Klinefelter Syndrome (KS) is a multisystemic disorder associated with a plethora of phenotypic features including mental retardation, cardiac abnormalities, osteoporosis, infertility, gynecomastia, type two diabetes and increased cancer risk. KS is the most common aneuploidy in humans (with a prevalence of 1:500 to 1:1000 born males) and is characterized by one or more supernumerary X-chromosomes (47-XXY, 48-XXXY, and 49-XXXXY karyotypes). While X-chromosome inactivation (XCI) represses extra Xs, few genes called “escape genes” elude the XCI mechanism and are actively transcribed from X inactive. The overdosage of escape genes has been considered the molecular landscape that underlies KS clinical features. In this project, we exploit an integration-free reprogramming method to generate the largest described cohort of iPSCs from seven patients with KS and healthy donor fibroblasts from two relatives. The unicity of this cohort relies on the derivation of 47-XXY iPSCs and their isogenic 46-XY healthy counterparts, along with multiple rare 48-XXXY and 49-XXXXY iPSC lines. Through X chromosome inactivation (XCI) assessment, we show consistent retention of n-1 XCI in all derived KS-iPSCs. We identify the genes within the PAR1 region as the most susceptible to dosage-dependent transcriptional dysregulation and therefore putatively responsible for the progressively worsening phenotype in higher grade X aneuploidies. Moreover, we explore the transcriptional impact of X overdosage on autosomes and identify that the X-dosage-sensitive autosomal transcription factor NRF1 is a master regulator of the X-linked escape gene ZFX. Finally, we dissect the potential pathological impact of the escape gene KDM6A on low- and high-grade supernumerary X iPSCs and differentiated derivatives. We highlight a considerable proportion of KDM6A targets that could be responsible for paradigmatic clinical manifestations of KS.

In this dissertation I describe the isolation and characterization of genomic clones for the human synapsin I gene, the establishment of a linkage map for the human synapsin I gene locus, and studies of the possible involvement of this gene in neurological disease. Synapsin I is a neuron-specific phosphoprotein which is concentrated at the presynaptic terminal. Evidence suggests that it plays a fundamental role in the regulation of neurotransmitter release. Altogether 27,500 bp of the human synapsin I gene have been isolated, and the gene structure has been partially determined. DNA sequence comparisons between human and rat genes show a high degree of conservation. Sequenced exons display an 87% identity to each other. The synapsin I genomic clones were employed in the search for a polymorphic marker. A compound (AC)n repeat located 1000 base pairs downstream from the human synapsin I gene and within the last intron of the A-raf-1 gene has been identified. DNA database comparisons of the sequences surrounding the repeat indicate that the synapsin I gene and the A-raf-1 gene lie immediately adjacent to each other, in opposite orientation. Polymerase chain reaction amplification of this synapsin I / A-raf-1 associated repeat using total genomic DNA from members of the 40 reference pedigree families of the Centre d'Etude du Polymorphisme Humaine showed it to be highly polymorphic, with a polymorphic information content value of 0.84 and a minimum of eight alleles. Because the synapsin I gene had been mapped previously to the short arm of the human X chromosome at Xp11.2, linkage analysis was performed with markers on the proximal short arm of the X chromosome. The most likely gene order is: DXS7 - SYN/ARAF1 - TIMP - DXS255 - DXS146 with a relative probability of 5 x 108 compared with the next most likely order. The SynI/Araf marker was next utilized in a linkage study aimed at establishing a more accurate placement of the genetic locus responsible for the ocular disorder Congenital stationary night blindness, which had been mapped previously close to DXS7. Our results confirm this prior localization and also exclude any placement proximal to the SYN/ARAF1 locus. Finally, the inheritance of the different alleles of the SynI/Araf marker in three families with Rett syndrome, a severe neurodegenerative disorder, which has been assigned to the X chromosome, was studied. In at least one of the families in which two half sisters with the same mother suffer from the disease, the inheritance of Rett syndrome was discordant with the inheritance of the same allele for the SynI/Araf marker. Thus, this highly informative repeat has proven already effective in the study of X-linked diseases and should serve as a valuable marker for disease loci mapped to the Xp11 region.

