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Methyl-CpG binding protein 2 deregulation: from Rett syndrome to MeCP2 duplication disorder

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Universitat de Barcelona. Facultat de Medicina

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[eng] NTRODUCTION To answer many complex and fascinating biological phenomena, we must go over or above (epi-) genetics because the DNA blueprint is identical in each of the abovementioned somatic cells. The mechanisms by which epigenetics affects so deeply the cell physiology are mediated by a large number of actors, most of them represented by covalently modified nucleotides and amino acids, non-coding RNAs and proteins. MeCP2 is an epigenetic reader able to bind to methylated and 5- hydroxymethylated cytosines. Despite all the initial evidences proposing a chromatin-repression role for MeCP2, many other functions have been demonstrated, including transcriptional activation, mRNA splicing regulation and protein synthesis modulation. Importantly, MeCP2 impairments are the primary responsible for RTT syndrome and have also been shown to be involved in several other disorders, albeit in very few patients, as Prader-Willi syndrome, Angelman syndrome, nonsyndromic mental retardation, and autism. AIMS To date, no MeCP2-regulated gene has been successfully targeted in order to improve the severe symptoms of RTT. In the present Doctoral Thesis we sought to identify new MeCP2 targets through different approaches with the purpose of expanding the knowledge of the impaired biological pathways in RTT. * In the first study we focused on a class of transcriptional regulators called long non-coding RNAs (lncRNAs). * In the second study we took advantage of RNA sequencing, a powerful high-throughput technique with the ability to detect very low amounts of transcript. Then, we proposed to investigate also the consequence of MeCP2 over-expression in a well-known developmental model such as the chicken embryo. RESULTS STUDY I DYSREGULATION OF THE LONG NON-CODING RNA TRANSCRIPTOME IN A RETT SYNDROME MOUSE MODEL * We found 701 lncRNAs that had a different expression pattern in wild-type and Mecp2-null brain with a score of <0.05 in the false discovery rate (FDR) test and a >1.5-fold expression change. Among the altered lncRNAs, downregulation of transcripts was predominant (520 of 701, 74%), whereas upregulation occurred in the minority of differentially expressed genes (181 of 701, 26%). * Following a selection of lncRNAs with a fold-change >2 that were associated with an annotated protein-coding gene involved in neuronal or glial functions, we validated two up-regulated lncRNAs, AK081227 and AK087060, in the Mecp2-null brain using qRT-PCR on independent samples. * We showed that AK081227 and AK087060 promoters were occupied by the MeCP2 protein in wild-type mouse brains. * We reported that the up-regulation of AK081227 in Mecp2-null mice was associated with a down-regulation of its host gene Gabrr2 in four brain regions (frontal cortex, hypothalamus, thalamus and cerebellum) (Pearson's correlation test = 0.44, p = 0.06). * In the case of AK087060, we found that the up-regulation of this 1ncRNA was correlated with an increase in the expression of its host gene Arhgef26 in the four studied brain regions (Pearson's correlation test = 0.41, p = 0.08). STUDY II RNA-SEQUENCING OF A RETT SYNDROME MOUSE MODEL REVEALS GLOBAL IMPAIRMENT OF IMMEDIATE-EARLY GENES EXPRESSION * We sequenced the transcriptome of Mecp2-null and control mice and we detected 1049 and 1154 differentially expressed genes in HIP and PFC, respectively. The ratio of up- and down-regulated genes was different between the two regions. In the HIP