Christian P. Bacher ¹, Michèle Guggiari ², Benedikt Brors ¹, Sandrine Augui ², Philippe Clerc ³, Philip Avner ³, Roland Eils ^{1, 4, 5}, and Edith Heard ^{2, 5}

¹ German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, German.
² CNRS UMR 218, Curie Institute, 26 rue d'Ulm, 75248 Paris Cedex 05, France.
³ Pasteur Institute, 25 rue du Docteur Roux, Paris 75015, France.
⁴ Institute of Pharmacy and Molecular Biotechnology (IPMB), University of Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany.
⁵ These authors contributed equally to this work.

Correspondence should be addressed to:
Edith Heard:    Edith.Heard@curie.fr    or,
Roland Eils:     r.eils@dkfz-heidelberg.de

News and Views Perspective:
   Fig. 1: A map of known genes and regulatory elements in the Xic region surrounding the Xist gene
   Fig. 2: A timecourse of the events that initiate XCI during ES cell differentiation:
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The initial differential treatment of the two X chromosomes during X-chromosome inactivation is controlled by the X-inactivation centre (Xic). This locus determines how many X chromosomes are present in a cell ('counting') and which X chromosome will be inactivated in female cells ('choice'). Critical control sequences in the Xic include the non-coding RNAs Xist and Tsix, and long-range chromatin elements. However, little is known about the process that ensures that X inactivation is triggered appropriately when more than one Xic is present in a cell. Using three-dimensional fluorescence in situ hybridization (FISH) analysis, we showed that the two Xics transiently colocalize, just before X inactivation, in differentiating female embryonic stem cells. Using Xic transgenes capable of imprinted but not random X inactivation, and Xic deletions that disrupt random X inactivation, we demonstrated that Xic colocalization is linked to Xic function in random X inactivation. Both long-range sequences and the Tsix element, which generates the antisense transcript to Xist, are required for the transient interaction of Xics. We propose that transient colocalization of Xics may be necessary for a cell to determine Xic number and to ensure the correct initiation of X inactivation.

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Alain Spatz, Christophe Borg & Jean Feunteun, "X-CHROMOSOME GENETICS AND HUMAN CANCER",
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Figure 1. Nuclear location of the Xic and Xist RNA in differentiating embryonic stem cells.

(a) Female embryonic stem cells differentiated for 2 days were analysed by DNA FISH using an X-chromosome paint (red) and an Xic probe (green). Nuclei were counterstained with DAPI (blue).

(b) Xic colocalization in female embryonic stem cells at day 1.5 after differentiation. The insert shows a three-dimensional projection of the original DAPI DNA counterstained image with Xist RNA signals (green).

(c) Raw data obtained by confocal microscopy on female embryonic stem cells following Xist RNA–Xic DNA FISH. Three of of the 60 sections acquired for this nucleus are shown. The distance between these sections along the z axis was approximately 0.4 mm.

(d) Before visualization, DAPI-stained nuclei were pre-processed and segmented to allow isosurface extraction (see Supplementary Information, Fig. S2). The nuclei were cut open to visualize the Xist–Xic signals (green and red, respectively). The insert shows a three-dimensional projection of original DAPI DNA counterstained images overlaid by the Xist RNA signal and Xic DNA signal (red; lower right only), respectively. Scale bars represent 2 mum.

Figure 2. Xic colocalization in differentiating embryonic stem cells.

Xic colocalization (or cross talk) events were detected in wildtype female (PGK1, HP310), female counting-deficient (D102, c.16.1), and transgenic male (53BL, L412) embryonic stem cells. A striking increase in Xic crosstalk events was observed for PGK1, HP310 and L412cell lines at days 1.5 and 2.0 after induction of X-chromsome inactivation. A general decrease in crosstalk events and an absence of Xic approximation events within the distance range of 0–1 mum was observed in 53Bl and D102 cells. S, simulated distribution of 1,000 calculated Xic distances from a random and uniform distribution within a simulated cell nucleus; MEF, control experiment using male or female MEF populations; d, days after the start of differentiation.

Figure 3. Analysis of inter-Xic distance distributions using quantile–quantile plots.

(a) Visualization of the Xic distance distributions using quantile–quantile plots for the indicated embryonic stem cell lines analysed (at days 1.5 and 2 of differentiation) against a simulation of 1,000 randomly determined Xic distance distributions. Closer than expected proximity between the two Xic's was observed in PGK, HP310 and c.16.1 cell lines (indicated by a shift of the points above the 45° reference line), whereas a complete lack of the short Xic inter-distance population was observed in the 53BL and D102 cell lines (indicated by a shift of the points below the 45° reference line).

(b) Comparison of wild type XX embryonic stem cell lines (HP310 and PGK) against the transgenic 53Bl (single copy) and L412 (multicopy) cell lines, and the deleted D102 and c.16.1 cell lines, at days 1.5 and 2 of differentiation. The quantile–quantile plots of PGK against HP310 distributions, for each day, lay approximately along the 45° reference line, and thus indicated that they have similar distributions. The slight shift towards shorter inter-Xic distances for PGK cells at day 1.5 after induction of differentiation was explained by the slightly slower inactivation kinetics observed for HP310 cells. The L412 line showed a very similar distribution to the other female lines. A lack of any close proximity between the two Xic loci was observed in 53BL and D102 cells when compared with wild-type PGK and HP310 cells. The complemented c.16.1 cells showed no shift when compared to the female HP310 cell lines, indicating that close proximity of the Xics was restored in this line.

