Transcript כרומוזומי הזוויג

‫כרומוזומי הזוויג‬
‫תורשת האדם‬
‫‪13.11.08 -‬‬
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SRY gene
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Sex determining factor (TDF)
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Intronless gene which initiates male sex determination
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Transcription factor (HMG-box family of DNA proteins)
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Mutations in SRY give rise to XY females with gondal dysgenesis
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Identified through Y-associated chromosomal aberrations:
– Translocations to X in rare XX males
– Deletions in rare XY females
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Was finally cloned by Sinclair (1990)
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The “home run” experiment by Koopman et al. (1991) used transgenic mice.
The transgenic Sry experiment:
How it was done
• Nuclei of fertilized XX eggs were injected with Sry gene, then the eggs
were transplanted to surrogate mothers.
• Sry gene then randomly incorporated into a chromosome and was
inherited in subsequent cell divisions.
• Animals karyotyped after development to adult.
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(Nature 351:117 (1991))
Genotypically female mice transgenic for Sry are
phenotypically male
XY male
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XX male
Human SRY
• Expression in the testis from 41d-18w of gestation
• Also expressed in brain, pancreas and heart
• Apart from SOX9, downstream targets are still largely unknown
Incidence of 15% of XY females have mutations in SRY
- 85% of these mutations are de novo
- 15% born to fertile fathers (fathers were gonadal mosaic for both
wild-type and mutant SRY alleles)
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Dosage Compensation
Sex determination in mammals: XX and XY
Chromosome X: relatively large, approximately 1100 genes, most are
active in somatic cells, critical for survival
Chromosome Y: very small, only 45 genes, required for male sex
determination and spermatogenesis, dispensable for
survival (XX)
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Dosage compensation
Problem: XX females produce twice the amount of X-linked gene
products (proteins) as XY males
Need in compensatory mechanism!
Potential mechanisms for dosage compensation between males and females:
1. X-linked genes in males are transcribed at twice the level of that in
females (fruit flies)
2. 2 fold decrease in expression level of X-linked genes in females
(nematodes)
3. Inactivation of a single X chromosome in each XX cell (mammals)
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Barr body in females
Neuronal nuclei of female cats
XX
XXXXX
Number of Barr bodies = n-1 rule
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Barr et al. 1949
Barr Bodies are Inactivated X Chromosomes in
Females
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Susumu Ohno 1959: two X chromosomes in mammals appear differently
All but one X chromosome are silenced per diploid set of chromosomes
Spotted phenotype of female mice heterozygous
for coat color
• XX mice are variegated for coat color
• XO female mice are viable and fertile,
uniform for coat color
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The Lyon Hypothesis of X Inactivation
(Mary Lyon and Liane Russell 1961)
• In every diploid cell of the female only one X chromosomes
is active.
• Inactivation of X chromosome occurs randomly in somatic
cells during embryogenesis.
• Progeny of cells all have same inactivated X chromosome
as original (clonality), creating mosaic individual.
• X inactivation is irreversible.
• Inactivation of X involves heterochromatinization and late
replication of the chromosome.
