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X-inactivation
Males are haploid for X-linked
genes
Barr body discovered (1949)
Lost twin?
Lyonization=X-inactivation
The hypothesis:
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The heteropyknotic
(..”condensed”) X chromosome
was inactive.
Paternal or maternal in origin.
The inactivation occurred early in
embryonic development.
The genetic facts that helped formulate
the hypothesis:
• X0 mice are normal fertile females (One
X is enough).
• Female mice heterozygous for coat colour
show mosaic phenotype.
Dosage compensation
• Dosage compensation
refers to a regulatory
mechanism that
ensures the
equalization of Xlinked gene products
in males and females.
Random X-inactivation (Placental
mammals)
Random Vs. Imprinted Xinactivation
• Imprinted refers to the
skewed inactivation of the
paternal X-chromosome.
• Imprinted X-inactivation is
found among marsupials
and also in the extraembryonic tissues in a
subset of placental
mammals.
(Duret et al., 2006)
Xic (X-inactivation center) locus
Ectopic expression of Xic transgene leads to X-inactivation in male cells.
Derivation of embryonic stem (ES)
cells from blastocysts:
Mouse embryonic stem cells derived and
cultured in-vitro- 1981 (Martin et al)
Human embryonic stem cells derived and
cultured in-vitro-1998 (Thomson et al)
ES cell give rise to the ~220 cell
types that comprise the human
body
I. Self renewalUnlimited division
while maintaining a
pluripotent
undifferentiated
state in-vitro.
II. DifferentiationThe ability to
differentiate to cells
from the three
embryonic germ layers
(in-vivo and in-vitro).
Targeted deletion of Xist in ES cells (1996)
Neo- positive
selection for
integration.
HSV-TKnegative selection
for recombination
that are not
homologous
Mutant clone – The probe does not recognize 129
allele but does recognize PGK allele (“B”)- the 129
Xist allele was deleted (“A).
Xist allele of 129 (From 129/PGK) was deleted
using Homologous Recombination.
Targeted deletion of Xist in ES cells
(1996)
• Option I: The mutant ES
cells will fail to undergo X
inactivation.
• Option II: The Xchromosome bearing the
Xist mutation (129) will fail
to x inactivate.
• Option III: The mutation will
have no effect at all on Xinactivation.
In activation is in CIS
X-related gene expression detected only in B -> no inactivation of the Xist
deleted X chromosome (129 is B). This leads to skewed monoallelic
expression between clones because A (PGK) is always inactivated!
Xist RNA coats the inactive X
(FISH )
Chromatin modification during initiation of
X-chromosome inactivation
And Loss of euchormatin-associated
histone modifications (H4 acetylation)
And DNA methylation
Xist spreading is LINE dependent
• The x-chromosome is
enriched for LINE
sequences compared to
autosomes.
•These may serve as “way
station” for Xist binding
(due to high affinity for
RNA binding).
• Tanslocation of X/4
chromosomes showed that
Xist spreading ended at
the translocation point
(correlates with a drop in
LINEs sequences in
chromosome 4).
Xic pairing in ES undergoing
differentiation (Bacher 2006)
• The pairing was
observed only in ES
undergoing
differentiation.
•In Xic-Del ES cell line
no pairing was observed.
•The X chromosome
harboring the deletion
was repeatedly the one
undergoing inactivation.
Tsix
• A long untranslated RNA transcribed in the anti-sense direction of
Xist which represses Xist by forming dsRNA hybrid that is processed
and degraded.
• Initially expressed on both X chromosomes (In ES) but persists on
the active X after differentiation. It is downregulated from the Inactive
X.
• Tsix is regulated by DXpas34 and Xite elements- Deletion of
DXpas34 leads to X inactivation.
• Disruption of Tsix induces X-inactivation in XY ES.
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Unresolved Questions
• What are the mechanisms for choosing and counting?
• How does the spreading along the chromosome occur?
• How does X inactivation maintained in the female soma?
• What is the difference between imprinted and random X inactivation?
• How is X inactivation coupled with cell differentiation?
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Nanog Oc4 and Sox2 are the master regulators
of the pluripotent undifferentiated state of ES
cells
Undifferentiated
pluripotent ES colony
(very small round cells)
cultured in-vitro.
Feeder cell
nucleus are
not stained for
Nanog (blue)
Oct4
Sox2
Nanog
ES nucleus
is positively
stained
(pink) for
Nanog
Molecular Coupling of Xist and
pluripotency (Navarro 2008)
Oct4, Sox2 and Nanog
transcription factors (TFs),
,which are the master
regulators of the pluripotent
state in ES, bind Intron1 of
Xist thus repressing its
transcription in ES cells.
When ES are induced to
differentiate, Oct4, Sox2 and
Nanog are downregulated and
Xist is freed from their binding
“grip” and rapidly accumulates
on one of the X-chromosomes.
WT ES=>Xist is repressed.
-/- Nanog ES=> Xist is
upregulated.
Binding site of Nanog in
intron1 of Xist
OCT4 controls pairing and counting
• Control XCI.
• Triggers pairing and
counting.
•Binds Xite and Tsix.
•Depletion- blocks
pairing and induces
XCI in both Xs.
Inconsistencies between syndromes and
X inactivation
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If normal XX female has one X inactivated, why is a X Turner female not
normal?
Similarly, if XXY male has one X inactivated, why does he have Klinefelter
syndrome?
Escape from X-inactivation ?
Carrel et al (2005) showed that ~15% of the genes on the inactive-X
escaped from inactivation, complicating the story even further..
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