1. X Chromosome Inactivation Nature Genetics 30, 77 – 80 (2002) Peters et al.

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. 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 X Chromosome Inactivation

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

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

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

12. Tet Repressor Model

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

14. No Choice after 48 hrs XIST X

15. No inactivation after 48 hours XIST

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

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 J Med Genet. 1995 Feb;32(2):113-6. 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.

28. Ubiquitin – Amino Acid Conservation

29. Ubiquitin – Nucleotide Conservation * * * * * * * * * * * * * * * * * * * *

30. Amino Acid Conservation in Critical Domains

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32. 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‡

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47. 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 )

48. 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…

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

50. Huntington’s Disease GFP-Huntingtin GFP-polyQ 138 -Huntingtin Xia et al., Human Molecular Genetics, 2003, Vol. 12, No. 12 1393-1403

51. Heterozygous knockouts are normal!

52. Transgenic Mouse

58. Trinucleotide Repeat Polymorphism DiseaseGene Location Repeat Sequence Normal Repeat Mutant Repeat Huntington4p16.3CAG9-3637-150 SCA16p23CAG19-3643-81 Myotonic Dystrophy 19q13CTG5-3650-4000