Thesis (Ph.D.)--Uppsala Universitet, 2008. / This website links to the complete document in PDF format. Title from title screen (viewed on November 22, 2009). Includes bibliographical references.

Meiosis is a specialized type of cell division essential for the production of four normal haploid gametes. In early prophase I of meiosis, the intimate synapsis between homologous chromosomes, and the formation of chiasmata, is facilitated by a proteinaceous structure known as the synaptonemal complex (SC). Ultrastructural analysis of germ cells of a number of organisms has disclosed that SC is a specialized tripartite structure composed of two lateral elements, one on each homolog, and a central element, which, in turn, are linked by transverse elements. Genetic studies have revealed that defects in meiotic chromosome alignment and/or segregation result in aneuploidy, which is the leading cause of spontaneous miscarriages in humans, hereditary birth defects such as Down syndrome, and are also, associated with the development and progression of certain forms of cancer. The mechanism(s) underlying the alignment/pairing of chromosomes at meiosis I differ among organisms. These can be divided into at least two broad pathways: one is independent of DNA double-strand breaks (DSB) and other is mediated by DSBs. In the DSB-dependent pathway, SC plays crucial roles in promoting homolog pairing and disjunction. On the other hand, the DSB-independent pathway involves the participation of telomeres, centromeres and non-coding RNAs in the pre-synaptic alignment, pre-meiotic pairing as well as pairing of homologous chromosomes. Although a large body of literature highlights the central role of SC in meiotic recombination, the possible role of SC components in homolog recognition and alignment is poorly understood. Genetic screens for Saccharomyces cerevisiae mutants defective in meiosis and sporulation lead to the isolation of genes required for interhomolog recombination, including those that encode SC components. In S. cerevisiae, ten meiosis-specific proteins viz., Hop1, Red1, Mek1, Hop2, Pch2, Zip1, Zip2, Zip3, Zip4 and Rec8 have been recognized as bona fide constituents of SC or associated with SC function. Mutations in any of these genes result in defective SC formation, thus leading to reduction in the rate of recombination. HOP1 (Homolog Pairing) encodes a ̴ 70 kDa structural protein, which localizes to the lateral elements of SC. It was found to be essential for the progression of meiotic recombination. In hop1Δ mutants, meiosis specific DSBs are reduced to 10% of that of wild type level and it fails to produce viable spores. It also displays relatively high frequency of inter-sister recombination over inter-homolog recombination. Bioinformatics analysis suggests that Hop1 comprises of an N-terminal HORMA domain (Hop1, Rev7 and Mad2), which is conserved among Hop1 homologs from diverse organisms. This domain is also known to be present in proteins involved in processes like chromosome synapsis, repair and sex chromosome inactivation. Additionally, Hop1 harbors a 36-amino acid long zinc finger 348374 motif (CX2CX19CX2C) which is critical for DNA binding and meiotic progression, and a putative nuclear localization signal corresponding to amino acid residues from 588-594. Previous studies suggested that purified Hop1 protein exists in multiple oligomeric states in solution and displays structure specific DNA binding activity. Importantly, Hop1 exhibited higher binding affinity for the Holliday junction (HJ), over other early recombination intermediates. Binding of Hop1 to the HJ at the core resulted in branch migration of the junction, albeit weakly. Intriguingly, Hop1 showed a high binding affinity for G4 DNA, a non-B DNA structure, implicated in homolog synapsis and promotes robust synapsis between double-stranded DNA molecules. Hop1 protein used in the foregoing biochemical studies was purified from mitotically dividing S. cerevisiae cells containing the recombinant plasmid over-expressing the protein where the yields were often found to be in the low-microgram quantities. Therefore, one of the major limitations to the application of high resolution biophysical techniques, such as X-crystallography and spectroscopic analyses for structure-function studies of S. cerevisiae Hop1 has been the non-availability of sufficient quantities of functionally active pure protein. In this study, we have performed expression screening in Escherichia coli host strains, capable of high level expression of soluble S. cerevisiae Hop1 protein. A new protocol has been developed +2 for expression and purification of S. cerevisiae Hop1 protein, using Ni-NTA and double-stranded DNA-cellulose chromatography. Recombinant S. cerevisiae Hop1 protein thus obtained was >98% pure and exhibited DNA binding activity with high-affinity for Holliday junction. The availability of the bacterial HOP1 expression vector and functionally active Hop1 