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.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

The STR markers linked to the X chromosome can be used for complement the analysis of autosomal markers, especially in complex cases of kinship testing, in cases of post-morten identification and in paternity testing, when the disputed child is a girl. The aim of this work was investigate five STR X-chromosome markers (DXS10079, DXS10074, DXS10075, DXS7424 and DXS101) in the population of Alagoas, Brazil and analyze their frequencies for forensic purposes. The sample was composed of 404 unrelated individuals, 203 males and 201 females. The DNA was extracted using Chelex procedure and amplification was performed by PCR in a pentaplex system and the fragments were separated by capillary electrophoresis. For the studied STR markers, it was calculated the allele and haplotype frequencies, the observed and expected Heterozygosity values, the Hardy Weinberg equilibrium (HWE), the genetic diversity, the Mean Exclusion Chance of trios involving daughters (MECT) as well as in father/daughter duos (MECD). Also, it was calculated Power of Discrimination in males (PDM) and in females (PDF) and the Polymorphism Information Content (PIC). The forensic efficiency values demonstrate that DXS101 is a highly informative marker, followed by DXS10074, DXS10079, DXS7424 and DXS10075. The polymorphism information content ranged from 0.7470 to 0.8858. For the pentaplex evaluated, the combined values of PDM and PDF were 0, 9998947 and 0, 9999998, respectively and the combined MEC in trios involving daughters and in father/daughter duos were 0,999817 and 0,998042, respectively. No deviations from the Hardy Weinberg equilibrium were observed. We concluded that the five ChrX STRs analyzed are highly informative markers for kinship testing and constitute a powerful tool for forensic practice in our population. / Fundação de Amparo a Pesquisa do Estado de Alagoas / Os marcadores STRs ligados ao cromossomo X podem ser utilizados para complementar as análises de marcadores autossômicos, especialmente em casos complexos de vínculo genético, em casos de identificação post-morten e em testes de paternidade, quando a criança analisada é uma menina. O objetivo desse trabalho foi investigar cinco marcadores STRs do cromossomo X (DXS10079, DXS10074, DXS10075, DXS7424 e DXS101) na população de Alagoas, Brasil, e analisar suas freqüências para propósitos forenses. A amostra foi composta de 404 indivíduos não aparentados, sendo 203 do sexo masculino e 201 do sexo feminino. O DNA foi extraído através do método Chelex-100 e a amplificação foi realizada por PCR em um sistema pentaplex, sendo os fragmentos separados por eletroferese de capilar. Para os marcadores STRs estudados, foram calculadas as freqüências alélicas e haplotípicas, Heterozigozidade esperada e observada, Equilíbrio de Hardy Weinberg (HWE), diversidade genética, Chance Média de Exclusão (MEC) em trios envolvendo filhas e em duplas de pai/filha. Também foram calculados Poder de Discriminação em homens (PDM) e mulheres (PDF) e Conteúdo de Informação Polimórfica (PIC). Os parâmetros forenses investigados demonstram que o STR DXS101 é o marcador mais informativo, seguido por DXS10074, DXS10079, DXS7424 e DXS10075. O Conteúdo de Informação Polimórfica variou de 0.7470 a 0.8858. Para o sistema pentaplex investigado, os valores combinados de PDM e PDF foram de 0,9998947 e 0, 9999998, respectivamente e o MEC combinado em trios envolvendo filhas e em duplas pai/filha foi de 0,999817 e 0, 998042, respectivamente. Nenhum desvio do Equilíbrio de Hardy Weinberg foi observado. Concluímos que os cinco marcadores analisados são altamente informativos para testes de parentesco e constituem uma poderosa ferramenta genética para a prática forense em nossa população.

