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. 2008 Sep;28(18):5583-94.
doi: 10.1128/MCB.02269-07. Epub 2008 Jul 14.

Xist RNA is confined to the nuclear territory of the silenced X chromosome throughout the cell cycle

Iris Jonkers  1 , Kim Monkhorst, Eveline Rentmeester, J Anton Grootegoed, Frank Grosveld, Joost Gribnau
Affiliations

Xist RNA is confined to the nuclear territory of the silenced X chromosome throughout the cell cycle

Iris Jonkers et al. Mol Cell Biol. 2008 Sep.

Abstract

In mammalian female cells, one X chromosome is inactivated to prevent a dose difference in the expression of X-encoded proteins between males and females. Xist RNA, required for X chromosome inactivation, is transcribed from the future inactivated X chromosome (Xi), where it spreads in cis, to initiate silencing. We have analyzed Xist RNA transcription and localization throughout the cell cycle. It was found that Xist transcription is constant and that the mature RNA remains attached to the Xi throughout mitosis. Diploid and tetraploid cell lines with an MS2-tagged Xist gene were used to investigate spreading of Xist. Most XXXX(MS2) tetraploid mouse embryonic stem (ES) cells inactivate the X(MS2) chromosome and one other X chromosome. Analysis of cells with two Xi's indicates that Xist RNA is retained by the Xi of its origin and does not spread in trans. Also, in XX(MS2) diploid mouse ES cells with an autosomal Xist transgene, there is no trans exchange of Xist RNA from the Xi to the autosome. We propose that Xist RNA does not dissociate from the Xi of its origin, which precludes a model of diffusion-mediated trans spreading of Xist RNA.

