Epigenetics and Rett Syndrome
Dr. Manel Esteller
Director Elect Josep Carreras Institute (IJC)
Director Cancer Epigenetics and Biology Program (PEBC) Bellvitge Biomedical Research Institute (IDIBELL) Genetics Professor, University of Barcelona ICREA Research Professor [email protected]
Urdinguio, Sanchez-Mut and Esteller, Lancet Neurology, 2009 miRNA PROCESSING Genetic syndromes caused by mutations in Epigenetic genes FMRP Fragile X Syndrome DNA METHYLATION DGCR8 DiGeorge Syndrome Goiter multinodular 1 Syndrome Amyotrophic lateral sclerosis (ALS) TARDBP DNA METHYLATION DICERDICER
miRNA/mRNA XPO5 DNMT1 Hereditary Sensory and Autonomic Drosha Pri-mRNA Neuropathy 1 (HSAN1) RISC DNMT3 Immunodeficiency-Centromeric Instability-Facilal Anomalies (ICF1)
miRNA Pre-mRNA MeCP2 Rett Syndrome Angelman Syndrome CH3
HISTONE MODIFICATIONSHISTONEHISTONE MODIFICATIONS MODIFICATIONS
Claes- Jensen X-linked Mental Retardation Syndrome JARIDC1JARIDC1 MLL2 Kabuki Syndrome 1 Siderius X-linked Mental Retardation Syndrome PHF8PHF8 MLL2 EZH2EZH2 Weaver Syndrome 2 Kabuki Syndrome JMJD3JMJD3 EHMT1EHMT1 KleefstraSyndrome Sotos Syndrome Weaver Syndrome 1 NSD1NSD1 CREBBP/CREBBP/ Rubinstein-Taybi Syndrome Beckwith-Wiedemann Syndrome P300P300 Berdasco and Esteller, Hum Genet. 2013 H4 H4 Brachydactyl- mental retardation Syndrome HDAC4HDAC4 MYST4MYST4 Genitopatellar Syndrome Say-Barber-Biesecker-Young-Sympson (SBBYS)
- N--Nterminal- terminal …K……K… DRLVKRHRKAGGKGLGKGGKGRGS DRLVKRHRKAGGKGLGKGGKGRGS 1 1 3 3 5 5 8 8 1212 1616 1818AcAc2020 9191AcAc CHROMATINPP MeMeAcAc AcAc MeMe- REMODELINGAcAc PP MeMe FACTORS ERCC6ERCC6 Alpha-Thalassemia X-linked mental retardation Syndrome Cockayne Syndrome, type A ATRXATRX Mental Retardation-hypotonic facies Syndrome, X-linked Cockayne Syndrome, type B ERCC8ERCC8 CDH7CDH7 CHARGE Syndrome Coffin- Siris Syndrome ARID1BARID1B SMARCB1SMARCB1 Coffin- Siris Syndrome Coffin- Siris Syndrome ARID1AARID1A SMARCAA4SMARCAA4 Coffin- Siris Syndrome Floating- Harbor Syndrome SRCAPSRCAP Coffin- Siris Syndrome SMARCA2SMARCA2 Nicolaides-Baraitser Syndrome Spinocerebellar ataxia 7 ATXN7ATXN7
CHROMATINCHROMATIN-REMODELING-REMODELING FACTORS FACTORS Rett Syndrome
Rett Syndrome (RS) is a neurodevelopmental disorder believed to Rett Syndrome (RS) is a neurodevelopmental disorder believed to affect 1 in 10,000 females..
Germline mutations in MECP2 (Methyl-CpG Binding Protein-2) found in 65% of Rett patients. MECP2 is located in Xq28.
Girls with Rett Syndrome appear to develop normally until 6 to 18 months of age. They then enter a period of regression, losing speech and hand skills they had acquired. Most girls develop seizures, repetitive hand movements, irregular breathing and motor-control problems. A slowing of the rate of head growth also becomes apparent. Most researchers agree that RS is a developmental disorder rather than a progressive, degenerative disorder. The girls can live to adulthood, but most never regain the ability to use their hands or to speak.
Germline mutations in MECP2 (Methyl-CpG Binding Protein-2) found in 65% of Rett patients. MECP2 is located in Xq28.