protein has enabled us to glean and understand several vital biological insights into the structure-function relationships of Hop1 as well as the generation of appropriate truncated mutant proteins. Mutational analyses in S. cerevisiae has shown that sister chromatid cohesion is required for proper chromosome condensation, including the formation of axial elements, SC assembly and recombination. Consistent with these findings, homolog alignment is impaired in red1hop1 strains and associations between homologs are less stable. red1 mutants fail to make any discernible axial elements or SC structures but exhibit normal chromosome condensation, while hop1 mutants form long fragments of axial elements but without any SCs, are defective in chromosome condensation, and produce in-viable spores. Using single molecule and ensemble assays, we found that S. cerevisiae Hop1 organizes DNA into at least four major distinct DNA conformations: (i) a rigid protein filament along DNA that blocks access to nucleases; (ii) bridging of non-contiguous segments of DNA to form stem-loop structures; (iii) intra-and intermolecular long range synapsis between double-stranded DNA molecules; and (iv) folding of DNA into higher order nucleoprotein structures. Consistent with B. McClintock’s proposal that “there is a tendency for chromosomes to associate 2-by-2 in the prophase of meiosis involving long distance recognition of homologs”, these results to our knowledge provide the first evidence that Hop1 mediates the formation of tight DNA-protein-DNA nucleofilaments independent of homology which might help in the synapsis of homologous chromosomes during meiosis. Although the DNA binding properties of Hop1 are relatively well established, comparable knowledge about the protein is lacking. The purification of Hop1 from E. coli, which was functionally indistinguishable from the protein obtained from mitotically dividing S. cerevisiae cells has enabled us to investigate the structure-function relationships of Hop1, which has provided important insights into its role in meiotic recombination. We present several lines of evidence suggesting that Hop1 is a modular protein, consisting of an intrinsically unstructured N-terminal domain and a core C-terminal domain (Hop1CTD), the latter being functionally equivalent to the full-length Hop1 in terms of its in vitro activities. Importantly, however, Hop1CTD was unable to rescue the meiotic recombination defects of hop1null strain, indicating that synergy between the N-terminal and C-terminal domains of Hop1 protein is essential for meiosis and spore formation. Taken together, our findings provide novel insights into the molecular functions of Hop1, which has profound implications for the assembly of mature SC, homolog synapsis and recombination. Several lines of investigations suggest that HORMA domain containing proteins are involved in chromatin binding and, consequently, have been shown to play key roles in processes such as meiotic cell cycle checkpoint, DNA replication, double-strand break repair and chromosome synapsis. S. cerevisiae encodes three HORMA domain containing proteins: Hop1, Rev7 and Mad2 (HORMA) which interact with chromatin during diverse chromosomal processes. The data presented above suggest that Hop1 is a modular protein containing a distinct N-terminal and C-terminal (Hop1CTD) domains. The N-terminal domain of Hop1, which corresponds to the evolutionarily conserved HORMA domain, although, discovered first in Hop1, its precise biochemical functions remain unknown. In this section, we show that Hop1-HORMA domain expressed in and purified from E. coli exhibits preferential binding to the HJ and G4 DNA, over other early recombination intermediates. Detailed functional analyses of Hop1-HORMA domain, using mobility shift assays, DNase I footprinting and FRET, have revealed that HORMA binds at the core of Holliday junction and induces marked changes in its global conformation. Further experimental evidence also suggested that it causes DNA stiffening and condensation. However, like Hop1CTD, HORMA domain alone failed to rescue the meiotic recombination defects of hop1 null strain, indicating that synergy between the N-and C-terminal domains of Hop1 is essential for meiosis as well as for the formation of haploid gametes. Moreover, these results strongly implicate that Hop1 protein harbours a second DNA binding motif, which resides in the HORMA domain at its N-terminal region. To our knowledge, these findings also provide the first insights into the biochemical mechanism underlying HORMA domain activity. In summary, it appears that the C-terminal (CTD) and N-terminal (HORMA) domains of Hop1 may perform biochemical functions similar (albeit less efficiently) to that of the full-length Hop1. However, further research is required to uncover the functional differences between these domains, their respective interacting partners and modulation of the activity of these domains.

