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Active chromatin marks are retained on X chromosomes lacking gene or repeat silencing despite XIST/Xist expression in somatic cell hybrids.

Thorogood NP, Brown CJ - PLoS ONE (2010)

Bottom Line: X-chromosome inactivation occurs early in mammalian development and results in the inactive X chromosome acquiring numerous hallmarks of heterochromatin.Cot-1 holes, regions of depleted RNA hybridization with a Cot-1 probe, were observed upon Xist expression; however, these were at reduced frequency and intensity in these somatic cells.These results are consistent with recent reports separating Cot-1 silencing from genic silencing, but also demonstrate repetitive element expression from an otherwise silent X chromosome in these hybrid cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, University of British Columbia, Vancouver, Canada.

ABSTRACT

Background: X-chromosome inactivation occurs early in mammalian development and results in the inactive X chromosome acquiring numerous hallmarks of heterochromatin. While XIST is a key player in the inactivation process, the method of action of this ncRNA is yet to be determined.

Methodology/principal findings: To assess which features of heterochromatin may be directly recruited by the expression and localization of the XIST RNA we have analyzed a mouse/human somatic cell hybrid in which expression of human and mouse XIST/Xist has been induced from the active X by demethylation. Such hybrids had previously been demonstrated to disconnect XIST/Xist expression from gene silencing and we confirm maintenance of X-linked gene expression, even close to the Xist locus, despite the localized expression of mouse Xist.

Conclusions/significance: Loss of the active chromatin marks H3 acetylation and H3 lysine 4 methylation was not observed upon XIST/Xist expression, nor was there a gain of DNA methylation; thus these marks of facultative heterochromatin are not solely dependent upon Xist expression. Cot-1 holes, regions of depleted RNA hybridization with a Cot-1 probe, were observed upon Xist expression; however, these were at reduced frequency and intensity in these somatic cells. Domains of human Cot-1 transcription were observed corresponding to the human chromosomes in the somatic cell hybrids. The Cot-1 domain of the X was not reduced with the expression of XIST, which fails to localize to the human X chromosome in a mouse somatic cell background. The human inactive X in a mouse/human hybrid cell also shows delocalized XIST expression and an ongoing Cot-1 domain, despite X-linked gene silencing. These results are consistent with recent reports separating Cot-1 silencing from genic silencing, but also demonstrate repetitive element expression from an otherwise silent X chromosome in these hybrid cells.

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X-linked gene expression in somatic cell hybrids before and after expression of XIST/Xist.(A–C) Shows the XIST/Xist expression in cell nuclei using RNA FISH probes for the human XIST RNA (green) and the mouse Xist RNA (red). (A) The XIST+/Xist+ (AHA-4C1) somatic cell hybrid; (B) The female mouse fibroblast control cell line, BMSL2; (C) The human female fibroblast line GM04626 (karyotype: 47,XXX). (D) Schematic diagram of both the human and mouse X chromosomes showing the location of the genes analyzed to the left of gel images of RT-PCR products of cDNA from the control female (GM7350 (human) and BMSL2 (mouse)); the hybrid not expressing AHA-11aB1 (XIST−/Xist−); and the derivative hybrid that expresses AHA-4C1 (XIST+/Xist+). (E) Quantitative RT-PCR of 4 X-linked genes. An intermediate hybrid AHA-A5-2b (XIST+/Xist−) was analyzed for expression along with the hybrid AHA-4C1 (XIST+/Xist+). The gene expression was normalized to Actin and expressed as a fold-change relative to the parent hybrid AHA-11aB1 (XIST−/Xist−). The error bars represent the standard deviation of three separate RNA isolations.
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pone-0010787-g001: X-linked gene expression in somatic cell hybrids before and after expression of XIST/Xist.(A–C) Shows the XIST/Xist expression in cell nuclei using RNA FISH probes for the human XIST RNA (green) and the mouse Xist RNA (red). (A) The XIST+/Xist+ (AHA-4C1) somatic cell hybrid; (B) The female mouse fibroblast control cell line, BMSL2; (C) The human female fibroblast line GM04626 (karyotype: 47,XXX). (D) Schematic diagram of both the human and mouse X chromosomes showing the location of the genes analyzed to the left of gel images of RT-PCR products of cDNA from the control female (GM7350 (human) and BMSL2 (mouse)); the hybrid not expressing AHA-11aB1 (XIST−/Xist−); and the derivative hybrid that expresses AHA-4C1 (XIST+/Xist+). (E) Quantitative RT-PCR of 4 X-linked genes. An intermediate hybrid AHA-A5-2b (XIST+/Xist−) was analyzed for expression along with the hybrid AHA-4C1 (XIST+/Xist+). The gene expression was normalized to Actin and expressed as a fold-change relative to the parent hybrid AHA-11aB1 (XIST−/Xist−). The error bars represent the standard deviation of three separate RNA isolations.