Orientador: Ana Silvia Alves Meira Tavares Moura / Banca: Luiz Lehmann Coutinho / Banca: Mônica Corrêa Ledur / Banca: Millor Fernandes do Rosário / Banca: José Roberto Sartori / Resumo: A partir de resultados de um estudo anterior, no qual foram mapeados QTLs para características de peso vivo, peso do coração e pulmões no GGA1, foi definida uma região no intervalo entre os marcadores ADL0234 e LEI0071, abrangendo 82,3 cM. Foram avaliadas três famílias de meios-irmãos paternos que compreendiam sete famílias de irmãos completos, num total de 652 F2 para as características: peso vivo aos 35 e 41 dias de idade, pesos do coração e pulmões e rendimentos de coração e pulmões. Os genótipos de seis marcadores microssatélites foram adicionados aos dez utilizados anteriormente. O mapa de ligação obtido da região compreendeu 110,8 cM com espaçamento médio entre os marcadores de 7,4 cM. Na análise de F2, em um único intervalo (LEI0146-LEI0174), compreendendo 28,8 cM, foram mapeados QTLs para todas as características estudadas, com exceção dos rendimentos de coração e pulmões. Neste intervalo estão localizados o gene IGF1 e o centrômero do cromossomo. A adição de seis marcadores confirmou os QTLs mapeados anteriormente, porém alguns em diferentes posições. A análise de meios-irmãos paternos indicou que os principais QTLs estavam segregando em apenas uma das famílias (7716), na qual cinco QTLs foram mapeados. Na análise de meios-irmãos maternos, duas famílias segregaram QTLs tanto na análise Individual como na Conjunta (7810 e 7971). As diferentes análises permitiram selecionar dois casais F1, que devem ser o alvo dos próximos estudos. Este estudo restringiu a busca por genes candidatos responsáveis pelas características de interesse a uma região de 28,8 cM (9,82 Mb) no GGA1. / Abstract: Based on the results from a previous study, in which QTL for body weight, heart and lungs weights and heart and lungs percentages were mapped to GGA1, a region was defined between markers ADL0234 and LEI0071, spanning 82.3 cM. Three paternal half-sib families, comprising seven full-sib families, totaling 652 F2 were evaluated for body weight at 35 and 41 days of age, heart and lungs weights and heart and lungs yields. Genotypes of six microsatellite markers were added to those of ten previously used. The linkage map of this region spanned 110.8 cM, with average spacing of 7.4 cM between markers. In a single interval (LEI0146-LEI0174), comprising 28.8 cM, QTLs for all traits, except for heart and lungs yields were mapped in the F2 analysis. In this same interval the IGF1 gene, and the chromosome centromere, are located. The use of six additional markers confirmed the same QTLs mapped previously, but some of them, in different positions. The paternal half-sib analysis indicated that the main QTLs were segregating in one of the families only (7716), in which five QTLs were mapped. In the maternal half-sib analysis, two families segregated QTLs both, in the across and within families analyses (7810 and 7971). These analyses allowed the selection of two F1 couples to be the target for future studies. This study restricted the search for candidate genes responsible for the traits of interest to a region of 28.8 cM (9.82 Mb) in GGA1. / Doutor

L'Indonésie a été l'objet de la dispersion Austronésienne qui a débuté il y a environ 5000 ans depuis Taiwan, se propager à travers les Philippines et l'Indonésie, puis toucher l'Océanie à l'est, et à Madagascar à l'ouest. Malgré de nombreuses recherches en génétique sur la dispersion Austronésienne vers l'est, il y a très peu de données sur la dispersion vers l'ouest, laissant sans réponse de nombreuses questions, liées notamment au peuplement de Madagascar. Reposant sur l'analyse des données culturelles et biologiques, les populations d'Indonésie semblent avoir joué un rôle majeur dans la colonisation de Madagascar, le premier millénaire de notre ère. Cependant, le peu de populations Indonésiennes étudiées à ce jour n'a pas permis jusqu'à présent d'identifier la population indonésienne source. Dans ce présent travail, j'ai réalisé des études en génétique des populations de 12 populations Indonésiennes, qui à priori devraient éclairer l'histoire des migrations austronésiennes dans l'Océan Indien. Parmi elles sont inclus le Ma'anyan du sud-est de Bornéo qui sont les plus proches linguistiquement des Malgaches. En utilisant différents marqueurs génétiques, ma recherche a amélioré nos connaissances de la diversité génétique Indonésienne, et du lien génétique entre