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O imprinting genômico, processo regulado epigeneticamente segundo o qual os genes se expressam de acordo com sua origem parental (paterna ou materna), está envolvido no desenvolvimento placentário. Na região cromossômica 11p15.5 encontram-se vários genes importantes para o desenvolvimento fetal e da placenta, os quais são regulados por duas principais regiões controladoras de imprinting (ICR1 e 2) onde se encontram as regiões diferencialmente metiladas H19DMR e KvDMR1, respectivamente. O imprinting genômico e a inativação aleatória do cromossomo X são processos epigenéticos presentes em mamíferos placentários. O presente trabalho teve como objetivo principal verificar a presença de mosaicismo do perfil epigenético entre tecidos extraembrionários de estágios precoces da gravidez (primeiro trimestre), e em vilosidade coriônica de placentas a termo (terceiro trimestre). Foram coletadas amostras de 10 gestações de primeiro trimestre (vilosidade coriônica, âmion, membrana de cordão umbilical e tecido embrionário) e 14 de terceiro trimestre (vilosidade coriônica), das quais 10 foram consideradas como controles e quatro utilizadas para estudo de mosaicismo restrito à vilosidade coriônica (coleta de amostras de todos os cotilédones). Após extração do DNA, foi utilizado o Método de Digestão Enzimática Sensível à Metilação Associada à PCR em Tempo Real para o estudo do padrão de metilação da KvDMR1 e da H19DMR em diferentes tecidos do primeiro trimestre gestacional e em tecido placentário do terceiro trimestre. O padrão de inativação do cromossomo X foi avaliado em todos os cotilédones de duas placentas a termo, de fetos do sexo feminino, por meio do ensaio do receptor de andrógeno humano (HUMARA assay), utilizando eletroforese capilar, e com acréscimo de um novo marcador de inativação do cromossomo X (ICX1). Na análise estatística foram utilizados o teste t não pareado, teste de Turkey e teste t pareado. A média de metilação da KvDMR1 das amostras de vilosidade coriônica do primeiro trimestre gestacional foi estatisticamente diferente da média de metilação do terceiro trimestre. Enquanto que a metilação da H19DMR não apresentou diferença estatística entre amostras de vilosidade coriônica do primeiro e do terceiro trimestre gestacionais. Com relação ao mosaicismo, a KvDMR1 não apresentou variação com relação ao tamanho ou a posição dos cotilédones, enquanto que a H19DMR apresentou diferença estatisticamente significativa na média de metilação com relação ao tamanho dos cotilédones e ao posicionamento nos quadrantes; em consequência da hipometilação em cotilédones pertencentes a uma das placentas estudadas. Não foram observadas diferenças estatisticamente significativas na média de metilação da KvDMR1 e da H19DMR entre diferentes tecidos das amostras do primeiro trimestre gestacional. No entanto, a comparação entre tecidos pareados de um mesmo indivíduo mostrou que a metilação não é correspondente entre os tecidos. Os dados obtidos mostram que o imprinting genômico provavelmente é um processo dinâmico, que evolui ao longo da gestação, estando relacionado a formação e ao amadurecimento da placenta. No presente estudo foi possível verificar que cotilédones de uma mesma placenta apresentam diferentes padrões de inativação do cromossomo X. Diferenças que podem ser explicadas pela expansão clonal das células trofoblásticas progenitoras com o cromossomo X paterno ou o cromossomo X materno inativo. Devido à variabilidade epigenética, exames em tecidos placentários devem considerar as diferenças intra-placentárias e as diferenças entre tecidos embrionários e extraembrionários. / Genomic imprinting, a mechanism of allele-specific expression depending on parental origin, is an epigenetic process that regulates the expression of many genes involved in placental development. Several important genes for fetal and placental growth are located on the human chromosome region 11p15.5, which are regulated by two imprinting control regions (ICR1 e 2), which have the differentially methylated regions H19DMR and KvDMR1, respectively. Genomic imprinting and random inactivation of X chromosome are two epigenetic processes present in placental mammals. The present study aimed to verify the presence of epigenetic mosaicism between extra-embryonic and embryonic tissues from early stages of pregnancy (first trimester), and in chorionic villi of term placentas (third trimester). Samples were collected from 10 pregnancies in the first trimester (chorionic villous, amnion, umbilical cord membrane, and embryonic tissue) and 14 from third trimester (chorionic villus sampling), of which 10 were considered as controls and four used to study mosaicism restricted to chorionic villi (sampling of all cotyledons). After DNA extraction, we used real time PCR associated to enzymatic restriction with a methylation sensitive enzyme to study the methylation pattern of KvDMR1 and H19DMR in different tissues from first trimester and placental third trimester tissue. The pattern of X chromosome inactivation was evaluated in all cotyledons from two term placentas of female fetuses, using the human androgen receptor (HUMARA) assay, capillary electrophoresis, and adding a new X chromosome inactivation (ICX1) marker. Unpaired and paired t and Turkey tests were used in statistical analysis. The average methylation of KvDMR1 of chorionic villi samples in first trimester was statistically different from average methylation of the third trimester. While the methylation of H19DMR showed no statistically significant difference between chorionic villi samples in the first and third trimester of pregnancy. In relation to the mosaicism, the KvDMR1 methylation did not vary in respect to the size or position of the cotyledons, while H19DMR showed statistically significant difference in average methylation relative to the size of the cotyledons, to the position in quadrants, due to the hypomethylation in cotyledons from one studied placenta. There were no statistically significant differences in the mean methylation KvDMR1 and H19DMR among different tissues from the first trimester of pregnancy, however, the comparison between paired tissues from the same individual showed that the methylation is different between tissues. The data from this study showed that genomic imprinting is probably a dynamic process and evolved across human pregnancy. This process is probable connected to placenta formation and maturation. We observed different patterns of X chromosome inactivation in cotyledons from the same placenta. This difference could be explained by clonal expansion of a limited number of trophoblastic progenitor cells with either an inactive maternal or paternal X chromosome. Due to the epigenetic variability, placental tissue examinations must consider the differences intra-placental and differences between embryonic and extra-embryonic tissues.