Thesis (PhD (Psychiatry))--Stellenbosch University, 2008. / BACKGROUND: Schizophrenia is a heterogeneous illness resulting from complex gene-environment interplay. The majority of molecular genetic work done has involved Caucasian populations, with studies in these and Asian populations showing 2-32% of sufferers to have chromosomal aberrations. So far the discovery of a specific susceptibility mechanism or gene still eludes us, but the use of endophenotypes is advocated as a useful tool in this search. No cytogenetic studies of this nature have been reported in any African schizophrenia population. AIM: The aim of the study was to combine genotypic and phenotypic data, collected in a homogenous population in a structured manner, with the hope of characterising an endophenotype that could be used for more accurate identification of individuals with possible chromosomal abnormalities. METHODOLOGY: A structured clinical interview was conducted on 112 Xhosa schizophrenia patients. (Diagnostic Interview for Genetic Studies, including Schedules for the Assessment of Negative and Positive Symptoms.) Blood samples (karyotyping and/or FISH analysis) as well as urine samples (drug screening) were obtained and nine head and facial measurements were performed. Descriptive statistics were compiled with reference to demographic, clinical and morphological variables. Comparisons between mean differences for these variables were made.

Human Artificial Chromosomes (HACs) have been confirmed as viable gene expression vectors and a potential tool for gene therapy. However, standard lipid-based delivery methods pose a developmental barrier. The work presented in this thesis includes the development of a novel and efficient HAC vector system for gene delivery into human cells using Herpes Simplex Virus-1 (HSV-1) amplicon technology. The development of HSV-1 amplicons for HAC delivery is a major step forward in the HAC field. In this study, utilising the technology allowed the generation of HACs at a high efficiency in a range of human cell types, which is a significant step in the development for HAC gene expression systems. Further work also showed a significant difference in HAC stability between cell lines. Real-time PCR analysis determined that Aurora B was over expressed in cell lines in which the HACs were unstable. This correlated with high levels of chromosomal instability and was confirmed by western blot analysis. Since Aurora B is a kinase involved in at least two cell cycle checkpoints, cellular phosphorylation levels were perturbed to mimic that observed in the unstable cells, using okadaic acid, which is both a protein phosphatase inhibitor and activates Aurora B. Treatment of cells showed an increase in both HAC and overall chromosomal instability and an increase in histone H3 Serine 10 and Serine 28 phosphorylation. The project also focussed on the development of a gene expression system using HSV-1 amplicons. Two different strategies were explored. Firstly, one approach involved engineering the HPRT genomic locus into an HSV-HAC vector, by Red mediated recombination for complementing the HPRT deficiency in HPRT- HT1080 cells. As an alternative approach, co-infection of two different HSV-1 HAC amplicons for generating a single HAC gene vector was investigated. Initial experiments utilising the latter approach were the most successful and show promise for generating HAC containing genes via this strategy.

Human response to ionising radiation (IR) shows a wide variation. This is most clearly seen in the radiation-response of cells as measured by frequencies of chromosomal aberrations. Different frequencies of IR-induced aberrations can be conveniently observed in phytohaemagglutin-stimulated peripheral blood T-lymphocytes from both normal individuals and sporadic cancer cases, in either metaphase chromosomes or as micronuclei in the following cell cycle. Metaphase cells show frequent chromatid breaks, defined as chromatid discontinuities or terminal deletions, if irradiated in the G 2 -phase of the cell cycle. It has been shown that the frequency of chromatid breaks in cells from approximately 40% of sporadic breast cancer patients, are significantly higher than in groups of normal individuals. This suggests that elevated radiation-induced chromatid break frequency may be linked with susceptibility to breast cancer. It is known that chromatid breaks are initiated by a double strand break (DSB), but it appears that the two are linked only indirectly as repair kinetics for DSBs and chromatid breaks do not match. Therefore, the underlying causes of the wide variation in frequencies of chromatid breaks in irradiated T-lymphocytes from different normal individuals and from sporadic breast cancer cases are still unclear but it is unlikely to be linked directly to DSB rejoining. My research has focused on the mechanism through which chromatid breaks are formed from initial DSBs. The lack of a direct association suggested that a signalling process might be involved, connecting the initial DSB and resulting chromatid break. The signal model, suggested that the initial