l'Indonésie et Madagascar. Résultats L'analyse des marqueurs uniparentaux (chr-Y et ADNmt) suggère que les Malgaches proviennent de plusieurs régions d'Indonésie, avec un lien privilégié avec le sud-est de Bornéo, le sud de Sulawesi et les îles de la Sonde. Etonnamment, les Ma'anyan partagent un nombre limité de lignées paternelles et maternelles avec les Malgaches, malgré leur proximité linguistique. Par ailleurs, en combinant l'analyse de fréquences des SNPs et l'analyse haplotypique à partir des données autosomales, il a été confirmé que la diversité génétique des Ma'anyan ne correspond pas à l'ancestralité asiatique des Malgaches. Cependant, en centrant l'analyse sur les populations du sud-est de Bornéo, l'origine de l'ancestralité asiatique des Malgaches est ancrée dans la population Banjar, un mélange de population Ma'anyan et Malaise, résultat des activités commerciales de l'empire Malais dans le sud-est de Bornéo, qui se sont poursuivies à travers l'océan Indien. Par ailleurs nos résultats ont aussi permis d'accroitre notre compréhension de la diversité génétique de l'Indonésie en identifiant (1) une nouvelle composante génétique austronésienne présente chez les Ma'anyan, et retrouvée à faible fréquence à travers l'Asie du Sud-Est, suggérant une plus grande complexité du modèle d'expansion austronésien dans la région et (2) le rôle joué par les nomades de la mer dans la structuration de la diversité génétique et les échanges entre populations dans l'Indonésie, soulignant l'histoire génétique complexe de populations suivant un mode de vie nomade. / Indonesia hosts a wide range of linguistic, ethnic and genetic diversity, comprising ~600 ethnic groups and 700 living languages. Indonesia has facilitated the last substantial wave of human migration was the Austronesian dispersal ~5,000 years ago, which is thought to have originated in Taiwan. Its influence spread through Philippines and Indonesia, ultimately impacting a wide geographical area, from Remote Oceania in the east and to Madagascar in the west. Despite considerable genetic research on the eastward Austronesian expansion, there is little equivalent research on the western edge, leaving major issues unresolved regarding the settlement of Madagascar. Based on cultural and biological studies, it has been suggested that Indonesian peoples played a major role in the colonization of Madagascar from around the mid-first millennium CE (Current Era). However, poor geographical coverage of Indonesian populations has prevented the Indonesian source populations from being identified. Here, I performed human population genetic studies on 12 new Indonesian populations, which were a priori expected to shed light on the westward migration of Austronesians across the Indian Ocean. This includes the Ma'anyan ethnic group from Southeast Borneo, who are the closest linguistic siblings to modern Malagasy. Using different genetic markers (Y-chromosome SNPs, mitochondrial DNA and genome-wide SNPs), my research has improved the description of Indonesian genetic diversity, and investigated the genetic links between Indonesia and Madagascar. Results Uniparental markers (Y-chromosome and mtDNA) analyses suggest that Malagasy derive from multiple regional sources in Indonesia, with a focus on southeastern Borneo, southern Sulawesi and the Lesser Sunda islands. Interestingly, the Ma'anyan share limited paternal and maternal lineages with the Malagasy, despite their linguistic connection. Furthermore, combining SNP frequency and haplotype-based analyses from autosomal genome-wide data, it was confirmed that the genetic diversity of the Ma'anyan does not match the Asian ancestry of the Malagasy. However, by focusing on Southeast Borneo populations, strong support was found for an origin of the Asian ancestry of Malagasy among the people of Banjar, an admixed population of Ma'anyan and Malay, likely resulting from trading activities by the Malay Empire in Southeast Borneo, and later continuing across the Indian Ocean arena. These results increase our understanding of genetic diversity across Indonesia by 1) identifying the unique and undiscovered Austronesian genetic component carried by the Ma'anyan, which occurs at low levels across Island Southeast Asia and suggests a more complex model for the Austronesian expansion in this region, and 2) describing the role played by sea-nomads in structuring genetic diversity and exchanges in central Indonesia, thus revealing the complex genetic history of populations living this rare nomadic lifestyle.

