כרומוזומי הזוויג -13.11.08תורשת האדם

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SRY gene

• Sex determining factor (TDF)• Intronless gene which initiates male sex determination • Transcription factor (HMG-box family of DNA proteins)• Mutations in SRY give rise to XY females with gondal dysgenesis• Identified through Y-associated chromosomal aberrations:

– Translocations to X in rare XX males– Deletions in rare XY females

• Was finally cloned by Sinclair (1990)• The “home run” experiment by Koopman et al. (1991) used transgenic mice.

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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.

(Nature 351:117 (1991))3

Genotypically female mice transgenic for Sry are phenotypically male

XY male XX male

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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 XYChromosome X: relatively large, approximately 1100 genes, most are

active in somatic cells, critical for survivalChromosome Y: very small, only 45 genes, required for male sex

determination and spermatogenesis, dispensable for survival (XX)

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Dosage compensationProblem: 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

Number of Barr bodies = n-1 ruleXX XXXXX

Neuronal nuclei of female cats

Barr et al. 19498

Barr Bodies are Inactivated X Chromosomes in Females

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2 3

Susumu Ohno 1959: two X chromosomes in mammals appear differentlyAll but one X chromosome are silenced per diploid set of chromosomes9

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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• 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.

The Lyon Hypothesis of X Inactivation(Mary Lyon and Liane Russell 1961)

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**Disconcordance in X-linked diseases between female monozygotic twins12

Heterozygous women for G6PD deficiency have two red cell populations of erythrocytes

mono-alleleic expression rather than down/up-regulation of X-linked genes

(Fialkow 1973)13

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

• Counting (x:autosomes ratio)• Choice• Initiation and spreading• Maintenance

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Embryonic stem (ES) cells as a model system for X inactivation

• 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

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XIC (X inactivation center)

• 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)18

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

(Penny et al. 1996)

Results: Xist is exclusively expressed from the inactive X in differentiated cells

of femalesConclusion: Inactivation fails to occur in cis on the X chromosome

bearing the deleted Xist allele (skewed inactivation)

Targeted deletion of the 129 strain Xist allele in PGK12.1 ES cells

PGK-1 expression in single cell differentiated clonesA = PGK12.1 allele B=129 allele

Deletion analysis for Xist:Targeted deletion into single Xist allele in XX ES cell lineIn vitro differentiation of targeted cellsExpression analysis of single cell clones for polymorphic X-linked genes

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Xist coats the inactive X

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X inactivation is triggered by Xist RNA stabilization

RNA FISH for XistA-XY ES, B-XX ES, C-XY fibroblasts, D- XX fibroblasts, E=7d XX embryo, F=XX diff. ES

(Panning et al. 1997, Sheardown et al. 1997)22

(Wutz et al. 2002)

Deletion analysis at the Xist gene:Generation of various mutant Xist transgenes (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% survivalXist: +/-Dox differentiation cell survival?

If +Dox has no effect than the deleted fragment is necessary for X inactivation

Silencing requires a conserved 5' element of Xist (RepA)

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Results:• Deletion at the 5' of Xist (RepA) had no effect on cell survival.•RepA 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.

(Wutz et al. 2000)

RepA: 5' region of Xisthighly conserved between human and mouseContains 7.5 repeat unitsEach repeat is predicted to form a secondary structure of 2 stem loops

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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

(Wutz et al. 2000)25

(Wutz et al. 2000)

Xist RNA and chromosome 11 DNAEctopic expression of Xist in Tg-ES cells by dox treatment

Reversible repression in Tg-ES cells

Ectopic expression of Xist in undifferentiated Tg-ES cells by dox treatment

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Xist RNA and chromosome 11 DNAMetaphase spreads of differentiated Tg-ES cellsXist-Tg is expressed from the autosome

Histone H4 acetylationMetaphase spreads of differentiated Tg-ES cells

(Wutz et al. 2000)

Brdu incorporation and DNA chromosome paintdifferentiated Tg-ES clone

Results:Tg-Xist induces autosomal late replication and histone H4 hypoacetylation as a result of differentiation

Ectopic expression of Xist in differentiated Tg-ES cells by dox treatment

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Xist-mediated silencing is restricted to the early stages of differentiation

(Wutz et al. 2000)

Xist RNA expression in Tg fibroblasts

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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

(Wutz et al. 2000)29

(Wutz et al. 2000)

Xist is crucial for initiating silencing, but has no role in Xist is crucial for initiating silencing, but has no role in maintaining the X inactive in the somamaintaining the X inactive in the soma

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Chaumeil et al. 2006

How Xist RNA coating leads to transcriptional silencing of X-linked genes?

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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

• 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

Lee et al. 1999

Xist RNA and Tsix RNA

Xist and Tsix expression during XX and XY ES differentiation

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Tsix negatively regulates Xist activityTargeted disruption of Tsix promoter in XY ES cellsIn vitro differentiation (4 days)Analysis of Xist expression and X inactivation markers

(Vigneau et al. 2006)

Results:Ectopic up-regulation of Xist and X inactivation in differentiating male ES cells

Xist RNA and histone H3K27methylationTargeted cells following 4 days of differentiation

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Tsix forms dsRNA duplexes with Xist, which are processed into small noncoding RNA molecules

((Ogawa et al 200836

Xist:Tsix sncRNAsDevelopmentally 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 duplexesXist and Tsix form duplex RNA moleculesdevelopmentally regulated, present in undifferentiated ES and down-regulated upon differentiationprimarily detected from the inactive X

(Ogawa et al 2008)

Suggested model for Tsix function -

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PRC2

PRC2 - a chromatin remodeling complex

• 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

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)Zhao et al. 2008(

• 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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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

)Zhao et al. 2008(40

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

((Navarro et al. 2008

• Xist intron 1 binds to three main transcription factors underlying pluripotency (Nanog, Oct3/4 and Sox2) in undifferentiated ES cells

• 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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• 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?

Inconsistencies between syndromes and X inactivation

Escape from X-inactivation ?

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