Reactivation of
            <i>XIST</i>
            in normal fibroblasts and a somatic cell hybrid: Abnormal localization of XIST RNA in hybrid cells

Hansen, R. Scott; Canfield, Th.K.; Stanek, Ann M.; Keitges, Elisabeth A.; Gartler, Stanley M.

doi:10.1073/pnas.95.9.5133

Cited by 45 publications

(44 citation statements)

References 47 publications

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“…The findings indicate that the lack of acetylation coincides with the presence of methylation at promoters of X-inactivated genes. In addition, these results confirm prior reports that human X chromosomes retain the same methylation status in hybrid cells as in intact human cells (29).…”

Section: Methylation Of Promoter Regions Of X-linked Genes Correlatessupporting

confidence: 82%

Promoter-specific hypoacetylation of X-inactivated genes

Gilbert

Sharp

1999

Proc. Natl. Acad. Sci. U.S.A.

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The histone H4 acetylation status of the active X (Xa) and inactive X (Xi) chromosomes was investigated at the level of individual genes. A moderate level of acetylation was observed along the lengths of genes on both the Xi and Xa, regardless of their X inactivation status. However, this moderate level of acetylation was modified specifically in promoter regions. Transcriptionally active genes showed elevated levels of acetylation at their promoters on both the Xi and Xa. In contrast, promoters of X-inactivated genes were markedly hypoacetylated, which coincided with the methylation of adjacent CG dinucleotides. This promoterspecific hypoacetylation may be a key component of an X inactivation machinery that operates at the level of individual genes. In mammals, X chromosome dosage is equalized between males and females by X inactivation, the transcriptional silencing of one of the two X chromosomes in female cells. The chromatin of the inactive X (Xi) is distinct from all other chromosomes, including the active X (Xa), in that it is late replicating (1, 2), methylated on CG dinucleotides (3, 4), enriched in the histone H2A variant macroH2A (5), and hypoacetylated on histones H2A, H3, and H4 (6-8). These unique features of the Xi chromatin are associated with, and may contribute to, the silencing of its several thousand resident genes.Although most genes on the Xi are transcriptionally repressed, there are clear exceptions. First, the XIST gene, which initiates X inactivation, is transcribed only from the Xi (9). Second, in both humans and mice, a number of genes escape X inactivation-that is, they are transcribed from the Xi as well as the Xa (for review see refs. 10 and 11). Genes that escape X inactivation typically have homologs on the Y chromosome, which in theory obviates their need for dosage compensation (12).Among the unique properties of the Xi chromatin, CG methylation and late replication have been studied at the level of individual genes and were found to correlate closely with gene expression. Where examined, these traits are associated with particular genes that are silenced, but not with those that escape X inactivation (2,13,14). In contrast, histone acetylation on the Xi has not been examined extensively at the level of individual genes. The prevailing view that the Xi is hypoacetylated comes from immunofluorescence microscopy analysis, which showed that all chromosomes except the Xi stained brightly with antisera against acetylated histones in female cells (6-8). However, these microscopy studies on metaphase chromosomes are very limited in resolution and thus cannot address the acetylation status of individual genes.A chromatin immunoprecipitation (IP) technique has been developed recently that allows the acetylation status of individual nucleosomes to be examined (15-17). We used this method to investigate the acetylation status of histone H4 associated with numerous genes on both the Xi and Xa. Our results indicate that nucleosomes on the Xi are in fact acetylated along the lengths of genes...

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Section: Methylation Of Promoter Regions Of X-linked Genes Correlatessupporting

confidence: 82%

Promoter-specific hypoacetylation of X-inactivated genes

Gilbert

Sharp

1999

Proc. Natl. Acad. Sci. U.S.A.

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“…Previous work has shown that depletion of the "maintenance" DNA methyltransferase Dnmt1 compromises Xist repression (17,36). We verified this in our fibroblasts using siRNA directed against the Dnmt1 transcript.…”

Section: Resultssupporting

confidence: 60%

“…Thus, the absence of Mbd2 increases the probability that Xist gene reactivation will occur. Previous studies reported that artificial demethylation reactivated Xist in about 1% of hypomethylated human fibroblasts (17), while in Dnmt1-deficient differentiated mouse embryonic stem cells, about 15% of cells carried Xist (36). Therefore, deficiency of either DNA methylation or Mbd2 increases the probability of Xist reactivation but does not cause Xist reexpression in every cell.…”

Section: Discussionmentioning

confidence: 99%

Mbd2 Contributes to DNA Methylation-Directed Repression of the Xist Gene

Barr

Hermann

Berger

et al. 2007

Molecular and Cellular Biology

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Transcription of the Xist gene triggers X chromosome inactivation in cis and is therefore silenced on the X chromosome that remains active. DNA methylation contributes to this silencing, but the mechanism is unknown. As methylated DNA binding proteins (MBPs) are potential mediators of gene silencing by DNA methylation, we asked whether MBP-deficient cell lines could maintain Xist repression. The absence of Mbd2 caused significant low-level reactivation of Xist, but silencing was restored by exogenous Mbd2. In contrast, deficiencies of Mbd1, MeCP2, and Kaiso had no detectable effect, indicating that MBPs are not functionally redundant at this locus. Xist repression in Mbd2-null cells was hypersensitive to the histone deacetylase inhibitor trichostatin A and to depletion of the DNA methyltransferase Dnmt1. These synergies implicate Mbd2 as a mediator of the DNA methylation signal at this locus. The presence of redundant mechanisms to enforce repression at Xist and other loci is compatible with the hypothesis that "stacking" of imperfect repressive tendencies may be an evolutionary strategy to ensure leakproof gene silencing.

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“…This contrasts with reports concerning human-rodent somatic cell hybrids, where the human XIST transcript was not found to be localized correctly to the human X chromosome and where it was suggested that species-specific factors presumably were necessary for cis localization (34,35). Because these studies concerned adult somatic cells rather than ES cells, it may be that murine factors present only during early development and in ES cells are able to ensure the correct cis localization of the human XIST transcript.…”

Section: Discussioncontrasting

confidence: 53%

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

Heard

Mongélard

Arnaud

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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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 ref lect 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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Reactivation of XIST in normal fibroblasts and a somatic cell hybrid: Abnormal localization of XIST RNA in hybrid cells

Cited by 45 publications

References 47 publications

Promoter-specific hypoacetylation of X-inactivated genes

Promoter-specific hypoacetylation of X-inactivated genes

Mbd2 Contributes to DNA Methylation-Directed Repression of the Xist Gene

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

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