Chez les Mammifères femelles, l’extinction transcriptionnelle d’un des deux chromosomes X pendant l’embryogénèse précoce compense le déséquilibre de dose des gènes liés à l’X entre les sexes. L’inactivation aléatoire du chromosome X est mise en place dans la masse cellulaire interne du blastocyste et maintenue jusqu’à l’âge adulte dans le soma. Chez certains Euthériens incluant la souris, les tissus extra-embryonnaires (trophectoderme et endoderme primitif) montrent une inactivation soumise à empreinte du X paternel. Le statut inactif du Xp peut être étudié ex vivo dans les cellules souches trophoblastiques (TS) dérivées du trophectoderme. Nous avons pu sélectionner des cellules TS montrant une réactivation partielle du Xp ou bien une inversion complète du profil d’inactivation. Ceci révèle une plasticité épigénétique accrue de l’inactivation dans le trophectoderme par au soma.L’inactivation aléatoire du chromosome X est récapitulée pendant la différenciation des cellules souches embryonnaires (ES), qui servent de modèle cellulaire. Ce processus est déclenché par l’accumulation en cis du long ARN non codant Xist qui crée un domaine nucléaire répresseur autour du futur chromosome X inactif. Avant la différenciation, l’accumulation de Xist est réprimée par un autre long ARN non codant, Tsix, qui est transcrit en antisens de Xist. Afin d’adresser la dynamique fonctionnelle des ARN Xist et Tsix, nous avons inséré différents motifs d’étiquetage au locus Xist/Tsix endogène. Incorporés dans l’ARN sens ou antisens, ces étiquettes sont reconnues spécifiquement par des molécules fluorescentes, permettant ainsi la visualisation de ces transcrits dans les cellules vivantes. / In female Mammals, the transcriptional silencing of one of the two X chromosomes during early embryogenesis compensates the dosage disequilibrium of X-linked genes between sexes. Random X chromosome inactivation occurs in the inner cell mass of the blastocyst and is maintained through adult life in the soma. In some Eutherian species including mice, extraembryonic tissues (trophectoderm and primitive endoderm) exhibit imprinted inactivation of the paternal X. The inactive state of the Xp can be extensively studied ex vivo in Trophoblast Stem (TS) cells derived from the trophectoderm. We were able to select from the general cell population, TS cells exhibiting partial reactivation of the Xp or showing a complete switch of imprinted X-inactivation pattern. This reveals an accrued epigenetic plasticity of imprinted X-inactivation in the trophectoderm as compared to random X-inactivation in the soma.Random X-chromosome inactivation is recapitulated during the differentiation of female Embryonic Stem (ES) cells – which serves as cellular model. This process is triggered by the cis-accumulation of Xist long non coding RNA molecules which create a nuclear repressive domain around the future inactive X chromosome. Before differentiation, the accumulation of Xist is repressed by another lncRNA, Tsix, that is transcribed antisense to Xist. In order to address the functional dynamics of Xist and Tsix RNAs, we inserted different types of tag sequences in the endogenous Xist/Tsix locus. Incorporated in the sense or antisense RNA, these tags are specifically recognized by fluorescent molecules, thereby allowing live cell imaging of these transcripts.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2015-12-11T11:16:50Z No. of bitstreams: 1 giselecardosodelemos.pdf: 1950421 bytes, checksum: 5d956c19538a640bfa5a8e77dcd04747 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2015-12-11T15:02:15Z (GMT) No. of bitstreams: 1 giselecardosodelemos.pdf: 1950421 bytes, checksum: 5d956c19538a640bfa5a8e77dcd04747 (MD5) / Made available in DSpace on 2015-12-11T15:02:15Z (GMT). No. of bitstreams: 1 giselecardosodelemos.pdf: 1950421 bytes, checksum: 5d956c19538a640bfa5a8e77dcd04747 (MD5) Previous issue date: 2015-08-31 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Este trabalho busca analisar as apropriações que o escritor indiano em língua inglesa Amitav Ghosh faz das noções ocidentais de ciência e religião em suas respectivas obras The Calcutta Chromosome e The Circle of Reason, por meio de diálogos, tensões e negociações destas noções com paradigmas filosófico-religiosos de caráter inclusivista e dialógico da civilização indiana, que são matrizes existenciais que perpassam gerações e influenciam inclusive a contemporaneidade da Índia. Para esse fim, esse trabalho privilegia a literatura ficcional como ferramenta crítica para as discussões sobre ciência e religião, uma vez que a ficção propicia a contextualização das coisas/seres, ou seja, a apreensão destes em sua totalidade. Com isso, também buscamos apresentar uma contextualização histórica, linguística, literária, científica e filosófico-religiosa para que sejam mais bem compreendidas algumas escolhas de Amitav Ghosh, a saber: a língua inglesa, o gênero literário romance, as temáticas da medicina tropical e da frenologia e a apropriação da doutrina da transmigração da alma (ātma), a lei do karma e a teoria dos guṇas, discutidas em fontes como os Upaniṣads e o Bhagavad-gītā. Como ferramentas de análise utilizamos, sobretudo, teorias pós-coloniais de subalternidade, tradições unitaristas da filosofia hindu, as obras não-ficcionais do próprio autor e as obras dos mais importantes críticos literários de Ghosh. Com as análises literárias mostramos que Ghosh, além de por em prática a tradição inclusivista indiana, ele