X Chromosome Inactivation
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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 Probe: anti-4x-methylH3-K9 What is this males karyotype? What Determines X-chromosome Inactivation? 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 – cisacting EMBO Rep. 2007 January; 8(1): 34–39. doi: 10.1038/sj.embor.7400871. X Chromosome Inactivation • X chromosome inactivation requires: • Initial XIST RNA expression and coating • Association of chromatin modifying proteins • DNA methylation 5’ of Xchromosome genes • Modification of histones by methyltransferases (HMTase) • Other chromatin modifying proteins X Chromosome Inactivation • Approaches for examining XIST biology 1) Knock it out! Nature, January 1996 XIST knockout in mouse ES cells ES cell Dffr or ES cell Dffr 100/0 50/50 X Chromosome Inactivation • Approaches for examining XIST biology 2) Knock it in! Tet Repressor Model XIST inactivation is Reversible up to 48 hours XIST X XIST No Choice after 48 hrs XIST X XIST No inactivation after 48 hours XIST XIST acts Early During Development and is Irreversible What Controls XIST Expression? TSIX is the Anti-Sense Strand of the XIST gene TSIX is the Anti-Sense Stand of the XIST gene Knock-down of TSIX Causes Skewed X-Chromosome Inactivation X 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 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. J Med Genet. 1995 Feb;32(2):113-6. Ubiquitin – Amino Acid Conservation * * Ubiquitin – Nucleotide Conservation * * * * * * * * * * * * * * * * * * Amino Acid Conservation in Critical Domains 11_03.jpg Errors in Protein Function • Eg. Cystic Fibrosis • Mutation causes loss-of-function • High occurrence of error may be a result of a heterozygote advantage Nature 393, 79 - 82 (07 May 1998) 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‡ 11_07.jpg 11_09.jpg 11_10.jpg 11_10_2.jpg 11_10_3.jpg 11_16.jpg 11_16_2.jpg 11_11.jpg 11_11_2.jpg 11_12.jpg 11_13.jpg 11_05.jpg 11_05_2.jpg 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 ) Huntington’s Disease MATLEKLMKA QPLLPQPQPP PEFQKLLGIA APRSLRAALW FGNFANDNEI LVPVEDEHST ELTLHHTQHQ SIVELIAGGG AASSGVSTPG FESLKSFQQQ QQQQQQQQQQ PPPPPPPPGP AVAEEPLHRP MELFLLCSDD AESDVRMVAD RFAELAHLVR PQKCRPYLVN KVLLKAFIAN LKSSSPTIRR LLILGVLLTL RYLVPLLQQQ DHNVVTGALE LLQQLFRTPP SSCSPVLSRK QKGKVLLGEE SAGHDIITE……… QQQQQQQQQQ KKELSATKKD ECLNKVIKAL LLPCLTRTSK TAAGSAVSIC VKDTSLKGSF PELLQTLTAV EALEDDSESR PPPPPPPPPP RVNHCLTICE MDSNLPRLQL RPEESVQETL QHSRRTQYFY GVTRKEMEVS GGIGQLTAAK SDVSSSALTA PQLPQPPPQA NIVAQSVRNS ELYKEIKKNG AAAVPKIMAS SWLLNVLLGL PSAEQLVQVY EESGGRSRSG SVKDEISGEL MATLEKLMKA QQQQQQQQQQ AVAEEPLHRP AESDVRMVAD PQKCRPYLVN LKSSSPTIRR RYLVPLLQQQ LLQQLFRTPP QKGKVLLGEE FESLKSFQQQ QQQQQQQQQQ KKELSATKKD ECLNKVIKAL LLPCLTRTSK TAAGSAVSIC VKDTSLKGSF PELLQTLTAV EALEDDSESR QQQQQQQQQQ PQLPQPPPQA NIVAQSVRNS ELYKEIKKNG AAAVPKIMAS SWLLNVLLGL PSAEQLVQVY EESGGRSRSG SVKDEISGEL QQQQQQQQQQ QPLLPQPQPP PEFQKLLGIA APRSLRAALW FGNFANDNEI LVPVEDEHST ELTLHHTQHQ SIVELIAGGG AASSGVSTPG QQQQQQQQQQ PPPPPPPPGP MELFLLCSDD RFAELAHLVR KVLLKAFIAN LLILGVLLTL DHNVVTGALE SSCSPVLSRK SAGHDIITE… QQQQQQQQQQ PPPPPPPPPP RVNHCLTICE MDSNLPRLQL RPEESVQETL QHSRRTQYFY GVTRKEMEVS GGIGQLTAAK SDVSSSALTA Huntington CAG Repeat P.Sudbery, Human Molecular Genetics 2nd ed, Prentice Hall. PCR analysis of CAG repeat length in family with Huntington’s disease Huntington’s Disease GFP-Huntingtin GFP-polyQ138-Huntingtin Xia et al., Human Molecular Genetics, 2003, Vol. 12, No. 12 1393-1403 Heterozygous knockouts are normal! Transgenic Mouse Trinucleotide Repeat Polymorphism Disease Gene Location Repeat Sequence Normal Repeat Mutant Repeat Huntington 4p16.3 CAG 9-36 37-150 SCA1 CAG 19-36 43-81 CTG 5-36 50-4000 6p23 Myotonic 19q13 Dystrophy