the ratio was favorable to the less expressed genes, being 388 (37%) and 661 (63%) the up- and down-regulated genes, respectively. On the other hand, in the PFC there were slightly more up-regulated genes, 630 (55%), compared to the down-regulated ones, 523 (45%). In addition we reported that only a small fraction of genes, 76 and 109, were up- and down-regulated, respectively, in both brain areas. * Gene Ontology (GO) analysis of differentially expressed transcripts revealed that both HIP and PFC up-regulated genes were enriched in neuronal function terms and, to a lesser extent, signal transduction ones. The scenario was similar for the down-regulated genes but in this case we found many inflammatory, apoptosis, oxidative stress and immune system-related terms. * We found several members of the immediate-early genes (IEGs) family to be up-regulated both in the PFC and HIP of Mecp2-null mouse. Consistent with the findings from the RNA-sequencing analysis in the HIP, qRT-PCR showed significant alterations in the expression of Fos, Junb, Egr2, Nr4a1, Npas4, Fosb and Egr1. Furthermore, Fos, Junb, Npas4 and Fosb were validated also in the PFC. * We demonstrated the binding of MeCP2 upon the regulatory regions of IEGs. In both PFC and HIP wild-type brain, we observed a reduction of MeCP2 occupancy upon the regions associated with high CpG content of Fos, Junb, Nr4a1, Npas4, Fosb and Egr1 promoters. We also found that the HIP chromatin was more accessible to MNase digestion in the Mecp2-null brain. * Then, we showed that four IEGs (Fos, Junb, Egr2, Npas4) displayed altered expression in Mecp2-null cultured neurons treated with forskolin. Precisely, this four IEG exhibited an aberrant kinetic of recovery to the basal state. One hour after forskolin withdrawal, Fos, Junb, Egr2 and Npas4 expression levels in the Mecp2-null hippocampal neurons continue to increase, while in wild-type they did not change or even decrease. The situation is the opposite in cortical neurons, where Fos, Junb, Egr2 and Npas4 are less expressed after forskolin withdrawal in Mecp2-null samples. * Finally, we evaluated whether the IEGs response was impaired in vivo as well. Indeed, we observed a significant increase of Junb expression in the hippocampus of Mecp2-null animals treated with kainic acid, when compared to treated wild type mice. STUDY III AN INCREASE IN MECP2 DOSAGE IMPAIRS NEURAL TUBE FORMATION * We detected the expression of both chicken MeCP2 (cMECP2) transcript and protein in a wide window of developmental stages. In addition, we showed that nuclear localization and the sequence of the region encompassing the methyl-CpG binding domain are conserved between human and chicken. * We found that the overexpression of MeCP2 in the neural tube of chicken embryos provokes an overall decrease in the number of proliferating BrdUpositive cells, with the most affected part being the ventricular zone. In addition, normal H3S1Op pattern along the lumen is disrupted upon MeCP2 overexpression. * Also, MeCP2 increase in dosage cause a clear decrease in the amounts of differentiated neuronal population located at the mantle zone, as it was demonstrated through immunostaining of neural tubes with TUJ1 and HUC/D, two neuronal-lineage restricted markers. Moreover, MeCP2 overexpression leads to a decrease of a neuroepithelial polarity marker such as N-cadherin. * Finally, we showed that one of the possible explanations of our phenotype is the increased cell death occurring upon MeCP2 increase in dosage. We reported an increment of apoptotic cells in MeCP2-overexpressing neural tubes immunostained with Caspase-3 and -8. Furthermore, we described also an increase of pyknotic cells number in MeCP2 electroporated neural tubes.