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Figures

FIG. 1.
FIG. 1.
Xist gene transcription during the cell cycle. (A) FACS analysis measuring the DNA content of day 4 differentiated diploid XX ES cells after release from a late G1 phase block. Samples were taken every 2 h. (B) RNA FISH on day 4 differentiated diploid XX ES cells after release from a late G1 phase block. DNA was stained with DAPI (blue), mature Xist RNA was stained with an exon probe (green with FITC), and primary Xist RNA transcripts were stained with an intron probe (rhodamine red). Primary Xist RNA transcript signals were categorized as none, weak, moderate, and high. (C) Percentage of day 4 differentiated diploid XX ES cells having no, weak, moderate, or high primary Xist RNA transcript signals at different time points after release from a late G1 phase block. (D) Q-PCR on day 4 differentiated diploid XX ES cells after release from a late G1 phase block. The threshold cycle (ΔCt) of primary and mature Xist RNA was taken at different time points and normalized with the β-actin control. neg, negative.
FIG. 2.
FIG. 2.
Xist clouds in day 4 differentiated F1 2-1 cells throughout the cell cycle. (A) Xist cloud in green in an interphase (inter) and a metaphase (meta) cell, followed by Xist clouds in two cells in telophase (telo) (Xist in FITC; DNA is DAPI stained). (B) Percentage of F1 2-1 cells with an Xist cloud in interphase, metaphase, or telophase cells.
FIG. 3.
FIG. 3.
Construction of ES cells expressing XistMS2 RNA. (A) A construct containing 16 repeats of the MS2 sequence next to a neomycin selection marker enclosed by Lox sites was integrated into exon 7 of the Xist gene. Positive clones were selected by Southern hybridization with the 5′ external (ext.) probe on BamHI-digested gDNA. Loop out of the neomycin resistance cassette was detected by Southern hybridization with the same digest and probe. (B) Allele-specific RT-PCR on Xist RNA isolated from day 7 differentiated ES cells using length polymorphism to distinguish whether Xist RNA originated from the XCast/Ei or the X129/Sv allele. (C) RNA FISH on day 10 differentiated XXMS2 cells. Xist RNA is in green (FITC), MS2 repeats are in red (rhodamine), and DNA is stained with DAPI. (D) Quantification of the number of cells containing either an Xist cloud or an XistMS2 cloud after 10 days of differentiation.
FIG. 4.
FIG. 4.
Tetraploid XXXXMS2 ES cells. (A) Schematic overview of the formation of tetraploid XXXXMS2 ES cells. (B) FACS analysis of the DNA content of undifferentiated diploid XX ES cells (2n, undiff), undifferentiated tetraploid XXXXMS2 ES cells (4n, undiff), and day 4 differentiated tetraploid XXXXMS2 ES cells (4n, 4d diff). The 2n peak in undifferentiated 4n cells represents male feeders. (C) RNA FISH with Xist and MS2 repeat probes was performed on day 10 differentiated tetraploid XXXXMS2 ES cells. The percentage of cells with a certain number of Xist clouds was determined and is depicted as black bars. The percentages of cells containing WT Xist RNA clouds only are depicted as light gray bars, and the percentages of cells containing no, one, or more WT Xist RNA clouds together with an XistMS2 RNA cloud are depicted as dark gray bars. (D) Xist clouds in tetraploid XXXXMS2 ES cells contain either Xist or XistMS2. RNA FISH on day 7 differentiated tetraploid XXXXMS2 ES cells after overnight incorporation of BrdU. The panels show incorporation of BrdU (blue with Cascade Blue), Xist RNA (green with FITC), and XistMS2 RNA (rhodamine red).
FIG. 5.
FIG. 5.
Xist and XistMS2 clouds in close proximity. (A, B, and C) The left panels show RNA FISH on day 7 differentiated tetraploid XXXXMS2 ES cells showing DNA (blue with DAPI), Xist RNA (green with FITC), and XistMS2 RNA (rhodamine red). The right panels show the intensities of the Xist and XistMS2 signals measured along the indicated white line across both Xist clouds. (A) A cell with two separate Xist clouds. (B) A cell with two Xist clouds in close proximity to each other but without overlap. (C) A cell with three Xist clouds, two of which are adjacent to each other and have overlapping Xist clouds. (D) Percentage of cells that have separate Xist clouds (light gray bars) or Xist clouds in close proximity to each other (dark gray bars). (E) Percentage of cells with Xist clouds in close proximity to each other that have nonoverlapping or overlapping Xist clouds.
FIG. 6.
FIG. 6.
Transgenic Xist RNA expression in the 30Δ1 8 cell line. (A) Interphase cells with either XistMS2 (in red) and autosomal Xist (green) clouds (left panel) or endogenous WT Xist and autosomal Xist clouds (both in green) (right panel). (B) Percentage of cells with one cloud of Xist or XistMS2, two clouds of either XistMS2 or autosomal Xist, or two clouds of either endogenous WT Xist or autosomal Xist. (C) Cells in telophase with XistMS2 (red) or autosomal Xist (green) still attached (left panel) or with Xist RNA detached and floating in the nucleoplasm (right panel). (D) Percentage of cells with XistMS2 cloud only or both XistMS2 and autosomal Xist clouds at interphase (inter), metaphase (meta), or telophase (telo). (E) Three metaphase cells showing XistMS2 (in red) and autosomal Xist (green) clouds.
FIG. 7.
FIG. 7.
Schematic models of Xist RNA spreading in M. musculus. (A) Xist RNA binds the chromosome directly, and the DNA folds to capture Xist. (B) Xist RNA binds to itself, creating Xist RNA clouds that are tethered to the chromosome. (C) A protein present in excess captures Xist RNA during transcription, allowing Xist to bind the chromosome.
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References

    1. Bailey, J. A., L. Carrel, A. Chakravarti, and E. E. Eichler. 2000. Molecular evidence for a relationship between LINE-1 elements and X chromosome inactivation: the Lyon repeat hypothesis. Proc. Natl. Acad. Sci. USA 976634-6639. - PMC - PubMed
    1. Branco, M. R., and A. Pombo. 2006. Intermingling of chromosome territories in interphase suggests role in translocations and transcription-dependent associations. PLoS Biol. 4e138. - PMC - PubMed
    1. Cattanach, B. M., and C. Rasberry. 1994. Identification of the Mus castaneus Xce allele. Mouse Genome 922.
    1. Clemson, C. M., J. A. McNeil, H. F. Willard, and J. B. Lawrence. 1996. XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure. J. Cell Biol. 132259-275. - PMC - PubMed
    1. Duthie, S. M., T. B. Nesterova, E. J. Formstone, A. M. Keohane, B. M. Turner, S. M. Zakian, and N. Brockdorff. 1999. Xist RNA exhibits a banded localization on the inactive X chromosome and is excluded from autosomal material in cis. Hum. Mol. Genet. 8195-204. - PubMed

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