Transcript Lyon stand
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