Transcript X-inactivation

X-inactivation
20/11/08
Males are haploid for X-linked genes
expression
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 X-linked gene products in
males and females.
Random X-inactivation (Placental
mammals)
Imprinted X-inactivation
• Imprinted refers to the
skewed inactivation of the
paternal X-chromosome.
• Imprinted X-inactivation is
found among marsupials and
also in the extra-embryonic
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-TK- negative
selection for
recombination that is
not homologous (enters
randomly on any
chromosome)
Mutant ES clone –Probe (2) does not detect 129
allele (“B”-9kb) because it was deleted but probe 1
does recognize the PGK allele (“A”-5KB”).
Xist allele of 129 (From 129/PGK heterozygote
cell line) 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 X-chromosome bearing
the Xist mutation (129) will fail to x
inactivate.
• Option III: The mutation will have no
effect at all on X-inactivation.
Bi-allelic expression because both Xchromosomes are active in ES.
Random X-inactivation of
both the PGK and 129 Xchromosomes in WT ES
because X-chromosome
related genes are expressed
randomly in sub-clones.
In-activation by Xist is in CIS
X-related gene expression (NOT Xist) detected only in B (129 allele deleted for
Xist) due to 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 and no X-related RNA is observed from it.
Xist RNA coats the inactive X (FISH
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Chromatin modification during initiation of
X-chromosome inactivation
And Loss of euchormatin-associated
histone modifications (H4ac, H3ac etc).
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
LINE 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 Oct4 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.
ES nucleus
is positively
stained
(pink) for
Nanog
Oct4 immunostaining
Sox2 immunostaining
Nanog immunostaining
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 to 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
Xist expression following
downregulation of three TFs
TC induce differentiation by down regulating Oct4 expression.
Inconsistencies between syndromes and
X inactivation
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If a 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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