Mentions: The mouse/human hybrid cell line AHA-11aB1 retains only the human Xa [14], and a subclone was previously described that stably expressed human XIST after three rounds of demethylation with 5aza (AHA-A5-2b) [15]. We have now subjected AHA-A5-2b to further rounds of demethylation to yield a new subclone designated AHA-4C1 that stably expresses both human XIST and mouse Xist. In these AHA-4C1 cells FISH analysis demonstrated that the human XIST RNA was not localized within the nucleus while mouse Xist RNA was localized (Figure 1A). 76% of cells had Xist localization and a diffuse XIST signal (n = 95), similar to numbers reported by Clemson et al when they analyzed a transiently-expressing hybrid [12]. Quantitative PCR analysis showed that mouse Xist was expressed equivalently in the hybrid cell line (AHA-4C1) and the female mouse cell line (BMSL2) used as a control (data not shown).


Active chromatin marks are retained on X chromosomes lacking gene or repeat silencing despite XIST/Xist expression in somatic cell hybrids.

Thorogood NP, Brown CJ - PLoS ONE (2010)

X-linked gene expression in somatic cell hybrids before and after expression of XIST/Xist.(A–C) Shows the XIST/Xist expression in cell nuclei using RNA FISH probes for the human XIST RNA (green) and the mouse Xist RNA (red). (A) The XIST+/Xist+ (AHA-4C1) somatic cell hybrid; (B) The female mouse fibroblast control cell line, BMSL2; (C) The human female fibroblast line GM04626 (karyotype: 47,XXX). (D) Schematic diagram of both the human and mouse X chromosomes showing the location of the genes analyzed to the left of gel images of RT-PCR products of cDNA from the control female (GM7350 (human) and BMSL2 (mouse)); the hybrid not expressing AHA-11aB1 (XIST−/Xist−); and the derivative hybrid that expresses AHA-4C1 (XIST+/Xist+). (E) Quantitative RT-PCR of 4 X-linked genes. An intermediate hybrid AHA-A5-2b (XIST+/Xist−) was analyzed for expression along with the hybrid AHA-4C1 (XIST+/Xist+). The gene expression was normalized to Actin and expressed as a fold-change relative to the parent hybrid AHA-11aB1 (XIST−/Xist−). The error bars represent the standard deviation of three separate RNA isolations.
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Related In: Results  -  Collection

