BI2332: Concepts of Disease - Epigenetics and Underlying Principles PDF

BI2332: Concepts of Disease Epigenetics and underlying principles Professor Ros John CONTENT WARNING: SOME GRAPHIC IMAGES AHEAD. Images are shown as representative samples in aid of teaching the material, not for shock value. Epigenetics and underlying...

BI2332: Concepts of Disease Epigenetics and underlying principles Professor Ros John CONTENT WARNING: SOME GRAPHIC IMAGES AHEAD. Images are shown as representative samples in aid of teaching the material, not for shock value. Epigenetics and underlying principles 3 Professor John X-inactivation  X Y p SRY = testes determining p factor q < 60 million base pairs < 200 genes (27 distinct Y-specific protein-coding genes) q > 150 million base pairs > 1,400 genes Electron 168 Mendelian diseases linked to mutations in Micrograph of 113 X-linked genes (Wellcome Trust Sanger X and Y Institute) chromosome  X-inactivation Mammals use epigenetic mechanisms to inactivate one of the X chromosomes in female cells = dosage compensation Y X Male Y X X X child Barr body X X X X Father Mother or Female children The inactive X chromosome adopts a heterochromatic structure ‘ The Barr Body’  Modified higher order chromatin organisation on the inactive X chromosome Active X chromosome Nucleus Xist RNA Inactive X chromosome Expressed gene Silenced gene Repeat element  The X inactivation centre (XIC); a cis-acting master switch locus that controls X inactivation Inactivation starts at the X-inactivation centre ] XIC = human Xic = mouse X chromosome X inactive specific transcript (Xist) underpins XIC function 15-17kb spliced and polyadenylated non-coding transcript Transcribed only from the inactive X chromosome elect Xist RNA coats the inactive X chromosome territory Xist is necessary and sufficient for X inactivation Xist present as a cloud-like structure in the nucleus DAPI (blue) Xist RNA (green) Xist is expressed from XIC Xist RNA Xist gene A second non-coding RNA called Tsix is expressed antisense to Xist Xist Tsix gene (antisense to Tsix RNA Xist gene) Mouse  Xist inactivation is a sequential process Differentiation /Development Xist domain Primary silencing Secondary silencing Maintenance formation factors factors phase Xist Xist Developmental window of opportunity  Chromatin features of the X chromosomes Active X chromosome (Xa) Histone tails acetylated Histone H3 lysine 4 tri-methylation on promoters Variant histones H3.3 and H2Abb enriched Polycomb histone modifications only at silent loci Gene promoters depleted of DNA methylation DNA methylation in gene bodies Inactive X chromosome (Xi) Histone tails hypo-acetylated Histone H3 lysine 4 methylation depleted Xist RNA Polycomb histone modifications enriched Variant histone macroH2A enriched DNA methylation of gene promoters DNA hypo-methylation in gene bodies Acetylated lysine Methylated H3 lysine 27 CpG dinucleotide KEY Methylated H3 lysine 4 Ubiquitylated H2A lysine 119 Methyl CpG dinucleotide  Xist is no longer required once inactivation is established Maintained by DNA methylation  a A a A a A a A a A a A a A Random a A a a A a A a A A a A a A X-inactivation A a a a A A Clonal A A a a inheritance of inactive X a a a a A A A A A A A A a a a a Inactivation of paternal X Inactivation of maternal X Calico cat  The first cloned cat, ‘Carbon Copy’, was created from a calico cat. Mum Clone Coat colour! Cloning reactivates both X-chromosomes. Inactivation recurs randomly resulting in an entirely different coat pattern even though the two individuals are genetically identical. Genetic disorders influenced by X-inactivation Some Mendelian genetics diseases are epigenetic because they involve genes on the X chromosome  X-linked recessive A person has a recessive mutation in one of the genes on the X. Parent-to-child transmission: If the mutant gene is passed from the female parent: 50% of male children will have the disease 50% of female children will be carriers without the disease If the mutant gene is passed from the male parent: None of male children will have the disease All female children will be carriers Rarely, some females will inherit two mutant X chromosomes and will have the disease (homozygous). Difference in sex of transmission and inheritance  A a A XA Y XA Xa Daughter Daughter Son Son A A a A A a Normal Unaffected carrier Normal Affected  A A a Xa Y XA XA Daughter Daughter Son Son A a A a A A Unaffected carrier Unaffected carrier Normal Normal  Mode of inheritance XY *XX *XX XY XX *XY *XY XX XY XY *XX *XX  Haemophilia A OMIM 306700 Estimated frequency at about 5 in 100,000 male live births. X-linked recessive Genetic deficiency of blood clotting factor VIII (HEMA gene). Symptoms - joint and muscle haemorrhages, easy bruising, and prolonged haemorrhage after surgery or trauma.  