IMP Research Report 2002

deletion analysis of the mouse Xist RNA. Using a Cre
recombinase based transgene homing strategy we
have integrated a panel of Xist cDNA constructs bearing
defined mutations into the Hprt locus on the X
chromosome in male ES cells, and characterized the
localization patterns of the resulting RNAs and their
ability to cause chromosomal silencing. Our results
show that chromosomal association of the RNA and
transcriptional repression can be separated by specific
mutations (Figure 2). Silencing is dependent on the Xist
result from the progressive heterochromatinisation of
the inactive chromosome in cellular differentiation.
Results of our experiments are expected to shed light
on the pathway by which Xist RNA localizes in cis to
chromatin and initiates silencing.
Xist mediated silencing in mice
Initiation of Xist mediated silencing is restricted to a
stage where cellular differentiation has not yet
progressed. Conversely, in differentiated somatic cells
Xist expression in general does no longer lead to
silencing despite the ability of Xist RNA to localize to
chromatin. We have generated an inducible Xist allele
to study the initiation of silencing in mouse development.
We are especially focusing our studies on the germline
stem cells, tumor cells and somatic stem cells. We
Figure 2: Chromosomal localization and silencing are mediated by
different sequences of Xist.
repeat A, a conserved repeat sequence located on the
expect to be able to epigenetically classify cells in the
developing mouse as Xist responsive or non-responsive
and thereby visualize this epigenetic transition in vivo.
5-prime end of Xist. Repeat A consists of seven perfect
and one imperfect repetitions of a sequence that can
be folded in two stem loops (Figure 3), whereby each
copy of the motif is separated by a variable spacer
region. Deletion of the repeat A results in an RNA that
still associates with chromatin, but does not affect
transcriptional repression. Localization of Xist RNA to
chromatin is a prerequisite for silencing and is mediated
by functionally redundant sequences that act
cooperatively and are dispersed throughout Xist, but
Functional studies of X inactivation in mice and ES cells
should provide insight into the epigenetic regulation of
gene expression in mammals. It appears that Xist
mediated silencing is a paradigm for a powerful
epigenetic system that is capable of heterochromatinising
an entire chromosome and determines
its specific nuclear localization. It is expected that similar
interactions underlie the regulation of other genes -
however, with less dramatic consequences.
share no homology or common sequence motifs.
We are interested in understanding the mechanism by
which Xist RNA associates in cis with chromatin and
mediates transcriptional repression. To this end we have
initiated experiments aiming at the biochemical
purification of proteins that interact with Xist RNA using
affinity purification and chromatin isolation methods as
competing and complementary approaches. Using
undifferentiated ES cells we hope to specifically identify
factors that interact with Xist RNA in the initiation of
chromosome wide silencing avoiding complications that
Figure 3: Secondary structure predicted for Xist repeat A. The motif
shown is repeated seven and a half times, whereby each copy is
separated by a variable spacer region.
Contact: wutz@imp.univie.ac.at
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