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**Disconcordance in X-linked diseases between female monozygotic twins
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Heterozygous women for G6PD deficiency have two red
cell populations of erythrocytes
mono-alleleic expression rather than down/up-regulation of X-linked genes
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(Fialkow 1973)
Inactive X – characteristics
• Transcriptionally inactive
• Late replicating during S phase
• Epigenetic modifications
(CpG DNA methylation, histone modifications like H4 hypoacetylation,
H3K9Me and H3K27Me, HP1 binding)
• Heterochromatic (barr body)
• Peripheral nuclear location
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Pattern of X inactivation during mouse
development
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Steps in the inactivation process
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Counting (x:autosomes ratio)
Choice
Initiation and spreading
Maintenance
Embryonic stem (ES) cells as a model
system for X inactivation
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Undifferentiated embryonic cell lines
Derived from the inner cell mass (ICM) of blastocyst embryos
Can be genetically manipulated in culture
Can be injected into blastocysts to generate chimeric mice
Recapitulate X inactivation as they differentiate in vitro
XIC (X inactivation center)
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80kb region (Xq13)
Necessary and sufficient to cause X inactivation
Contains a regulatory element that affects the choice of X to inactivate (Xce)
Includes two noncoding RNA genes (Xist and Tsix)
(Lee at al. PNAS 1999)
Xist (X inactivation specific transcript)
• A large (17kb in human) untranslated RNA transcript
in the nucleus
• Exclusively expressed from the inactive X
• Coats the inactive X in somatic cells of females
• Required in cis for the initiation of X inactivation
• Contains a short repeat (RepA) at it’s 5' end
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Xist is exclusively expressed from the inactive X
Deletion analysis for Xist:
Targeted deletion into single Xist allele in XX ES cell line
In vitro differentiation of targeted cells
Expression analysis of single cell clones for polymorphic X-linked genes
Targeted deletion of the 129 strain Xist
allele in PGK12.1 ES cells
PGK-1 expression in single cell differentiated clones
A = PGK12.1 allele B=129 allele
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Results:
Xist is exclusively expressed from the inactive X in differentiated cells
of females
Conclusion: Inactivation fails to occur in cis on the X chromosome
bearing the deleted Xist allele (skewed inactivation)
(Penny et al. 1996)
Xist coats the inactive X
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X inactivation is triggered by Xist RNA
stabilization
RNA FISH for Xist
A-XY ES, B-XX ES, C-XY fibroblasts, D- XX fibroblasts,
E=7d XX embryo, F=XX diff. ES
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(Panning et al. 1997, Sheardown et al. 1997)
Silencing requires a conserved 5' element of
Xist (RepA)
Deletion analysis at the Xist gene:
Generation of various mutant Xist transgenes (D)
Transgene under the regulation of inducible promoter (Dox)
Targeted integration to the X-linked gene HPRT (single copy)
Introduction into male ES cells
Biologicl assay:
full transcript:
+Dox (Xist induction)
X inactivation in XY ES
100% cell death
-Dox (no Xist )
single X is active in XY
100% survival
DXist:
+/-Dox
differentiation
cell survival?
If +Dox has no effect than the deleted fragment is necessary for X
inactivation
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(Wutz et al. 2002)
Results:
• Deletion at the 5' of Xist (RepA) had no effect on cell survival.
• DRepA construct expressed a transcript that clusters to the X chromosome,
indicating that RepA is not responsible for proper localization on Xi.
Conclusion:
- The RepA containing region is responsible for X inacivation.
- Transcriptional silencing and chromosomal localization are functionally
separated.
RepA:
5' region of Xist
highly conserved between human and mouse
Contains 7.5 repeat units
Each repeat is predicted to form a secondary structure of 2 stem loops
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(Wutz et al. 2000)
Ectopic expression of Xist is sufficient
for chromosome-wide silencing
Established a tissue culture-based inducible expression system:
Deoxycycline-inducible 15kb Xist transgene (rtTA-Tg)
introduced into a XY ES cell line
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(Wutz et al. 2000)
Ectopic expression of Xist in undifferentiated Tg-ES cells
by dox treatment
Xist RNA and chromosome 11 DNA
Ectopic expression of Xist in Tg-ES cells by dox
treatment
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Reversible repression in Tg-ES cells
(Wutz et al. 2000)
Ectopic expression of Xist in differentiated Tg-ES cells by
dox treatment
Brdu incorporation and DNA chromosome paint
differentiated Tg-ES clone
Xist RNA and chromosome 11 DNA
Metaphase spreads of differentiated Tg-ES cells
Xist-Tg is expressed from the autosome
Histone H4 acetylation
Metaphase spreads of differentiated Tg-ES cells
Results:
Tg-Xist induces autosomal late replication and histone H4 hypoacetylation
as a result of differentiation
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(Wutz et al. 2000)
Xist-mediated silencing is restricted to the early
stages of differentiation
Xist RNA expression in Tg fibroblasts
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(Wutz et al. 2000)
Conclusions:
• Xist RNA expression in ES cells:
- Is sufficient for establishing chromosome-wide silencing
- Silencing is reversible
- Does not involve changes in replication timing and histone
hypoacetylation (data not shown)
• Xist expression in differentiated cells:
- Does not lead to silencing
- Is not required for maintaining the inactive state
• Xist expression during differentiation:
- Leads to irreversible inactivation
- Is accompanied by heterochromatinization
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(Wutz et al. 2000)
Xist is crucial for initiating silencing, but has no role in
maintaining the X inactive in the soma
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(Wutz et al. 2000)
How Xist RNA coating leads to transcriptional
silencing of X-linked genes?