Table 1: Embryonic stem cell lines
 

Cell line	Genotype	Counting and choice	Tsix transcription	Cis inactivation	Reference or source
HM1	XY	Yes	Yes	NA	Gift from E.Wagner
CK35	XY	Yes	Yes	NA	Ref. 7
PGK1  (PGK12.1)	XX	Yes	Yes	Yes	Ref. 27
HP310	XX	Yes	Yes	Yes	Ref. 5
D102	XXD65kb	No	No *	Yes	Ref. 5
c.16.1	XXD65kb+16kb	No	Yes	Yes	Ref. 16
53BL	XY+XicTgn=1	No	Yes	No	Ref. 7
L412	XY+XicTgn>1	Yes	Yes	Yes	Ref. 7

NA, not applicable.
*No Tsix transcription on the deleted (XX^D65kb) allele only.

http://www.nature.com/ncb/journal/v8/n3/suppinfo/ncb1365_S1.html

"Sealed with a X".

Céline Morey & Wendy Bickmore

MRC Human Genetics Unit, Crewe Road, Edinburgh EH4 2XU, UK.

E:mail:  Celine.Morey@hgu.mrc.ac.uk

Figure 1. A map of known genes and regulatory elements in the Xic region surrounding the Xist gene.

The Tsix transcript (brown arrow) is antisense to Xist (green arrow), mainly initiates 16 kb downstream of Xist and extends over at least 40 kb. The DNA regulatory element Xite (purple) and the DXPas34 minisatellite (purple) have been shown to enhance (+) Tsix expression16, which in turn represses Xist (-). The structures of the 65-kb deleted (65^D) and 16kb-complemented alleles, and the Xic YAC used by Bacher et al. are shown below. The occurrence of X–X association in female-deleted cells and of X-autosome association in male transgenic cells is indicated to the left.

Figure 2. A timecourse of the events that initiate XCI during ES cell differentiation.

In undifferentiated ES cells, transcription of Tsix (brown lines) from both chromosomes maintains Xist expression at low levels and restricts Xist RNA to the transcription site (day 0). Induction of differentiation triggers X–X colocalization, counting and choice. Tsix is then downregulated on the presumptive Xi, thereby allowing Xist RNAs (blue lines) to accumulate in cis (day 2). On Xa, the repression of Xist is maintained through the persistence of Tsix expression promoted by the Xite locus. Subsequently, the Xi accumulates heterochromatic epigenetic marks to maintain the silent state (day 4).

Alain Spatz, Christophe Borg & Jean Feunteun, "X-CHROMOSOME GENETICS AND HUMAN CANCER",
Nature Reviews Cancer 4, 617-629 (2004); doi:10.1038/nrc1413
http://www.nature.com/nrc/journal/v4/n8/abs/nrc1413_fs.html

These links to content published by NPG are automatically generated:
You are viewing results 1 to 4 of 4

X-Chromosome Genetics and Human Cancer
Nature Reviews Cancer Review (01 Aug 2004)

The kangaroo genome
EMBO Reports Review (01 Feb 2003)

X-CHROMOSOME INACTIVATION: COUNTING, CHOICE AND INITIATION
Nature Reviews Genetics Review (01 Jan 2001)

Chromatin modification and epigenetic reprogramming in mammalian development
Nature Reviews Genetics Review (01 Sep 2002)

REVIEWS:
X-CHROMOSOME INACTIVATION: COUNTING, CHOICE AND INITIATION
Nature Reviews Genetics Review (01 Jan 2001)

NEWS AND VIEWS:
Anti-Xistentialism
Nature Genetics News and Views (01 Apr 1999)

RESEARCH:
Evidence for de novo imprinted X-chromosome inactivation independent of meiotic inactivation in mice
Nature Letters to Editor (17 Nov 2005)

Tsix, a gene antisense to Xist at the X-inactivation centre
Nature Genetics Letters (01 Apr 1999)

Role of the region 3' to Xist exon 6 in the counting process of X-chromosome inactivation
Nature Genetics Letters (01 Jul 1998)

1. Frenster JH, and Hovsepian JA, "Ultrastructure of Euchromatin Contact Points between the Closed Loops of Adjacent Interphase Chromosomes".

2. Hovsepian JA, and Frenster JH, "Sense and Antisense during RNA Initiation of the DNA Transcription Bubble".

3. Frenster JH, and Hovsepian JA, "Ultrastructure  of Closed Loops within Euchromatin of Isolated Lymphocyte Nuclei".

4. Xu N, Tsai C-L, and Lee JT, "Transient Homologous Pairing Marks the Onset of X Inactivation", Science vol. 311, no. 5764, pp. 1149-1152 (February 24, 2006).

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