragmentary homology to a protein-coding Gene Lnx3, emerged de novo in early eutherians by integration of mobile elements which gave rise to simple tandem repeats. The XIST Gene promoter region and four out of ten exons found in eutherians retain homology to exons of the Lnx3 Gene. The remaining six XIST exons including those with simple tandem repeats detectable in their structure have similarity to different transposable elements. Integration of mobile elements into XIST accompanies the overall evolution of the Gene and presumably continues in contemporary eutherian species. Additionally we showed that the combination of remnants of protein-coding sequences and mobile elements is not unique to the XIST Gene and is found in other XIC Genes producing non-coding nuclear RNA.
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enox a novel Gene that maps 10 kb upstream of XIST and partially escapes x inactivation
Genomics, 2002Co-Authors: Colette M Johnston, Neil Brockdorff, Alistair E T Newall, Tatyana B NesterovaAbstract:Dosage compensation in mammals is accomplished by the transcriptional silencing of a single X chromosome in female cells, a process termed X inactivation. A cytoGenetically defined region of the X chromosome, the X-inactivation center (Xic), is necessary in cis for this process. Although the precise nature of the Xic remains unknown, a key component, the XIST Gene, has been shown to be essential for X inactivation. In XX somatic cells, XIST RNA is specifically transcribed from the inactive X chromosome, which is otherwise essentially heterochromatic. Previous studies aimed at defining the proximal limit of the Xic have indicated that it lies within 30 kb upstream of the XIST promoter. Here we describe a novel Gene, Enox (expressed neighbor of XIST), that maps to an unmethylated CpG island 10 kb upstream of XIST. Enox transcripts are antisense relative to XIST, highly heteroGeneous, and apparently noncoding. In female somatic tissue Enox partially escapes from X inactivation. We discuss the implications of these findings in relation to our understanding of the Xic.
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x chromosome inactivation closing in on proteins that bind XIST rna
Trends in Genetics, 2002Co-Authors: Neil BrockdorffAbstract:X inactivation is the developmentally regulated silencing of a single X chromosome in XX female mammals. In recent years, the XIST Gene has been revealed as the master regulatory switch controlling this process. Parental imprinting and/or counting mechanisms ensure that XIST is expressed only on the inactive X chromosome. Chromosome silencing then results from the accumulation of the XIST RNA silencing signal, in cis, over the entire length of the X chromosome. A key issue has been to identify the factors that interact with XIST RNA to initiate heritable Gene silencing. This review discusses recent progress that has put this goal in sight.
Jinwu Nam - One of the best experts on this subject based on the ideXlab platform.
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en bloc and segmental deletions of human XIST reveal x chromosome inactivation involving rna elements
Nucleic Acids Research, 2019Co-Authors: Hyeon J Lee, Jeannie T Lee, Hongjae Sunwoo, Ramu Gopalappa, Seo Won Choi, Suresh Ramakrishna, Hyongbum Kim, Jinwu NamAbstract:The XIST RNA is a non-coding RNA that induces X chromosome inactivation (XCI). Unlike the mouse XIST RNA, how the human XIST RNA controls XCI in female cells is less well characterized, and its functional motifs remain unclear. To systematically decipher the XCI-involving elements of XIST RNA, 11 smaller XIST segments, including repeats A, D and E; human-specific repeat elements; the promoter; and non-repetitive exons, as well as the entire XIST Gene, were homozygously deleted in K562 cells using the Cas9 nuclease and paired guide RNAs at high efficiencies, followed by high-throughput RNA sequencing and RNA fluorescence in situ hybridization experiments. Clones containing en bloc and promoter deletions that consistently displayed no XIST RNAs and a global up-regulation of X-linked Genes confirmed that the deletion of XIST reactivates the inactive X chromosome. Systematic analyses of segmental deletions delineated that exon 5 harboring the non-repeat element is important for X-inactivation maintenance, whereas exons 2, 3 and 4 as well as the other repeats in exon 1 are less important, a different situation from that of mouse XIST. This Cas9-assisted dissection of XIST allowed us to understand the unique functional domains within the human XIST RNA.
Sohaila Rastan - One of the best experts on this subject based on the ideXlab platform.
