Upload
fordon
View
50
Download
0
Tags:
Embed Size (px)
DESCRIPTION
כרומוזומי הזוויג תורשת האדם 13.11.08 -. 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 - PowerPoint PPT Presentation
Citation preview
כרומוזומי הזוויג -13.11.08תורשת האדם
1
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.
2
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
4
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)
5
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)
6
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)
7
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
0 1
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
10
• 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)
11
**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
14
Pattern of X inactivation during mouse development
15
Steps in the inactivation process
• Counting (x:autosomes ratio)• Choice• Initiation and spreading• Maintenance
16
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
17
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
19
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
20
Xist coats the inactive X
21
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)
23
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
24
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
26
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
27
Xist-mediated silencing is restricted to the early stages of differentiation
(Wutz et al. 2000)
Xist RNA expression in Tg fibroblasts
28
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
30
Chaumeil et al. 2006
How Xist RNA coating leads to transcriptional silencing of X-linked genes?
31
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
32
Tsix RNA overlaps with Xist gene and is transcribed in an antisense orientation
33
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
34
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
35
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 -
37
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
38
)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(
39
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
41
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?
42
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)
43
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
44
• 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 ?
45