Presentation on theme: "X-inactivation 20/11/08. Males are haploid for X-linked genes expression."— Presentation transcript:
Males are haploid for X-linked genes expression
Barr body discovered (1949)
Lyonization=X-inactivation The hypothesis: 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 (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 renewal- Unlimited division while maintaining a pluripotent undifferentiated state in-vitro. II. Differentiation- The ability to differentiate to cells from the three embryonic germ layers (in-vivo and in-vitro).
Neo- positive selection for integration. HSV-TK- negative selection for recombination that is not homologous (enters randomly on any chromosome) Targeted deletion of Xist in ES cells (1996) Xist allele of 129 (From 129/PGK heterozygote cell line) was deleted using Homologous Recombination. 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 ” ).
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. 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. In-activation by Xist is in CIS Bi-allelic expression because both X- chromosomes are active in ES. Random X-inactivation of both the PGK and 129 X- chromosomes in WT ES because X-chromosome related genes are expressed randomly in sub-clones.
Xist RNA coats the inactive X (FISH )
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. ?
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? 20
Nanog Oct4 and Sox2 are the master regulators of the pluripotent undifferentiated state of ES cells Oct4 immunostainingSox2 immunostainingNanog immunostaining Undifferentiated pluripotent ES colony (very small round cells) cultured in-vitro. ES nucleus is positively stained (pink) for Nanog
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. Binding site of Nanog in intron1 of Xist WT ES=>Xist is repressed. -/- Nanog ES=> Xist is upregulated.
Xist expression following downregulation of three TFs TC induce differentiation by down regulating Oct4 expression.
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? Inconsistencies between syndromes and X inactivation Escape from X-inactivation ? 24 Carrel et al (2005) showed that ~15% of the genes on the inactive-X escaped from inactivation, complicating the story even further..