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regulatory elements in the minimal promoter region of the mouse XIST Gene
Gene, 1997Co-Authors: Steven A Sheardown, Sohaila Rastan, Alistair E T Newall, D P Norris, Neil BrockdorffAbstract:The XIST Gene plays a central role in regulating X chromosome inactivation and XIST transcription has recently been shown to be necessary for X inactivation in mouse. We are currently analysing regulation of the XIST Gene in order to determine the mechanisms underlying initiation of XIST expression and X inactivation. Sequence comparisons indicate that a region of approximately 0.4 kb upstream of the the major transcriptional start site comprises the XIST minimal promoter. Analysis of reporter constructs demonstrates that the minimal promoter region is active both in embryonic stem (ES) cells and in differentiated derivatives, indicating that sequences either further upstream or downstream are required for appropriate developmental control of XIST transcription. We have examined the minimal promoter region in detail, and in addition to common promoter elements have identified two previously uncharacterised transcription-factor binding sites. Mutation of these sites in reporter constructs indicates that they are functionally important.
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requirement for XIST in x chromosome inactivation
Nature, 1996Co-Authors: Graeme D Penny, Sohaila Rastan, Steven A Sheardown, Neil BrockdorffAbstract:The XIST Gene has been proposed as a candidate for the X inactivation centre, the master regulatory switch locus that controls X chromosome inactivation. So far this hypothesis has been supported solely by indirect evidence. Here we describe Gene targeting of XIST, and provide evidence for its absolute requirement in the process of X chromosome inactivation.
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x chromosome inactivation and the XIST Gene
Current Opinion in Genetics & Development, 1994Co-Authors: Sohaila RastanAbstract:X chromosome inactivation in mammals was first described over 30 years ago. The biological problem is how to achieve Gene dosage equivalence between XX females and XY males; the solution is to Genetically silence one whole X chromosome in each cell of the early developing female embryo. The molecular mechanism by which this is achieved, however, remains a mystery. Recently, through the discovery of the XIST Gene, it appears that we may be on the brink of learning how this unique phenomenon is mediated. Here, I discuss the developmental regulation of X inactivation and the candidacy of XIST as the X chromosome inactivation centre, with particular reference to its possible role in the initiation, spread and maintenance of X inactivation.
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expression of XIST during mouse development suggests a role in the initiation of x chromosome inactivation
Cell, 1993Co-Authors: Graham F Kay, Neil Brockdorff, Graeme D Penny, Alan Ashworth, Dipika Patel, Sohaila RastanAbstract:The mouse XIST Gene maps to the X inactivation center (Xic) region and is expressed exclusively from the inactive X chromosome. It is thus a candidate Gene for the Xic. We show that the onset of XIST expression in mouse development precedes X chromosome inactivation and may therefore be a cause rather than merely a consequence of X inactivation. The earliest XIST expression in morulae and blastocysts is imprinted, resulting in specific expression of the paternal XIST allele. Imprinted XIST expression may thus be the cause of nonrandom inactivation of the paternal X in trophectoderm. Strong Xce alleles can act to reduce the effect of imprinted XIST expression in the trophectoderm. The imprint on XIST expression is lost shortly before gastrulation when random X inactivation occurs. Our data support a direct role for XIST in the initiation of X inactivation.
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the product of the mouse XIST Gene is a 15 kb inactive x specific transcript containing no conserved orf and located in the nucleus
Cell, 1992Co-Authors: Neil Brockdorff, Alan Ashworth, D P Norris, Graham F Kay, Veronica M Mccabe, Penny Cooper, Sally Swift, Sohaila RastanAbstract:Abstract The XIST Gene maps to the X inactivation center region in both mouse and human, and previous analysis of the 3′ end of the Gene has demonstrated inactive X-specific expression, suggesting a possible role in X inactivation. We have now analyzed the entire mouse XIST Gene. The mature inactive X-specific transcript is 15 kb in length and contains no conserved ORF. The XIST sequence contains a number of regions comprised of tandem repeats. Comparison with the human XIST Gene demonstrates significant conservation of sequence and Gene structure. XIST RNA is not associated with the translational machinery of the cell and is located almost exclusively in the nucleus. Together with conservation of inactive X-specific expression, these findings support a role for XIST in X inactivation, possibly as a functional RNA or as a chromatin organizer region.
