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כרומוזומי הזוויג תורשת האדם 13.11.08 - 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 2 • Was finally cloned by Sinclair (1990) • 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. 3 (Nature 351:117 (1991)) Genotypically female mice transgenic for Sry are phenotypically male XY male 4 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) 5 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) 6 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) 7 Barr body in females Neuronal nuclei of female cats XX XXXXX Number of Barr bodies = n-1 rule 8 Barr et al. 1949 Barr Bodies are Inactivated X Chromosomes in Females 9 0 1 2 3 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 10 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. 11 **Disconcordance in X-linked diseases between female monozygotic twins 12 Heterozygous women for G6PD deficiency have two red cell populations of erythrocytes mono-alleleic expression rather than down/up-regulation of X-linked genes 13 (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 14 Pattern of X inactivation during mouse development 15 Steps in the inactivation process • • • • 16 Counting (x:autosomes ratio) Choice Initiation and spreading Maintenance Embryonic stem (ES) cells as a model system for X inactivation • • • • • 17 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) • • • • 18 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 19 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 20 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 21 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 22 (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 23 (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 24 (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 25 (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 26 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 27 (Wutz et al. 2000) Xist-mediated silencing is restricted to the early stages of differentiation Xist RNA expression in Tg fibroblasts 28 (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 29 (Wutz et al. 2000) Xist is crucial for initiating silencing, but has no role in maintaining the X inactive in the soma 30 (Wutz et al. 2000) How Xist RNA coating leads to transcriptional silencing of X-linked genes? 31 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 32 Tsix RNA overlaps with Xist gene and is transcribed in an antisense orientation 33 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 34 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 35 (Vigneau et al. 2006) Tsix forms dsRNA duplexes with Xist, which are processed into small noncoding RNA molecules 36 (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 - 37 (Ogawa et al 2008) PRC2 - a chromatin remodeling complex PRC2 • • • 38 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) 39 (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 40 (Zhao et al. 2008) 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 • 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 (Navarro et al. 2008( Inconsistencies between syndromes and X inactivation • • 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 ? 45