Girls with Rett Syndrome appear to develop normally until 6 to 18 months of age. They then enter a period of regression, losing speech and hand skills they had acquired. Most girls develop seizures, repetitive hand movements, irregular breathing and motor-control problems. A slowing of the rate of head growth also becomes apparent. Most researchers agree that RS is a developmental disorder rather than a progressive, degenerative disorder. The girls can live to adulthood, but most never regain the ability to use their hands or to speak. MeCP2
➢ Protein with affinity for methylated DNA (CpG pairs). ➢ i.e. Fraga et al. NAR 2003; Ballestar et al. EMBO J 2003 ➢ Transcriptional repressor. ➢ i.e. Fournier et al. EMBO J 2002; Ballestar et al. EMBO J 2003; Lopez-Serra et al. Cancer Res 2006; Lopez-Serra et al., Oncogene 2008
Two main functional domains: MBD
TRD Lopez-Serra et al., British Journal of Cancer 2008 Establishment of Immortalized Lymphocyte Cell Lines
Healthy Donor MeCP2 Rett Patient
Lymphoblastoid Cell Lines Available
MeCP2 CDKL5 NTG1 FOXG1 n=23 n=7 n=3 n=2
+ + 103 N2 B MPP1 57 ºC 57 Annealing Silencing NAB UB - B MeCP2 UB Ballestar et al., Human Mutation, Mutation, Human al., 2005 Ballestar et I 5´-CTTAAAAATCATCTCRCAAAAACT-3´ 5´-YGAATTTGGTTAGGGTATTYGGG-3´ Control R106W MPP1 Primer sequence Primer Forward Reverse 264 - in Rett Syndrome Patients Rett Syndrome in N2 Gene MPP1 N1 release of MeCP2releaseof
Clustering expression analysis andanalysis expressionClustering NoMut3
NoMut1
NoMut4
NoMut2
R255X
R306C
R294X
P391X
T158M
R106W 36 36 38 38 35 35 49 49 36 36 41 41 105 105 108 108 84 84 9090 125 125 125 123 123 126 126 112 112 125 125 120 120 56 56 53 53 77 77 7171
Mouse Model of Rett
1
3 2
7.5gr/10.25cms 1.-Encéfalo 299mg 2.-Mesencéfalo 3.-Cerebelo
1 3 2
8.1gr/11.05cms 310mg Looking for transcriptional changes
Wild type mice Mecp2-null mice Cortex Cortex Midbrain Midbrain Cerebellum Cerebellum
RNA Extraction RNA Extraction
Amplification Amplification Candidate overexpressed genes Reverse Reverse in Mecp2-null mice transcription and transcription and labeling labeling
Hybridization
Image scanning
Data analysis Mobp Dlk1 – Gtl2 Myelin-associated oligodendrocytic basic protein
Family of abundant CNS myelin proteins Localizes to major dense line of compact myelin
Plagl1 Ddc
zinc finger protein regulator of apoptosis DOPA decarboxylase and cell cycle arrest (Zac1) L-Dopa
Dopamine
L-5-HTP
Serotonine
Urdinguio et al., PLoS ONE, 2008 Effective drugs for Parkinson's reduce symptoms of Rett syndrome in mice
100 8 vehicle
80 L-Dopa L-Dopa / Ddci 6 60 * 4 * * 40
Percent survival 2
20 ** *** Total ScoreSymptom
0 0 60 70 80 90 100 6 7 8 9 10 Life span (days) Time (weeks)
*** *** * ** *** *** ** *** *** *** *** ** 1.0 * ** 1.0 * * 1.0 * * * * * *** * ** ** * ***
0.5 0.5 0.5
Tremor
Mobility Breathing
0.0 0.0 0.0
7 w eeks 8 w eeks 9 w eeks 10 w eeks 7 w eeks 8 w eeks 9 w eeks 10 w eeks 7 w eeks 8 w eeks 9 w eeks 10 w eeks
Szczesna et al., Neuropsychopharmacology 2014
An Impact of MeCP2 Disruption on lncRNA Transcription: The Mouse Model of Rett Syndrome
Wild type MeCP2 KO Wild-type MeCP2 KO Brain Brain
Mouse LncRNA Microarray
GAMMA-AMINOBUTYRIC ACID RECEPTOR, RHO-2 AK08122 7
AK081227/RPL 5 38** Gabrr2/RPL38 W… 2,0 K… 4 W… 3 K… * 1,0 * 2 * * 1 0,0 0 BO FC HIP HTA TH C
Petazzi et al., RNA Biology, 2013 MECP2 duplication syndrome
Intellectual disability, weak muscle tone in infancy, feeding difficulties, poor or absent speech, seizures, delayed development of motor skills, such as sitting, loss of previously acquired skills (developmental regression), one third cannot walk without assistance. An Increase in MECP2 Dosage Impairs Neural Tube Formation
•We report a new in vivo model of MeCP2 overexpression. •The increase in MeCP2 dosage prevents the correct formation of the neural tube. •MeCP2-electroporated cells show an ectopic neuronal localization. •Neuroblasts overexpressing MeCP2 undergo decreased proliferation and increased death.
Petazzi et al., Neurobiology of Disease, 2014 Circadian Cycle-Dependent MeCP2 and Brain Chromatin Changes
Martínez de Paz et al, PLoS One (2015) Genomics Girls with Clinical Features of Rett Syndrome but without any mutation in the “usual suspect” genes
Whole-Exome Sequencing Mutations in JMJD1C (jumonji domain containing 1C) are involved in Rett syndrome and intellectual disability
Saez et al., Genetics in Medicine , 2015 Other Mutations in Histone Modifiers Are Also Involved in Intellectual Disability Dravet syndrome Pitt-Hopkins syndrome SCN1A mutation TCF4 mutation
Lucariello et al., Human Genetics 135, 1343-1354, 2016
Final Update Mllt2h Eya2
Suggested transcriptional factor Transcriptional coactivator
Specifically expressed in Purkinje cell (Af4) Expressed during development of several organs
S100a9 S100 calcium binding protein A9 (calgranulin B)
IRAK1 !!!!
Marenholz I, Heizmann CW and Fritz G. (2004) Biochem Biophys Res Commun. 322:1111-22 Urdinguio et al., PLoS ONE, 2008 Inhibition of Gsk3b reduces NFk-B signaling and rescues synaptic activity to improve the Rett syndrome phenotype in Mecp2-knockout mice
Jorge-Torres et al., Cell Reports 2018 Treatment with a GSK3β inhibitor (SB216763) Improves Neuronal Functionality
WT KO VEH KO+SB216763 Merge Psd95 Dapi
WT
Map2
KO VEH KO
KO + + KO
Thresholding SB216763
Branching Postsynaptic
Jorge-Torres et al., Cell Reports 2018 Future Genetic Edition of MeCP2 in Neurons
Normal Neurons Neurons with MeCP2 Loss Genetic Edition of MeCP2 in Neurons
MECP2DAPITUJ Obtaining GABAergic Neurons
CTR Protocol
GABADAPITUJ Thanks to all the families and research supporters….
Sonia Guil Raul Delgado-Morales
Catalan Rett Syndrome Association
Valencian Rett Syndrome Association