DSB is located within a chromatin loop that leads to an intra- or interchromatid rearrangement resulting in incomplete mis-joining of chromatin ends during the decatenation of chromatids during G 2 . It was therefore proposed that topoisomerase II alpha (topo IIα) might be involved, mainly because of its ability to incise DNA and its role in sister chromatid decatenation. During my PhD research I have used a strategy of altering topo II activity or expression and studying whether this alters IR-induced chromatid break frequency. The first approach involved cell lines that varied in topo IIα expression. The frequency of IR-induced chromatid breaks was found to correlate positively with topo IIα expression level, as measured in three different cell lines by immunoblotting, i.e. two cell lines with lower topo IIα expression exhibited lower chromatid break frequency. Topo II activity in these three cell lines was also estimated indirectly by the ability of a topo IIα poison to activate the G 2 /M checkpoint, and this related well with topo IIα expression. A second approach involved ‘knocking down’ topo IIα protein expression by silencing RNA (siRNA). Lowered topo IIα expression was confirmed by immunoblotting and polymerase chain reaction. SiRNA-lowered topo IIα expression correlated with a decreased IR-induced chromatid break frequency. In a third series of experiments cells were treated with ICRF-193, a topo IIα catalytic inhibitor. It was shown that inhibition of topo IIα also significantly reduced IR-induced chromatid breaks. I also showed that lowered chromatid break frequency was not due to cells with high chromatid break frequencies being blocked in G 2 as the mitotic index was not altered significantly in cells with lowered topo IIα expression or activity. These experiments show that topo IIα is involved in IR-induced chromatid break formation. The final experiments reported here attempted to show how topo II might be recruited in the process of forming IR-induced chromatid breaks. Hydrogen peroxide was used as a source of reactive oxygen species (reported to poison topo IIα) and it was shown that topo IIα under these conditions is involved in the entanglement of metaphase chromosomes and formation of chromatin ‘dots’ as well as chromatid breaks. Experiments using atomic force microscopy attempted to confirm these dots as excised chromatin loops. The possible role of topo IIα in both radiation- and hydrogen peroxide-induced primary DNA damage was also tested. It was shown that topo IIα does not affect radiation-induced DSBs, even though it does affect chromatid break frequency. Also, topo IIα does not affect hydrogen peroxide-induced DNA damage at low doses. The results support the idea that topo IIα is involved in the conversion of DSBs to chromatid breaks after both irradiation and treatment with hydrogen peroxide at a low concentrations. I have demonstrated that topo IIα is involved in forming IR-induced chromatid breaks, most likely by converting the initial DSBs into chromosomal aberrations as suggested by the signal model.

En France, le cancer de sein héréditaire représente environ 2500 nouveaux cas par an, dont prés de la moitié est attribuée à la mutation du gène BRCA1.La recherche de la mutation par biologie moléculaire est un travail fastidieux, coûteux et long (8 mois d’attente environ actuellement).Pour trouver une solution à ce délai, nous avons étudié en immunohistochimie une série initiale de 21 anticorps répartis en 5 groupes : anticorps antiBrca1 du commerce, liés à la perte de l’inactivation de l’X, liés à la signature basale ou myoépithéliale, anticorps dits classiques du cancer de sein et finalement dérivés de signatures établies par cDNAarray.Nous avons utilisé la technique de’ tissue microarrays’ en utilisant de manière comparative une population de 27 cas de cancer de sein présentant une mutation germinale de BRCA1, et 81 cas témoins de cancer de sein sporadiques appariés à l’âge, ainsi qu’à des lignées cellulaires d’origine mammaires. Dans une deuxième série indépendante de validation nous avons appliqué les résultats obtenus de la première série sur 28 cas de cancer mammaire muté, et 28 cas du cancer mammaire sporadique dans les mêmes conditions initiales.Nos résultats montrent pour la première fois sur des tissus tumoraux une probabilité forte d’une association entre la mutation Brca1 et la perte de l’inactivation de l’X ; confirment la valeur de MS110 comme un bon anticorps prédictif d’une mutation de Brca1 ; apportent un argument pour une participation myoépithéliale dans l’oncogenèse de cancer mammaire Brca1 muté; appuient la relation entre ce dernier et les récepteurs RE,RP ainsi que P53 , Bcl2,Ki67 et valident en protéomique la valeur discriminant de CDC47 correspondant à un des gènes de la signature génomique.Après confirmation des mêmes résultats dans la série de validation, nous soutenons en analyses multivariés un modèle qui comprend seulement Grade 3, MS110, Lys27H3 négative, Vimentine et KI67 positive. Cette équation correspond à une sensibilité de 82% et spécificité de 81% et propose une approche rapide économique