The X-inactivation trans-activator Rnf12 is negatively regulated by pluripotency factors in embryonic stem cells
Pablo Navarro
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Michael Moffat
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Nicholas P. Mullin
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Ian Chambers
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Present Address: M. Moffat MRC Human Genetics Unit, Western General Hospital, University of Edinburgh
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Edinburgh, Scotland, UK
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P. Navarro (&) M. Moffat N. P. Mullin I. Chambers (&) MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh
, King's Buildings, West Mains Road, Edinburgh EH9 3JQ,
Scotland, UK
X-inactivation, the molecular mechanism enabling dosage compensation in mammals, is tightly controlled during mouse early embryogenesis. In the morula, X-inactivation is imprinted with exclusive silencing of the paternally inherited X-chromosome. In contrast, in the post-implantation epiblast, X-inactivation affects randomly either the paternal or the maternal X-chromosome. The transition from imprinted to random X-inactivation takes place in the inner cell mass (ICM) of the blastocyst from which embryonic stem (ES) cells are derived. The trigger of X-inactivation, Xist, is specifically downregulated in the pluripotent cells of the ICM, thereby ensuring the reactivation of the inactive paternal X-chromosome and the transient presence of two active X-chromosomes. Moreover, Tsix, a critical cis-repressor of Xist, is upregulated in the ICM and in ES cells where it imposes a particular chromatin state at the Xist promoter that ensures the establishment of random X-inactivation upon differentiation. Recently, we have shown that key transcription factors supporting pluripotency directly repress Xist and activate Tsix and thus couple Xist/Tsix control to pluripotency. In this manuscript, we report that Rnf12, a third X-linked gene critical for the regulation of X-inactivation, is under the control of Nanog, Oct4 and Sox2, the three factors lying at the heart of the pluripotency network. We conclude that in mouse ES cells the pluripotency-associated machinery exerts an exhaustive control of X-inactivation by taking over the regulation of all three major regulators of X-inactivation: Xist, Tsix, and Rnf12.
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In her seminal manuscripts proposing random
X-inactivation as the mechanism underlying dosage compensation in
mammals (Lyon 1961; Lyon 1962), Mary Lyon suggested
this process must occur early during female embryonic
development, around the formation of the late blastocyst.
This first approximation was confirmed by means of single
cell blastocyst injections (Gardner and Lyon 1971),
cytogenetical observation of the heterochromatic
X-chromosome (Plotnick et al. 1971; Takagi and Sasaki 1975), and
biochemical analysis of X-linked genes encoding
metabolic enzymes at different developmental stages (Monk
and Kathuria 1977): these studies established that cells of
the inner cell mass (ICM) of the blastocyst carry two active
X-chromosomes, whilst in the trophectoderm the paternally
inherited X-chromosome is inactivated. The idea that
emerged from this was that X-inactivation is initially
established upon differentiation, either imprinted in
extraembryonic tissues or randomly in the embryo proper. This
enduring concept was well supported by analysis of
embryonic stem (ES) cells: random X-inactivation is only
established upon differentiation (Rastan and Robertson
1985). The delivery of important molecular insights of the
X-inactivation process, and their study in the
pre-implantation embryo challenged this initial dogma (reviewed in
Navarro and Avner 2009). In particular, the discovery of
the X-linked non-coding Xist gene (Brown et al. 1991;
Borsani et al. 1991; Brockdorff et al. 1991) and of the
mechanisms by which it triggers X-inactivation in cis, were
critical milestones.
Xist produces a 17 kb-long non-coding RNA exclusively
expressed from the inactive X-chromosome (Xi) of female
cells that mediates X-wide silencing. Xist RNA structures a
nuclear compartment from which the transcriptional
machinery is excluded and members of the Polycomb
group recruited, leading to the silencing of X-linked genes
and the initiation of a cascade of chromatin events that end
up with the heterochromatinisation of the X-chromosome
(reviewed in Ng et al. 2007). In both male and female ES
cells, Xist is consistently repressed and this might be
sufficient to keep all X-chromosomes active (Xa) (reviewed in
Navarro and Avner 2009). Indeed, forced expression of
Xist in ES cells leads to X-inactivation even before
differentiation (Wutz and Jaenisch 2000), indicating that Xist
repression is the most critical event required to abolish
X-inactivation in undifferentiated cells. Notably, upon
differentiation, Xist is monoallelically upregulated
exclusively in female cells, thereby triggering random
X-inactivation (reviewed in Navarro and Avner 2009). The
analysis of Xist RNA and associated heterochromatin
marks in early mouse embryos radically changed our
conception of the developmental dynamics of
X-inactivation (Mak et al. 2004; Okamoto et al. 2004). Although all
cells of the ICM appeared to la (...truncated)