Systemic injection of Mecp2Bnull/y mice with scAAV9/MeCP2 virus results in MeCP2 expression in different cell types in brain. Systemic injection of Mecp2Bnull/y mice with scAAV9/MeCP2 virus results in MeCP2 expression in different cell types in brain. A, Schematic of scAAV9/MeCP2 vector. Mouse Mecp2-e1 is cloned downstream of 730 bp fragment of Mecp2 promoter. Other abbreviations are as noted in the legend to Figure 1. B, Experimental paradigm. C, Immunostaining of MeCP2 expression in different brain regions. Cell counts are relative to total DAPI-positive cells (36 sections, n = 2 mice). D, Expression of MeCP2 in neurons and non-neuronal cells varies with brain region. Cell counts are relative to indicated cell-specific marker (12 sections, n = 2 mice). *p < 0.05, **p < 0.01, ***p < 0.001, NS by one-way ANOVA (Newman–Keuls multiple-comparison test). Data are means ± SEM. E, Western blot of MeCP2 protein in different brain regions. Lane 1, Mecp2+/y; Lane 2, Mecp2Bnull/y; and Lane 3, Mecp2Bnull/y-AAV9/MeCP2. Garg S K et al. J. Neurosci. 2013;33:13612-13620 ©2013 by Society for Neuroscience

MeCP2 expressed from virus binds to DNA, restores normal neuronal somal size, and improves survival. MeCP2 expressed from virus binds to DNA, restores normal neuronal somal size, and improves survival. A, Ectopic MeCP2 localizes to DAPI+ heterochromatin puncta in Mecp2Bnull/y-scAAV9/MeCP2–injected mice. Shown is colocalization of DAPI and MeCP2 in olfactory neuron (top), CA3 pyramidal neuron (middle), and dentate gyrus astrocyte (bottom). Pearson's correlation coefficient = 0.943, 0.932, and 0.985, respectively. Scale bar, 5 μm. B, Image (left) shows representative MeCP2-positive (arrow) and MeCP2-negative (arrowhead) CA3 pyramidal neurons. Scale bar, 10 μm. Average somal diameters (right) of MeCP2-positive (purple) and MeCP2-negative (red) CA3 pyramidal neurons and olfactory bulb mitral cells from Mecp2Bnull/y-scAAV9/MeCP2–injected mice (n = 2). Also shown are measurements from WT mice (black bars; n = 2). The number of cells analyzed is indicated above each bar. ***p < 0.001 by one-way ANOVA (Newman–Keuls multiple-comparison test). Data are means ± SEM. C, Kaplan-Meier survival curve. D, Observational scores. Mecp2Bnull/y-scAAV9/MeCP2 (n = 5), Mecp2Bnull/y-AAV9/Control (n = 6), and Mecp2+/y (n = 6). Data are means ± SEM. E, Field pixel intensities of MeCP2-Cy3 immunofluorescence measured from brainstem sections of Mecp2+/y and Mecp2Bnull/y-scAAV9/MeCP2 mice. Traces represent pixel intensities from individual fields and each field is indicated by a differently colored trace, n = 2 mice per genotype, 5 fields per mouse. Garg S K et al. J. Neurosci. 2013;33:13612-13620 ©2013 by Society for Neuroscience

Inappropriate Silencing of Genes Fragile-X Syndrome

Completely methylated Fragile-X Syndrome Length Methylation Females Males Stable 6 to ~45 Unmethylated Not affected Gray zone ~45 to ~55 Premutation ~55 to ~200 Usually not affected Full mutation >200 Completely methylated ~50% affected All affected

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Skewed X-Chromosome inactivation in a family with Fragile X

Southern Blot Analysis Blood sample Digest genomic DNA with EcoRI and EagI Electrophoresis and transfer to membrane Hybridize with FMR1 specific probe “A normal female will show an unmethylated 2.8-kb band and a 5.2-kb methylated band that correspond to the normal FMR1 gene present in the active and inactive X chromosome, respectively.”

DNA Methylation Beckwith-Wiedemann syndrome

DNA Methylation Beckwith-Wiedemann syndrome Above average birth weight Increase growth after birth (>95% growth curve) Enlarged organs Hypoglycemic following birth Increase risk of cancers Imprinting defect located at 11p15.5

Beckwith-Wiedemann syndrome Genetic causes of BWS: Maternal DMR hypermethylation UPD Remainder unknown

Reading of imprinted gene marks and regulation of imprinted gene expression. (A) A scheme showing the function of CTCF binding and barrier function at the IGF2/H19 locus. Transcriptional enhancer elements are indicated by gray circles. The imprinting control region (ICR) is indicated by a checkered box. CTCF bound to the non‐methylated maternal ICR is shown to block enhancer access to the IGF2 promoter. (B) A scheme of the KCNQ1 locus showing maternally and paternally expressed genes. The untranslated RNA transcript on the paternal allele is indicated by a dotted line. Black arrows indicate the absence of transcription, while white arrows indicate active transcription. Red dots indicate sites of allele‐specific ICR methylation. The genes indicated by abbreviations are given in full below each part of the figure © IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSION' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. The Journal of Pathology Volume 211, Issue 3, pages 261-268, 18 DEC 2006 DOI: 10.1002/path.2116 http://onlinelibrary.wiley.com/doi/10.1002/path.2116/full#fig1