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pone-0010787-g001: X-linked gene expression in somatic cell hybrids before and after expression of XIST/Xist.(A–C) Shows the XIST/Xist expression in cell nuclei using RNA FISH probes for the human XIST RNA (green) and the mouse Xist RNA (red). (A) The XIST+/Xist+ (AHA-4C1) somatic cell hybrid; (B) The female mouse fibroblast control cell line, BMSL2; (C) The human female fibroblast line GM04626 (karyotype: 47,XXX). (D) Schematic diagram of both the human and mouse X chromosomes showing the location of the genes analyzed to the left of gel images of RT-PCR products of cDNA from the control female (GM7350 (human) and BMSL2 (mouse)); the hybrid not expressing AHA-11aB1 (XIST−/Xist−); and the derivative hybrid that expresses AHA-4C1 (XIST+/Xist+). (E) Quantitative RT-PCR of 4 X-linked genes. An intermediate hybrid AHA-A5-2b (XIST+/Xist−) was analyzed for expression along with the hybrid AHA-4C1 (XIST+/Xist+). The gene expression was normalized to Actin and expressed as a fold-change relative to the parent hybrid AHA-11aB1 (XIST−/Xist−). The error bars represent the standard deviation of three separate RNA isolations.
Mentions: The mouse/human hybrid cell line AHA-11aB1 retains only the human Xa [14], and a subclone was previously described that stably expressed human XIST after three rounds of demethylation with 5aza (AHA-A5-2b) [15]. We have now subjected AHA-A5-2b to further rounds of demethylation to yield a new subclone designated AHA-4C1 that stably expresses both human XIST and mouse Xist. In these AHA-4C1 cells FISH analysis demonstrated that the human XIST RNA was not localized within the nucleus while mouse Xist RNA was localized (Figure 1A). 76% of cells had Xist localization and a diffuse XIST signal (n = 95), similar to numbers reported by Clemson et al when they analyzed a transiently-expressing hybrid [12]. Quantitative PCR analysis showed that mouse Xist was expressed equivalently in the hybrid cell line (AHA-4C1) and the female mouse cell line (BMSL2) used as a control (data not shown).

Bottom Line: X-chromosome inactivation occurs early in mammalian development and results in the inactive X chromosome acquiring numerous hallmarks of heterochromatin.Cot-1 holes, regions of depleted RNA hybridization with a Cot-1 probe, were observed upon Xist expression; however, these were at reduced frequency and intensity in these somatic cells.These results are consistent with recent reports separating Cot-1 silencing from genic silencing, but also demonstrate repetitive element expression from an otherwise silent X chromosome in these hybrid cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, University of British Columbia, Vancouver, Canada.

ABSTRACT

Background: X-chromosome inactivation occurs early in mammalian development and results in the inactive X chromosome acquiring numerous hallmarks of heterochromatin. While XIST is a key player in the inactivation process, the method of action of this ncRNA is yet to be determined.

Methodology/principal findings: To assess which features of heterochromatin may be directly recruited by the expression and localization of the XIST RNA we have analyzed a mouse/human somatic cell hybrid in which expression of human and mouse XIST/Xist has been induced from the active X by demethylation. Such hybrids had previously been demonstrated to disconnect XIST/Xist expression from gene silencing and we confirm maintenance of X-linked gene expression, even close to the Xist locus, despite the localized expression of mouse Xist.

Conclusions/significance: Loss of the active chromatin marks H3 acetylation and H3 lysine 4 methylation was not observed upon XIST/Xist expression, nor was there a gain of DNA methylation; thus these marks of facultative heterochromatin are not solely dependent upon Xist expression. Cot-1 holes, regions of depleted RNA hybridization with a Cot-1 probe, were observed upon Xist expression; however, these were at reduced frequency and intensity in these somatic cells. Domains of human Cot-1 transcription were observed corresponding to the human chromosomes in the somatic cell hybrids. The Cot-1 domain of the X was not reduced with the expression of XIST, which fails to localize to the human X chromosome in a mouse somatic cell background. The human inactive X in a mouse/human hybrid cell also shows delocalized XIST expression and an ongoing Cot-1 domain, despite X-linked gene silencing. These results are consistent with recent reports separating Cot-1 silencing from genic silencing, but also demonstrate repetitive element expression from an otherwise silent X chromosome in these hybrid cells.

Show MeSH
Related in: MedlinePlus