de pré- ciblage de la mutation Brca1 ; ce qui améliorait la prise en charge préventive, thérapeutique et globale des patients et leurs familles. / Family structure, lack of reliable information, cost and delay are usual concerns faced with when deciding to perform BRCA analyses. Testing the breast cancer tissues with four antibodies (MS110, lys27H3, Vimentin, KI67) in addition to grade evaluation enabled to rapidly select patients to carry out genetic testing identification. We constituted an initial breast cancer tissue micro-array, considered as a learning set comprising 27 BRCA1 and 81 sporadic tumours. A second independent validation set of 28 BRCA1 tumours was matched to 28 sporadic tumours using the same original conditions.We have investigated morphological parameters and 21 markers by immunohistochemistry.A logistic regression model was used to select the minimal number of markers providing the best model to predict BRCA1 status. The model was applied to the validation set to estimate specificity and sensibility.In the initial set, the univariate analysis identified 11 markers significantly associated with BRCA1 status. Then the best multivariate model comprised only Grade 3, MS110, Lys27H3, Vimentin and KI67. When applied to the validation set, BRCA1 tumours were correctly classified with a sensitivity of 83% and a specificity of 81%. The performance of this model was superior when compared to other profiles.This work offers a new rapid and economic method for the pre-screening of patients at high risk of being BRCA1mutation carriers, then to guide genetic testing, and finally to provide appropriate preventive measure, advices and treatments including targeted therapy to patients and their families.

De par leur diversité de mécanismes de déterminisme du sexe et de chromosomes sexuels, les poissons téléostéens représentent d’excellents modèles pour mieux comprendre les bases moléculaires et évolutives du contrôle du développement sexuel chez les vertébrés. Grâce à l’analyse de chromosomes artificiels bactériens couvrant les chromosomes sexuels du platy Xiphophorus maculatus, trois copies d’un nouveau gène nommé teximY ont été découvertes dans la région de déterminisme du sexe du chromosome Y mais pas du chromosome X. Un gène texim1 très apparenté à teximY ainsi que trois gènes plus divergents ont été identifiés sur les autosomes. Les gènes teximY sont préférentiellement exprimés dans les testicules, au niveau des cellules germinales lors des étapes tardives de la spermatogénèse, alors que texim1 est également transcrit dans les gonades femelles. Des gènes texim ont été détectés chez d'autres poissons téléostéens mais pas chez le poisson-zèbre, ainsi que chez des céphalocordés, des urocordés et des échinodermes mais pas chez les tétrapodes. Les gènes texim codent pour des estérases putatives à domaine SGNH apparentées à des protéines cellulaires procaryotes et eucaryotes ou codées par des retrotransposons animaux. Les gènes texim sont associés à des transposons Helitron chez les poissons mais pas chez les autres animaux, suggérant capture et mobilisation du gène ancestral texim par un transposon à la base de la radiation des téléostéens. TeximY pourrait jouer un rôle dans la transposition du transposon Helitron dans la lignée germinale mâle, ou correspondre à un gène de spermatogenèse mobilisé par le transposon Helitron sur les nouveaux chromosomes sexuels de poissons. / The molecular and evolutionary basis of sex determination in vertebrates needs to be unveiled via comparison of different systems. Fish exhibit hypervariability of sex determination mechanisms. Thanks to the analysis of the Bacterial Artificial Chromosome (BAC) library covering the sex chromosomes of the platyfish Xiphophorus maculatus (Rio Jamapa population, XX /XY), three copies of a new gene have been identified in the sex-determining region of the Y but not the X chromosome, and named teximY. Four autosomal counterparts of teximY have been also detected in the genome of the platyfish with one of them, texim1 presenting 95% of cDNA sequence identity with the Y-linked copies. RT-qPCR expression analyses have been performed for each copy in male and female tissues. Two Y-linked teximY copies were preferentially expressed in testis, whereas the autosomal copy texim1 showed preferential expression in male and female gonads. In situ hybridizations with a teximY/1 probe revealed expression in late spermatids and spermatozeugmata. Texim sequences were detected in several fish species, but not in zebrafish, as well as in cephalochordates, urochordates and sea echinoderms but not in tetrapods. Predicted Texim proteins are related to proteins from different origins. Interestingly, texim genes are associated with a Helitron transposon in fish but neither in cephalochordates nor in echinoderms, suggesting capture and mobilization of an ancestral texim gene at the base of the bony fish lineage. TeximY proteins may play a role in Helitron transposition in the male germ line in fish, or texim genes are spermatogenesis genes mobilized and spread by transposable elements in fish genomes.