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Chaumeil et al. 2006
Tsix
• 40kb antisense transcript
• Starts 12kb downstream to Xist and spans the entire length of Xist, and
beyond
• Negatively regulates Xist activity by overlap transcription
• Blocks inactivation on the future XA in both imprinted and random
inactivation
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Tsix RNA overlaps with Xist gene and is
transcribed in an antisense orientation
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Dynamic relationship between Tsix and Xist
Xist and Tsix expression during XX and XY ES differentiation
Xist RNA and Tsix RNA
• Exprssion is specific to undifferentiated ES regardless of sex
• Persists briefly at the onset of X inactivation, appearing only on the future
active X
• Disappears after X inactivation is established
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Lee et al. 1999
Tsix negatively regulates Xist activity
Targeted disruption of Tsix promoter in XY ES cells
In vitro differentiation (4 days)
Analysis of Xist expression and X inactivation markers
Xist RNA and histone H3K27methylation
Targeted cells following 4 days of differentiation
Results:
Ectopic up-regulation of Xist and X inactivation in differentiating male ES cells
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(Vigneau et al. 2006)
Tsix forms dsRNA duplexes with Xist, which are
processed into small noncoding RNA molecules
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(Ogawa et al 2008(
Xist:Tsix sncRNAs
Developmentally regulated:
Undetected in ES and fully differentiated cells (before and after X inactivation)
Present in ES cells while differentiating (during X inactivation)
Dicer-dependent (data not shown)
Xist:Tsix RNA duplexes
Xist and Tsix form duplex RNA molecules
developmentally regulated, present in undifferentiated ES and
down-regulated upon differentiation
primarily detected from the inactive X
Suggested model for Tsix function -
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(Ogawa et al 2008)
PRC2 - a chromatin remodeling complex
PRC2
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multimeric protein complex, termed Polycomb Repressive Complex 2
(PRC2) responsible for di- and tri-methylation of histone 3 at lysine 27
(H3K27)
core components (SUZI12, EED and EZH2) are conserved between fly and
vertebrates
PRC2 components and tri-methylated H3K27 (H3K27-3Me) are enriched
within the promoters of transcriptionally repressed genes
• A 1.6-kb noncoding RNA within Xist
• Contains the Repeat A region
• Present in both male and female before differentiation, but restricted to
females after differentiation and X inactivation
• Induces full-length Xist transcription and histone H3K27Me (data not shown)
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(Zhao et al. 2008)
Tsix competes with RepA on PRC2 binding
RepA:
• Direct target of PRC2 (Ezh2 as a direct binding unit)
• Tsix RNA inhibits RepA interaction with PRC2
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(Zhao et al. 2008)
Proposed model
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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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Xce (X chromosome controlling element) –
responsible for choosing
• Different alleles vary in their tendency to undergo X inactivation
(skewed inactivation)
• Deletions downstream to Xist result in skewed inactivation, only
inactivation of the deleted allele (Clerc and Avner 1998)
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Coupling X inactivation and differentiation
• Xist intron 1 binds to three main transcription factors underlying
pluripotency (Nanog, Oct3/4 and Sox2) in undifferentiated ES cells
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Release of all three factors from Xist triggers ectopic accumulation
of Xist RNA
Inappropriate Xist up-regulation in XY ES cells upon drastic silencing of Oct3/4
Xist RNA and Tsix RNA
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(Navarro et al. 2008(
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 ?
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