X-linked Hypohidrotic Ectodermal Dysplasia Arise from OMIM 305100 ectodermal Occurs in 1 in 100,000 lineages X-linked recessive disorder apparent in males and females Localised at Xq12-q13.1 Mutation in ectodysplasin-A (EDA) Symptoms - defect in the hair, teeth and sweat glands = inability to sweat which can result in life-threatening and brain-damaging episodes of hyperthermia.. Starch-iodine test: Functional sweat glands = areas of brown colouration. Yellow = area without sweatglands  X-linked dominant A person has a dominant mutation in one of the genes on the X Parent-to-child transmission: If the mutant gene is passed from the female parent (who is affected): 50% of all children will have the disease If the mutant gene is passed from the male parent: None of male children will have the disease All of female children will be affected Difference in sex of transmission and inheritance Dominant  Rett Syndrome OMIM 312750 Frequency 5 in 100,000 primarily affecting females (lethal in males) X-linked dominant Mutations in the methyl-CpG-binding protein-2 (MECP2) Maps to Xq28 Vast majority of cases are sporadic Symptoms appear after normal development from 7-18 months Autism, dementia, ataxia, and loss of purposeful hand use, regression of acquired skills, loss of speech, stereotypical movements of the hands, microcephaly, seizures, and mental retardation. Shorter lifespan  Recessive Heterozygous for XY *XX recessive conditions but has phenotype *XX XY XX *XY *XY XX XY XY *XX *XX Dominant Heterozygous for dominant mutation but has no phenotype Females with a recessive X-linked condition can manifest some symptoms. Females with a dominant X-linked condition can be phenotypically normal  Recessive a A A = normal allele a A a A a = mutated allele a A a A a A a A Random a A a a A a A a A A a A a A X-inactivation A a a a A A Clonal A A a a inheritance of inactive X a a a a A A A A A A A A a a a a Cells mutant Cells normal (inactivation of normal allele) (inactivation of mutant allele) Where the phenotype depends on a circulating product, as in hemophilia, there is an averaging effect between the normal and abnormal cells. Female carriers have an intermediate phenotype, and are usually clinically unaffected but biochemically abnormal. Ex. Haemophillia A Where the phenotype is a localised property of individual cells, as in hypohidrotic ectodermal dysplasia or DMD, female carriers can show patches of normal and abnormal tissue. Ex. Hypohidrotic ectodermal dysplasia Hypohidrotic ectodermal dysplasia and heterozygous females Mutation  X-linked disorders and heterozygous females Females with same mutation can have very different phenotypes Will depend on with X-chromosome predominantly inactivated Dominant mutations If their mutant X inactivated = “normal” phenotype Recessive mutations If their normal X inactivated = “disease phenotype” Skewed X-inactivation  X-linked disorders and heterozygous females Monozygotic female twins can be discordant for X-linked conditions This can be explained if both twins carry the same mutation on one of their X-chromosome but one twin has predominantly inactivated their mutant X (normal phenotype) while the affected twin has predominantly inactivated their normal X Skewed X-inactivation  Cure for Rett Syndrome? Rett syndrome is caused by mutations in methyl-CpG- binding protein 2 (MECP2) 70 kD Binds single symmetrically methylated CpG *CG GC* MECP2 encodes a nuclear protein (MECP2) which is especially abundant within neurons MECP2 functions as a global transcriptional regulator by binding specifically to methylated DNA Knock-out mouse recapitulates a broad spectrum of phenotypes seen in human patients Milestone study - restoring MECP2 expression reversed neurological symptoms and normalised lifespan (Professor Adrian Bird - led the team which first identified CpG islands) Is X chromosome reactivation possible? Normally X chromosome reactivation can only during very early stages of embryonic development Reprogrammed somatic cells into iPS cells in vitro results in both X being active but……… The "dual modality" in vivo approach 1) Xist-binding antisense oligonucleotide (a synthetic nucleic acid strand that binds to and degrades an RNA molecule) 2) DNA methylation inhibitor called 5-aza-2'- deoxycytidine (AzaC) Combined treatment increased MECP2 expression from the inactive chromosome up to 30,000-fold ……in cell lines But mice lacking Xist expression in their blood develop cancer Brain-specific approach Knock-out Xist only in brain cells in mice (using a conditional Xist allele and a brain-specific cre recombinase) But not applicable to humans And AzaC is toxic when administered over a long time course Potential avenue of treatment is introducing a normal copy of MECP2 into cells by gene therapy  Learning outcomes/Summary Inequality between X and Y resolved through X-inactivation X-inactivation is a sequential epigenetic process originating at the XIC X-inactivation involves non-coding RNAs, histone modification, DNA methylation and histone variants X-inactivation is clonal X-linked genetic conditions show Mendelian patterns of inheritance BUT some females carrying dominant mutations can be phenotypically normal and some females carrying recessive mutations can be phenotypically abnormal due to skewed X-inactivation