Model for the establishment of male germline imprinting marks Model for the establishment of male germline imprinting marks. A proposal for male germline imprinting establishment is presented. The proposal derives from Jelinic et al. (2006). Individual histones are indicated by green spheres. The zinc finger region of CTCFL is indicated by small projections which recognize specific sequences within the H19 ICR. Methylation of adjacent histones is indicated by red stars. Methylation of cytosines within the dinucleotide sequence CpG is indicated by red stars within the indicated ICR. The DNA strand is indicated in blue. SDMA = symmetrical dimethylated arginine; PRMT7 = protein arginine methyltransferase 7; CTCFL = CTCF like; Dnmt3s = denovo methyltransferases 3a, b, and L. The proposed scheme of events is described in greater detail in the text © IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSION' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. The Journal of Pathology Volume 211, Issue 3, pages 261-268, 18 DEC 2006 DOI: 10.1002/path.2116 http://onlinelibrary.wiley.com/doi/10.1002/path.2116/full#fig3

Genes Dev. Vol. 11, No. 23, pp. 3128-3142, December 1, 1997 Mouse mutant embryos overexpressing IGF-II exhibit phenotypic features of the Beckwith-Wiedemann and Simpson-Golabi-Behmel syndromes Jonathan Eggenschwiler,1 Thomas Ludwig,2 Peter Fisher,3 Philip A. Leighton,4,5 Shirley M. Tilghman,4 and Argiris Efstratiadis1,6

Nat Rev gene. 7: 21–33 doi:10.1038/nri1748 Figure 2 The epigenetic progenitor model of cancer. Feinberg AP et al. (2005) The epigenetic progenitor origin of human cancer Nat Rev gene. 7: 21–33 doi:10.1038/nri1748

Prader-Willi and Angelman Syndrome Mild mental retardation Severe impairment and loss of speech endocrine abnormalities seizures and ataxia temper tantrums unprovoked laughter Obesity hyperactivity 1 in 15,000

Prader-Willi and Angelman Syndrome Genes/proteins involved Prader-Willi syndrome 15q11-13

Prader-Willi and Angelman Syndrome UBE3A is paternally silenced This primarily occurs in brain, other tissues show biallelic expression

Prader-Willi and Angelman Syndrome What happens in each pathologies? If the maternal copy of chromosome 15 is missing, then genes normally expressed from this parental origin are not expressed Consequences…

Prader-Willi and Angelman Syndrome If paternal chromosome 15 is missing, then only the maternally expressed proteins are made Consequence: UBE3A is ok, but other genes in the region are not expressed…Prader-Willi syndrome

Prader-Willi and Angelman Syndrome Thus, two different diseases based on the cells “memory” of methylation – alter the memory, alter the phenotype

Prader-Willi and Angelman Syndrome How do you “lose” chromosome 15? Microdeletion of 15q11-13 on one chromsome – 70% Single gene mutation – 15% of AS Defect in imprinting centre (IC) – 5% Uniparental Disomy – 30% of PWS, 5% AS

Prader-Willi and Angelman Syndrome

Uniparental Disomy Receive two chromosomes from one parent Eg. Paternal disomy – both of chromosome 15 are from father, thus both have silenced UBE3A

Uniparental Disomy How does it happen? Trisomic Rescue - majority Monosomic Duplication

Meiosis I

Meiosis II

Meiosis I Non-disjunction

Meiosis I Non-disjunction Fertilization Meiosis I non-disjunction always creates a problem Trisomy

Meiosis II Non-disjunction 2/3 gametes following Meiosis II non-disjunction are normal Fertilization Fertilization Trisomy Normal

Trisomy Trisomy for most autosomal chromosomes is lethal BIG exception: Trisomy 21, smallest autosomal chromosome, fewest genes, not lethal Under rare conditions, some autosomal trisomies can escape – Trisomic Rescue

Trisomic Rescue following Meiosis I Non-disjunction Two copies of homologous, but not identical, chromosomes + Maternal Anaphase lag Maternal Paternal M,M 1/3 M,P 1/3 M,P 1/3

Trisomic Rescue following Meiosis II Non-disjunction Two copies of identical chromosomes + Anaphase lag M,M 1/3 M,P 2/3

Uniparental Disomy How does it happen? Trisomic Rescue - majority Monosomic Duplication

Monosomic Duplication + Two identical copies of paternal chromosome - isodisomy P,P

Parental Origin Determines Phenotype M,M { 15 Prader-Willi Syndrome M,M Prader-Willi Syndrome P,P Angelman Syndrome

Prader-Willi and Angelman syndrome Non-disjunction is more common in Meiosis I in females In human females, Meiosis I starts before birth but is arrested at diplotene stage (late prophase I) Oocytes sit like this for decades Complete meiosis II once each month While arrested at the diplotene stage, the tetrad chromosomes are held together by chiasmata (formed during recombination) If a pair of chromosomes don’t undergo recombination, the lack of chiasmata can contribute to non-disjunction Uniparental Disomy – 30% of PWS, 5% AS Maternal non-disjunction and trisomic rescue leading to the pair of maternal chromosomes

Uniparental Disomy and Human Disease Eric Engel. Some lessons from uniparental disomy (UPD) in the framework of contemporary cytogenetics and molecular biology. Atlas Genet Cytogenet Oncol Haematol. December 2003.

Trisomic rescue of Meiosis II non-disjunction can have other problems Anaphase lag { Pair of identical chromosomes (isochromosomes)

Harboring CFTR mutation Uniparental disomy as a mechanism for human genetic disease. Spence JE, Perciaccante RG, Greig GM, Willard HF, Ledbetter DH, Hejtmancik JF, Pollack MS, O'Brien WE, Beaudet AL. Am J Hum Genet. 1988 Feb;42(2):217-26. CFTR-/+ CFTR+/+ CFTR-/- Uniparental isodisomy and reduction to homozygosity { Pair of identical Chromosome 7 Harboring CFTR mutation