demonstra a superioridade do ―domínio espiritual‖ sobre o ―domínio material‖, (conceitos cunhados por Partha Chatterjee) e reabilita a noção de uma racionalidade ocidental excludente tornando-a uma razão iluminadora e libertadora. / This study analyzes the appropriations of Western notions of science and religion by the Indian writer in English Amitav Ghosh, in his respective works The Calcutta Chromosome and The Circle of Reason, through dialogues, tensions, and negotiations between these notions and religious and philosophical paradigms of the Indian civilization, characterized by its inclusive and dialogical characteristics. These paradigms form an existential matrix that crosses generations and even influences contemporary India. To this end, this work focuses on fictional literature as a critical tool for the discussion on science and religion, since fiction provides contextualization for things/beings, that is, the comprehention of these in their entirety. With this, we also seek to provide a historical, linguistic, literary, scientific, philosophical and religious context in order to better understand some of Amitav Ghosh‘s choices, namely the English language, the novel as literary genre, the themes of tropical medicine and phrenology and the appropriation of the doctrine of transmigration of the soul (saṃsāra), the law of karma and the theory of guṇas discussed in sources such as the Upaniṣads and the Bhagavad-gītā. As tools of analysis, we use especially postcolonial theories of subalternity, unitarian traditions of Hindu philosophy, nonfictional works of the author himself and the works of the most important literary critics of Ghosh‘s work. With literary analysis we show that Ghosh, besides using the inclusivist Indian tradition, demonstrates the superiority of ―spiritual domain‖ over the ―material domain‖ (concepts coined by Partha Chatterjee) and also rehabilitates the notion of an exclusionary Western rationality transforming it into an enlightening and liberating reason.

La dystrophie Facio-Scapulo-Humérale est caractérisée par une faiblesse musculaire progressive et asymétrique. Elle affecte principalement les muscles faciaux, scapulaires et huméraux. L’association de plusieurs évènements épigénétiques à trois facteurs génétiques de la région subtélomérique du chromosome 4 résulte en un changement dans l’organisation chromatinienne la rendant permissive à l’expression aberrante des gènes de la région 4q35. Les myoblastes DFSH présentent des défauts de différenciation in vitro et des dérégulations dans des voies majeures comme celle de la réponse cellulaire au stress oxydant et de la différenciation myogénique. L’enjeu génétique et épigénétique complexe dans la DFSH et les limitations de la thérapie cellulaire dans son contexte laissent la DFSH jusque-là incurable. Toutefois les avancées dans les thérapies cellulaires et génétiques des myopathies ouvrent des horizons pour de futures applications dans le cadre de la DFSH.Le travail de thèse s’articule autour de trois thématiques. Premièrement, nous démontrons la faisabilité de la correction phénotypique et fonctionnelle des myotubes DFSH in vitro par la fusion de 50% de myoblastes normaux avec des myoblastes DFSH. Ensuite, nous évaluons deux approches d’édition génomique. Dans la première approche, nous ciblons le site de rattachement du chromosome 4 à la matrice nucléaire, FR-MAR avec la protéine CTCF à l’aide du système CRISPR/dCas9 en vue du rétablissement de l’organisation chromatinienne et de la fonction isolatrice de FR-MAR. Dans la deuxième, nous échangeons par translocation les régions homologues 4q35 et 10q26 dans le but de corriger les myoblastes DFSH comme les trois facteurs génétiques du locus 4q35 ne sont pathogéniques que sur un fond génétique lié au chromosome 4. Finalement, nous étudions le rôle du stress oxydant dans la DFSH. / Facio-Scapulo-Humeral dystrophy is characterized by progressive and asymmetrical muscle weakness. It mainly affects the facial, scapular and humeral muscles. The association of several epigenetic events with three genetic factors of the subtelomeric region of chromosome 4 results in a chromatin organization modification making it permissive to the aberrant expression of genes in the 4q35 region. FSHD myoblasts exhibit differentiation defects in vitro and dysregulations in major pathways such as the cellular response to oxidative stress and myogenic differentiation. The limitations of cell therapy and the complex genetic and epigenetic interplay in FSHD leave it, till now, incurable. However advances in cellular and genetic therapies of myopathies open up new horizons for future applications in the FSHD context. The thesis work is structured around three themes. First, we demonstrate the feasibility of phenotypic and functional correction of FSHD myotubes in vitro by fusing 50% of normal myoblasts with FSHD myoblasts. Next, we evaluate two genomic editing approaches. In the first one, we target the site of attachment of chromosome 4 to the nuclear matrix, FR-MAR with the CTCF protein using the CRISPR / dCas9 system for the purpose of restoring the chromatin organization and the insulating function of FR-MAR. In the second one, we exchange the homologous regions 4q35 and 10q26 by translocation in order to correct the FSHD myoblasts as the three genetic factors of the 4q35 locus are pathogenic only on a genetic background linked to chromosome 4. Finally, we study the role of the oxidative stress in the FSHD.