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[spa] Introducción: El síndrome de Rett (RTT, OMIM#312750) fue por primera vez descrito en 1966 por el pediatra austriaco Andreas Rett. El síndrome de Rett causa retraso mental en 1 de cada 10000 niñas, lo que hace que sea la segunda causa de retraso mental en niñas. En 1999 en el laboratorio de Huda Zoghbi descubrieron las bases genéticas de la enfermedad. El 95% de los casos de Rett clásico se produce por mutaciones en MeCP2. Es interesante el hecho de que mutaciones que provocan el incremento de copias del gen MECP2 también llevan a enfermedades neurológicas, como es el caso del trastorno provocado por la duplicación de MeCP2. MeCP2 es una proteína nuclear, que se expresa en diferentes tejidos, pero es especialmente abundante en neuronas del sistema nervioso maduro. MeCP2 es una proteína con capacidad para unirse a dinucleótidos CpG. Entre las varias funciones biológicas propuesta para MeCP2 se encuentran: 1) Silenciamento transcripcional; 2) activador transcripcional; 3) regulador de splicing; 4) Regulador de la cromatina. Objetivos del estudio: El principal objetivo de esta tesis es evaluar el impacto del incremento o disminución de expresión de MeCP2 , tanto a nivel transcripcional como de desarrollo, al fin de caracterizar las vías moleculares desreguladas en las manifestaciones clínicas relacionadas con MeCP2. En los primeros dos estudios se buscarán nuevos targets de MeCP2 a través de dos diferentes tecnologías, secuenciación del ARN y microarray. En ambos estudios utilizaremos un modelo murino bien establecido (MeCP2-null), obtenido mediante supresión del gen MeCP2, que simula el síndrome de Rett. Las diferencias entre los primeros dos estudios es que mientras en el primero se buscarán solo "long non-coding RNA" relacionados con MeCP2, el segundo será enfocado en todos los ARN codificantes. En el tercer estudio evaluaremos el efecto de la sobreexpresión de MeCP2 en un bien establecido modelo de desarrollo embrionario como es el embrión de pollo. Resultados y conclusiones: Parte 1 * Se han encontrado 701 lncRNAs diferencialmente expresados entre el cerebro del ratón Mecp2-null y el control (salvaje). * MeCP2 está unido a los promotores de los lncRNAs AK081227 y AK087060. * El incremento de expresión de AK081227 en ratones Mecp2-null está asociado con la bajada de expresión de su gen huésped Gabrr2 en cuatro regiones del cerebro. * La sobre regulación de AK087060 se correlaciona con un aumento en la expresión de su gen huésped Arhgef26 en las cuatro regiones cerebrales estudiadas. Parte 2 * Hemos encontrados 1049 y 1154 transcritos diferencialmente expresado en el hipocampo (HIP) y la corteza pre-frontal (PFC), respectivamente, del ratón Mecp2- null. * Los genes "immediate early genes" (IEGs) Fos, JunB, EGR2, NR4A1, Npas4, FosB y Egrl están sobreexpresados en el HIP de Mecp2-null. Además, Fos, JunB, Npas4 y FosB están sobreexpresados también en el PFC. * En tanto la PFC como en el HIP del ratón wild-type, la unión de MeCP2 se reduce en las regiones asociadas con alto contenido de CpG de los genes Fos, JunB, NR4A1, Npas4, FosB y Egr1. Además, los promotores de Fos, JunB y Npas4 son más accesibles a la digestión con nucleasas micrococales (MNase) en el HIP de ratones Mecp2-null. * Cuatro IEGs (Fos, JunB, Egr2, Npas4) muestran un patrón de expresión alterado en neuronas derivadas de animales Mecp2-null y tratadas con forskolina. * La expresión de JunB es incrementada significativamente en el hipocampo de los animales Mecp2-null tratados con ácido kaínico, en comparación con ratones controles tratados. Parte 3 * El transcrito y la proteína de MeCP2 de pollo se expresan en varios estadio del desarrollo embrionario y especialmente en el tubo neural * La sobreexpresión de MeCP2 en el tubo neural de embriones de pollo provoca una disminución general en el número de células proliferantes. Además, el patrón de localización del marcador mitótico H3S1Op es aberrante en tubos neurales que sobreexpresan MeCP2. * Una dosis elevada de MeCP2 provoca una clara disminución de las neuronas diferenciadas localizadas en la zona del mantel. Por otra parte, la sobreexpresión de MeCP2 conduce a una disminución del marcador de polaridad neuroepitelia Ncadherin. * La sobreexpresión de MeCP2 en tubos neurales provoca un aumento de apoptosis.

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Methyl-CpG binding protein 2 deregulation: from Rett syndrome to MeCP2 duplication disorder