Em mamíferos a inativação do cromossomo X (ICX) consiste no silenciamento gênico de um dos dois X presentes nas células somáticas normais das fêmeas, garantindo a compensação de dose transcricional em relação aos machos. Existem duas formas de ICX: aleatória, na qual a escolha do cromossomo X inativado se dá ao acaso (X paterno ou materno); e de maneira completamente desviada, na qual a atividade do cromossomo X dependerá de sua origem parental. Nas fêmeas marsupiais a inativação ocorre de forma completamente desviada, sendo o X paterno preferencialmente inativado em todas as células, já nas células embrionárias de eutérios, o que se observa é a ICX aleatória. Entretanto, naquelas células que darão origem aos tecidos extra-embrionários, de camundongos e bovinos, a ICX se dá de forma equivalente à dos marsupiais, ou seja, o X paterno é preferencialmente inativado. Há mais de 30 anos o padrão de ICX em tecidos extra-embrionários humanos tem sido alvo de intenso debate. A crítica que se faz aqui é que tais estudos foram realizados com base na expressão de apenas um ou dois genes ligados ao X com amostras de tecidos extra-embrionários em diferentes idades gestacionais e, por vezes, em poucas amostras, o que deve ter levado às contradições entre as conclusões. O diferencial deste trabalho foi a utilização de técnicas de genotipagem de SNPs presentes em regiões codificadoras, para analisar o padrão de atividade alelo-específica de um grande número de genes presentes ao longo de todo o cromossomo X, gerando um panorama mais representativo da ICX em placenta humana. Neste estudo é comprovado o padrão aleatório de ICX em placenta humana a termo e demonstrado que este órgão se apresenta como um 65 mosaico em relação à escolha do X inativo. A análise global da atividade gênica no cromossomo X indicou ainda que a manutenção do estado epigenético do X inativo parece ser heterogêneo. Em conjunto, os dados gerados são capazes de explicar as incongruências entre as conclusões previamente publicadas. Este trabalho também ilustra as diferenças nos mecanismos de ICX entre humanos e camundongos e reforça a importância de se avaliar esse tema em outras espécies de mamíferos eutérios na tentativa de se elucidar os processos evolutivos envolvidos na compensação de dose em mamíferos / Imprinted inactivation of the paternal X chromosome in marsupials is the primordial mechanism of dosage compensation for X-linked genes between females and males in Therians. In Eutherian mammals, X chromosome inactivation (XCI) evolved into a random process in cells from the embryo proper, where either the maternal or paternal X can be inactivated. However, species like mouse and bovine maintained imprinted XCI exclusively in extraembryonic tissues. The existence of imprinted XCI in humans remains controversial, with studies based on the analyses of only one or two X-linked genes in different extraembryonic tissues. Here we readdress this issue in human term placenta by performing a robust analysis of allele-specific expression of 23 X-linked genes, including XIST, using 28 SNPs in transcribed regions. We show that XCI is random in human placenta, and that this organ is arranged in relatively large patches of cells with either maternal or paternal inactive X. In addition, this chromosome-wide analysis indicated heterogeneous maintenance of the epigenetic state along the inactive X, which combined with the extensive mosaicism found in placenta, can explain the lack of agreement among previous studies. Our results illustrate the differences of XCI mechanism between humans and mice, and highlight the importance of addressing the issue of imprinted XCI in other species in order to understand the evolution of dosage compensation in placental mammals

Le développement embryonnaire précoce des mammifères femelles s’accompagne de l’inactivation transcriptionnelle d’un de leurs deux chromosomes X. Cet évènement est initié suite à l’expression mono-allélique de l’ARN non codant Xist, qui est contrôlée par de nombreux éléments cis-régulateurs présents dans le centre d’inactivation du chromosome X (Xic) – tel son anti-sens répresseur Tsix. Mon travail de thèse a consisté à développer des approches permettant d’appréhender le paysage structural dans lequel s’exerce cette régulation. La caractérisation de l’architecture tridimensionnelle du Xic, par des techniques basées sur la capture de conformation chromosomique (3C) et l’hybridation in situ en fluorescence (FISH), m’a permis de mettre en évidence que