Yes / Abstract The budding yeast genome contains regions where meiotic recombination initiates more frequently than in others. This pattern parallels enrichment for the meiotic chromosome axis proteins Hop1 and Red1. These proteins are important for Spo11-catalyzed double strand break formation; their contribution to crossover recombination remains undefined. Using the sequence-specific VMA1-derived endonuclease (VDE) to initiate recombination in meiosis, we show that chromosome structure influences the choice of proteins that resolve recombination intermediates to form crossovers. At a Hop1-enriched locus, most VDE-initiated crossovers, like most Spo11-initiated crossovers, required the meiosis-specific MutLγ resolvase. In contrast, at a locus with lower Hop1 occupancy, most VDE-initiated crossovers were MutLγ-independent. In pch2 mutants, the two loci displayed similar Hop1 occupancy levels, and VDE-induced crossovers were similarly MutLγ-dependent. We suggest that meiotic and mitotic recombination pathways coexist within meiotic cells, and that features of meiotic chromosome structure determine whether one or the other predominates in different regions.

The intimate relationship between the higher-order chromatin organisation and the regulation of gene expression is increasingly attracting attention in the scientific community. Thanks to high-resolution microscopy, genome-wide molecular biology tools (3C, ChIP-on-chip), and bioinformatics, detailed structures of chromatin loops, territories, and nuclear domains are gradually emerging. However, to fully reveal a comprehensive map of nuclear organisation, some fundamental questions remain to be answered in order to fit all the pieces of the jigsaw together. The underlying mechanisms, precisely organising the interaction of the different parts of chromatin need to be understood. Previous work in our lab hypothesised and verified the “transcription factory” model for the organisation of mammalian genomes. It is widely assumed that active polymerases track along their templates as they make RNA. However, after allowing engaged polymerases to extend their transcripts in tagged precursors (e.g., Br-U or Br-UTP), and immunolabelling the now-tagged nascent RNA, active transcription units are found to be clustered in nuclei, in small and numerous sites we call “transcription factories”. Previous work suggested the transcription machinery acts both as an enzyme as well as a molecular tie that maintains chromatin loops, and the different classes of polymerases are concentrated in their own dedicated factories. This thesis aims to further characterise transcription factories. Different genes are transcribed by different classes of RNA polymerase (i.e., I, II, or III), and the resulting transcripts are processed differently (e.g., some are capped, others spliced). Do factories specialise in transcribing particular subsets of genes? This thesis developed a method using replicating minichromosomes as probes to examine whether transcription occurs in factories, and whether factories specialise in transcribing particular sets of genes. Plasmids encoding the SV40 origin of replication are transfected into COS-7 cells, where they are assembled into minichromosomes. Using RNA fluorescence in situ hybridisation (FISH), sites where minichromosomes are transcribed are visualised as discrete foci, which specialise in transcribing different groups of genes. Polymerases I, II, and III units have their own dedicated factories, and different polymerase II promoters and the presence of an intron determine the nuclear location of transcription. Using chromosome conformation capture (3C), minichromosomes with similar promoters are found in close proximity. They are also found close to similar endogenous promoters and so are likely to share factories with them. In the second part of this thesis, I used RNA FISH to confirm results obtained by tiling microarrays. Addition of tumour necrosis factor alpha (TNF alpha) to human umbilical vein endothelial cells induces an inflammatory response and the transcription of a selected sub-set of genes. My collaborators used tiling arrays to demonstrate a wave of transcription that swept along selected long genes on stimulation. RNA FISH confirmed these results, and that long introns are co-transcriptionally spliced. Results are consistent with one polymerase being engaged on an allele at any time, and with a major checkpoint that regulates polymerase escape from the first few thousand nucleotides into the long gene.