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X Chromosome Inactivation
The Big Epigenetic Event
• Males – 46,XY
• Females – 46,XX
• Compensate for gene dosage, all mammals
inactivate extra X chromosome
• Discovered by Mary Lyon
–
Lyon, M.F., 1961. Gene action in X-chromosome of the mouse (Mus musculus L).
Nature 190, pp. 372–373.
X Chromosome Inactivation
• Inactivated X chromosome called the Barr
body
• Highly condensed, heterochromatinized
• Euchromatin – relaxed, open – transcriptionally
active
• Heterochromatin – compact, condensed – poor
access by transcription factors
• Highly methylated
X Chromosome Inactivation
Peters et al. Nature Genetics 30, 77 – 80 (2002)
X Chromosome Inactivation
• Males are hemizygous for X chromosome
genes
• Females, upon inactivation of an X
chromosome, become functionally
hemizygous
X Chromosome Inactivation
• Some regions of the X chromosome are
not inactivated
• Pseudoautosomal regions between X and Y
• Small number of nonpseudoautosomal genes are
not inactivated
– ZFX
– RPS4X
– UBE1
• Homologous recombination
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
• X chromosome
inactivation is random
• Initial decision is then
clonal
• Females are thus
mosaics, comprised of
clonally derived tissue
expressing either Xp or
Xm.
Xm Xp
Xm Xp
Xm Xp
Xm Xp
Xm Xp
Xm Xp
Xm Xp
Xm Xp
Xm Xp
X Chromosome Inactivation
• Blood samples provide a source of
material to study mosaic X chromosome
assortment
Southern Blot
M F
XmXp
XmXp
XmXp
XmXp XmXp
XmXp
XmXp
XmXp
XmXp
XmXp m p
X X
XmXp
XmXp
XmXp
m
p
X X
50:50 m:p inactivation ratio
X Chromosome Inactivation
• Two separate controlling mechanisms:
Counting
Maintain 2:1
autosome/X
ratio
Choice
Nonrandom
Random
Klinefelter Syndrome
• Males 46,XY / 47,XXY Mosaic; 48,XXXY;
49XXXXY
• 1 in 500 to 1 in 1,000 male births
•
•
•
•
•
•
•
•
•
infertility
osteoporosis
learning, emotional, and mental disorders
autoimmune disorders such as lupus
low energy
low self esteem
communication difficulties
outbursts
developmental delays
Turner’s Syndrome
• Females – 45,X
•
•
•
•
1:2500
Short stature (mean height of 4'7")
infertility
high blood pressure
• 1:4 spontaneous abortions
• Females – 47,XXX
• Normal
X Chromosome Inactivation
Probe: anti-4x-methylH3-K9
What is this males karyotype?
X Chromosome Inactivation
• Two separate controlling mechanisms:
Counting
Maintain 2:1
autosome/X
ratio
Choice
Nonrandom
Random
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
What Controls TSIX Expression?
Molecular Cell, Vol 11, 731-743, March 2003
Xite, X-Inactivation Intergenic Transcription Elements that
Regulate the Probability of Choice
Yuya Ogawa and Jeannie T. Lee
Howard Hughes Medical Institute, Department of Genetics, Harvard Medical
School,Department of Molecular Biology, Massachusetts General Hospital, Boston, USA
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.
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
PLoS Genet. 2015 Aug; 11(8): e1005409.
Identifying Disease-Causing
Gene Variations
• Linkage analysis and Positional Cloning
– Clone disease gene without knowing anything
except the approximate chromosomal location
Recombination
• Recombination during meiosis separates loci
– More often when they are farther apart
– Less often when they are close
• Recall discussion of the Genetic Map
– Loci on separate chromosomes segregate
independently
– Loci on the same chromosome segregate as a
function of recombination
Recombination
13-1
13_06.jpg
Linkage analysis
• Linkage analysis locates the disease gene
locus
– Linkage analysis requires
• Clear segregation patterns in families
• Informative markers close to the locus
– Utilize LOD analysis to verify linkage
– Calculate cM distance between Loci
Positional Cloning
• Widely used strategy in human genetics
for cloning disease genes
• No knowledge of the function of the gene
product is necessary
• Strong for finding single-gene disorders
Positional Cloning
• Linkage analysis with polymorphic
markers establishes location of disease
gene
• LOD score analysis, and other methods
are employed
• Once we know the approximate location…
– The heavy molecular biology begins
Positional Cloning
• Example - Huntington’s disease
– CAG…
– Autosomal dominant
– 100% penetrance
– Fatal
– Late onset means patients often have children
Finding the Huntington Gene –
1981-1983
• Family with Huntington's disease found in
Venezuela
• Originated from a single founder - female
• Provided:
– Traceable family pedigree
– Informative meiosis
– Problem was… only a few polymorphic markers
where known at the time
Finding the Huntington Gene
• Blood samples taken
• Check for disease symptoms
• Paternity verified
Finding the Huntington Gene
• By luck, one haplotype segregated very
closely with Huntington disease
• Marker was an RFLP called G8 (later
called D4S10)
Finding the Huntington Gene
Finding the Huntington Gene
• Locate the region to the tip of the short
arm of chromosome 4 by linkage with G8
(D4S10)
• Maximum LOD score occurred at about 4
cM distance, i.e. 4 in 100 meiosis
Finding the Huntington Gene
• Together this started an international effort
to generate YAC clones of the 4 Mb region
• More polymorphisms were found
Finding the Huntington Gene
• Next, find an unknown gene in an
uncharacterized chromosome location
• Locate CpG islands
• Cross-species comparisons
• Further haplotype analysis suggested a 500 Kb
region 3’ to D4S10
Finding the Huntington Gene
• Exon trapping was key
• Compare cloned exons between normal
and Huntington disease patients
Finding the Huntington Gene
Finding the Huntington Gene
• One exon, called IT15, contained an
expanded CAG repeat….
• Mapping to 4 cM – 1983
• Cloning of Huntington gene – 1993
Complex Disease and
Susceptibility
Single gene disorders
Gene
Mendelian Inheritance
High penetrance
Low environmental influence (but
sometimes significant)
Gene
LOD-based linkage analysis works great
Genetic heterogeneity
Disease
Low population incidence
Complex Disease and
Susceptibility
Gene
Gene
Gene
Gene
Environment
Disease A
Disease B
Disease C
Multifactorial disorders