X Chromosome Inactivation

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

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X Chromosome Inactivation
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Probe anti-4x-methylH3-K9 What is this males
karyotype?
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What Determines X-chromosome Inactivation?
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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

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EMBO Rep. 2007 January 8(1) 3439. doi
10.1038/sj.embor.7400871.
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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

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X Chromosome Inactivation

• Approaches for examining XIST biology
• 1) Knock it out!

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XIST knockout in mouse ES cells
ES cell
Dffr
or
50/50
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X Chromosome Inactivation

• Approaches for examining XIST biology
• 2) Knock it in!

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Tet Repressor Model
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XIST inactivation is Reversible up to 48 hours
X
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No Choice after 48 hrs
X
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No inactivation after 48 hours
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XIST acts Early During Development and is
Irreversible
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What Controls XIST Expression?
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TSIX is the Anti-Sense Strand of the XIST gene
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TSIX is the Anti-Sense Stand of the XIST gene
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Knock-down of TSIX Causes Skewed X-Chromosome
Inactivation
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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

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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 Feb32(2)113-6.
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Ubiquitin Amino Acid Conservation
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Ubiquitin Nucleotide Conservation
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Amino Acid Conservation in Critical Domains
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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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Huntingtons 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 )

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Huntingtons 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
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Huntington CAG Repeat
P.Sudbery, Human Molecular Genetics 2nd ed,
Prentice Hall.
PCR analysis of CAG repeat length in family with
Huntingtons disease
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Huntingtons Disease
GFP-Huntingtin
GFP-polyQ138-Huntingtin
Xia et al., Human Molecular Genetics, 2003, Vol.
12, No. 12 1393-1403
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Heterozygous knockouts are normal!
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Transgenic Mouse
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Trinucleotide Repeat Polymorphism
Disease Gene Location Repeat Sequence Normal Repeat Mutant Repeat
Huntington 4p16.3 CAG 9-36 37-150
SCA1 6p23 CAG 19-36 43-81
Myotonic Dystrophy 19q13 CTG 5-36 50-4000