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X Chromosome Inactivation

X Chromosome Inactivation. Peters et al. Nature Genetics 30 , 77 – 80 (2002). X Chromosome Inactivation. X chromosome inactivation occurs early during development – around 24 cell Thus, females embryos have two active X chromosomes until one is inactivated. X Chromosome Inactivation.

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X Chromosome Inactivation

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  1. X Chromosome Inactivation Peters et al. Nature Genetics30, 77 – 80 (2002)

  2. X Chromosome Inactivation • X chromosome inactivation occurs early during development – around 24 cell • Thus, females embryos have two active X chromosomes until one is inactivated

  3. X Chromosome Inactivation

  4. Probe: anti-4x-methylH3-K9 What is this males karyotype?

  5. What Determines X-chromosome Inactivation?

  6. X Chromosome Inactivation • Mechanism of X Chromosome inactivation • XIC – X chromosome Inactivation Center • XIC controls expression of the XIST gene • XIST: X-inactive-specific transcript • XIST produces a non-coding 17 kb RNA molecule • “Coats” the entire local X-chromosome – cis-acting

  7. EMBO Rep. 2007 January; 8(1): 34–39. doi: 10.1038/sj.embor.7400871.

  8. X Chromosome Inactivation • X chromosome inactivation requires: • Initial XIST RNA expression and coating • Association of chromatin modifying proteins • DNA methylation 5’ of X-chromosome genes • Modification of histones by methyltransferases (HMTase) • Other chromatin modifying proteins

  9. Nature, January 1996 X Chromosome Inactivation • Approaches for examining XIST biology 1) Knock it out!

  10. ES cell Dffr 100/0 XIST knockout in mouse ES cells ES cell Dffr or 50/50

  11. X Chromosome Inactivation • Approaches for examining XIST biology • 2) Knock it in!

  12. Tet Repressor Model

  13. XIST XIST XIST inactivation is Reversible up to 48 hours X

  14. XIST XIST No Choice after 48 hrs X

  15. XIST No inactivation after 48 hours

  16. XIST acts Early During Development and is Irreversible

  17. What Controls XIST Expression?

  18. TSIX is the Anti-Sense Strand of the XIST gene

  19. TSIX is the Anti-Sense Stand of the XIST gene

  20. X Knock-down of TSIX Causes Skewed X-Chromosome Inactivation

  21. TSIX Asymmetry Governs Choice • TSIX must be downregulated for XIST expression on the (future) inactivated X Chromosome • TSIX expression must remain for XIST downregulation on the (future) activated X Chromosome

  22. Human Pathology • Without XIST, Human X Chromosome aneuploidy is Severe Molecular cytogenetic characterisation of a small ring X chromosome in a Turner patient and in a male patient with congenital abnormalities: role of X inactivation.Callen DF, Eyre HJ, Dolman G, Garry-Battersby MB, McCreanor JR, Valeba A, McGill JJ.

  23. Ubiquitin – Amino Acid Conservation

  24. * * * * * * * * * * * * * * * * * * * * Ubiquitin – Nucleotide Conservation

  25. Amino Acid Conservation in Critical Domains

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  27. Nature393, 79 - 82 (07 May 1998) Errors in Protein Function • Eg. Cystic Fibrosis • Mutation causes loss-of-function • High occurrence of error may be a result of a heterozygote advantage Salmonella typhi uses CFTR to enter intestinal epithelial cells GERALD B. PIER*, MARTHA GROUT*, TANWEER ZAIDI*, GLORIA MELULENI*, SIMONE S. MUESCHENBORN*, GEORGE BANTING†, ROSEMARY RATCLIFF‡, MARTIN J. EVANS§ & WILLIAM H. COLLEDGE‡

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  41. Huntington’s Disease • CAG repeat codes for glutamine (Q) • polyQ located near the N-terminus of Huntingtin protein • Expansion in the coding region of the gene (unlike, for eg. FMR1 – Fragile X syndrome - expansion is in 5’ UTR )

  42. Huntington’s Disease MATLEKLMKA FESLKSFQQQ QQQQQQQQQQ QQQQQQQQQQ PPPPPPPPPP PQLPQPPPQA QPLLPQPQPP PPPPPPPPGP AVAEEPLHRP KKELSATKKD RVNHCLTICE NIVAQSVRNS PEFQKLLGIA MELFLLCSDD AESDVRMVAD ECLNKVIKAL MDSNLPRLQL ELYKEIKKNG APRSLRAALW RFAELAHLVR PQKCRPYLVN LLPCLTRTSK RPEESVQETL AAAVPKIMAS FGNFANDNEI KVLLKAFIAN LKSSSPTIRR TAAGSAVSIC QHSRRTQYFY SWLLNVLLGL LVPVEDEHST LLILGVLLTL RYLVPLLQQQ VKDTSLKGSF GVTRKEMEVS PSAEQLVQVY ELTLHHTQHQ DHNVVTGALE LLQQLFRTPP PELLQTLTAV GGIGQLTAAK EESGGRSRSG SIVELIAGGG SSCSPVLSRK QKGKVLLGEE EALEDDSESR SDVSSSALTA SVKDEISGEL AASSGVSTPG SAGHDIITE……… MATLEKLMKA FESLKSFQQQ QQQQQQQQQQ QQQQQQQQQQ QQQQQQQQQQ QQQQQQQQQQ QQQQQQQQQQ QQQQQQQQQQ PPPPPPPPPP PQLPQPPPQA QPLLPQPQPP PPPPPPPPGP AVAEEPLHRP KKELSATKKD RVNHCLTICE NIVAQSVRNS PEFQKLLGIA MELFLLCSDD AESDVRMVAD ECLNKVIKAL MDSNLPRLQL ELYKEIKKNG APRSLRAALW RFAELAHLVR PQKCRPYLVN LLPCLTRTSK RPEESVQETL AAAVPKIMAS FGNFANDNEI KVLLKAFIAN LKSSSPTIRR TAAGSAVSIC QHSRRTQYFY SWLLNVLLGL LVPVEDEHST LLILGVLLTL RYLVPLLQQQ VKDTSLKGSF GVTRKEMEVS PSAEQLVQVY ELTLHHTQHQ DHNVVTGALE LLQQLFRTPP PELLQTLTAV GGIGQLTAAK EESGGRSRSG SIVELIAGGG SSCSPVLSRK QKGKVLLGEE EALEDDSESR SDVSSSALTA SVKDEISGEL AASSGVSTPG SAGHDIITE…

  43. Huntington CAG Repeat P.Sudbery, Human Molecular Genetics 2nd ed, Prentice Hall. PCR analysis of CAG repeat length in family with Huntington’s disease

  44. Huntington’s Disease GFP-Huntingtin GFP-polyQ138-Huntingtin Xia et al., Human Molecular Genetics, 2003, Vol. 12, No. 12 1393-1403

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