les promoteurs respectifs de Xist et Tsix sont engagés dans des interactions physiques intimes avec des loci distaux, localisés au sein du Xic, et de montrer qu’au moins certaines de ces régions exercent un effets régulateurs à longue-distance. Les éléments du Xic contactés par les régions promotrices de Xist et de Tsix sont en outre fondamentalement différents, chacune engageant des associations chromosomiques sur plusieurs centaines de kilobases dans leur direction 5’ respective.Ce travail a également permis de révéler des propriétés insoupçonnées de l’architecture chromosomiques. En effet, le Xic apparaît scindé en plusieurs sous-régions, couvrant chacune entre 200kb et 1Mb, à l’intérieur desquelles les interactions chromosomiques sont préférentiellement établies. L’existence de ces domaines d’interaction s’intègre avec d’autres propriétés structurales du génome, tels la composition de la chromatine sous-jacente et l’association à la lamine nucléaire, mais n’apparaît pas en dépendre directement. En étudiant la dynamique de la conformation chromosomique du Xic au cours de la différenciation cellulaire, j’ai pu constater la robustesse de cette organisation, sauf sur le chromosome X inactif, qui se distingue par la perte des contacts chromosomiques préférentiels détectables sur son homologue actif.Enfin, j’ai pu mettre en évidence que la variabilité du repliement général du chromosome X amène à un instant donné chaque allèle de Tsix à contacter physiquement des jeux de séquences distales différents, suggérant que l’environnement structural instantané de chacun de ces allèles à l’orée de l’activation mono-allélique de Xist est différent. Ce travail, combinant des approches à l’échelle de la population cellulaire d’une part et de la fibre de chromatine unique d’autre part, apporte une nouvelle vision du paysage structural et régulateur dans lequel s’inscrit le contrôle de l’activité transcriptionnelle de Xist, et fourni de nouvelles perspectives concernant les principes fondamentaux de l’organisation topologique des chromosomes chez les mammifères. / Early development of female mammals is accompanied by transcriptional inactivation of one of their two X chromosomes. This event is initiated following mono-allelic expression of the Xist non-coding RNA – what is achieved by the interplay of numerous cis-regulatory elements present within the X inactivation center (Xic), such as its repressive antisense Tsix. Our work aimed at throwing light on the structural landscape that underlies such long-range regulation. Characterization of the three-dimensional architecture of the Xic, by the means of Chromosome Conformation Capture (3C)-based techniques and in situ fluorescence hybridization (FISH), revealed that the respective promoters of Xist and Tsix contact many distal genomic elements within the Xic, and that at least one of such interacting region exerts long-range cis-transcriptional control. Noticeably, Xist and Tsix promoters associate with different sets of elements in their respective 5’ direction that are spread out over several hundreds of kilobases These experiments also revealed unforeseen properties of chromatin architecture. Indeed, the Xic appears to be partitioned in several sub-regions, each spanning between 200kb and 1Mb, inside which chromosomal interactions are preferentially established. The existence of these interaction domains integrates with other structural features of the genome, such as underlying chromatin composition and association with the nuclear lamina, but does not seem to directly depend on them. By analyzing chromosome conformation of the Xic during cell differentiation we document the robustness of this organizational principle, with the noticeable exception of the inactive X chromosome that assumes a folding pattern that is more random than its active homolog. Finally we also bring evidence that variability in the folding pattern of the two X chromosomes in the same cell brings each Tsix allele in association with different sets of chromosomal partners at a given moment, suggesting that the instantaneous structural environment of each allele at the onset of mono-allelic Xist up-regulation is different.By combining approaches at the scale of cell populations on the one hand, and at the single chromatin fiber level on the other, this study provides a first vision of the structural landscape in which Xist regulation takes place, and brings new insights concerning fundamental properties of chromosome organization in mammals.