No description available.

A inativação do cromossomo X (ICX) em fêmeas é um exemplo de regulação epigenética. O silenciamento de um dos cromossomos X leva à formação estável da heterocromatina facultativa através da aquisição de múltiplas modificações na cromatina que são mantidas nas subsequentes divisões celulares. Atualmente, algumas características epigenéticas associadas à manutenção da ICX têm sido descritas, contudo os mecanismos de ação e a identidade dos diferentes fatores envolvidos na manutenção da ICX ainda são desconhecidos ou pouco compreendidos. Nosso laboratório realizou uma triagem funcional genômica por bibliotecas de shRNAs (short harpin RNAs) para encontrar genes envolvidos na manutenção da ICX em humanos. A partir deste estudo foram identificados 20 novos genes candidatos a estarem envolvidos na manutenção da ICX. Assim, o objetivo deste trabalho foi validar o grau de envolvimento de dois destes genes candidatos (H3F3B e ASF1A) no processo de controle epigenético do cromossomo X. Para isto, foi realizado o silenciamento dos genes candidatos através da utilização de partículas lentivirais portando shRNAs específicos em fibroblastos primários femininos heterozigotos para uma mutação no gene HPRT e com desvio total de ICX, onde o único alelo normal do gene HPRT está no Xi. A reativação do Xi nestas células foi avaliada por cultivo das mesmas em meio HAT, que seleciona células HPRT+. Só sobreviveram os fibroblastos que foram silenciados para o gene H3F3B. Nestes, as células transduzidas com o shH3F3B.2 expressam o alelo selvagem do gene, presente no Xi, além do gene mutante. Ensaios de RNA-FISH e trimetilação de histonas foram feitos nessas células para avaliar a perda das marcas de cromatina inativa. Foi observada uma perda da nuvem de XIST nas células transduzidas com o shH3F3B.2 e selecionadas em HAT em passagens altas. Por último, análises de expressão alelo-específica de genes ligados ao X comprovaram que dois genes que são submetidos à ICX apresentaram expressão do alelo inativado (FLNA e FHL1). Porém, também foi observada uma mudança no padrão de expressão alelo-específica em genes autossômicos. Finalmente, as análises de expressão geral do cromossomo X mostraram que as células transduzidas com o shH3F3B.2 e selecionada em HAT tinham uma expressão gênica aumentada em relação ao controle. Em conclusão, nossos resultados sugerem uma descondensação da cromatina no cromossomo Xi e portanto um provável envolvimento do gene H3F3B na manutenção da ICX. / The X chromosome Inactivation (XCI) in females is an example of epigenetic regulation. Silencing of one of the X chromosomes leads to the stable formation of the facultative heterochromatin through the acquisition of multiple modifications in the chromatin that are maintained in the subsequent cell divisions. Currently, some epigenetic features associated with the maintenance of XCI have been described. Nonetheless, the mechanisms of action and the identity of the different factors involved in the maintenance of XCI are still unknown or poorly understood. Our laboratory performed a genomic functional screening by shRNA (short harpin RNAs) libraries to find genes involved in the maintenance of XCI in humans. From this study, we identified 20 new candidate genes to be involved in the maintenance of XCI. Thus, the objective of this work was to validate the degree of involvement of two of these candidate genes (H3F3B and ASF1A) in the epigenetic process control of the X chromosome. For this, the silencing of the candidate genes was performed in female heterozygous primary fibroblasts for a mutation of the HPRT gene and with a total XCI shift through the use of lentiviral particles carrying specific shRNAs, where the only normal allele of the HPRT gene is in the Xi (inactivated X). Xi reactivation was evaluated in these cells by culturing them in HAT medium, which selects HPRT + cells. Only the fibroblasts that were silenced for the H3F3B gene survived. Furthermore, the cells transduced with shH3F3B.2 express the HPRT wild gene allele, present in Xi, in addition to the mutant gene. RNA-FISH and histone trimethylation assays were performed on these cells to evaluate the loss of inactive chromatin marks. A loss of the XIST cloud was observed in cells transduced with shH3F3B.2 and selected in HAT at high passages. Finally, allele-specific expression analyzes of X-linked genes showed that two genes that undergo XCI showed expression of the inactivated allele (FLNA and FHL1). However, a change in allele-specific expression pattern was also observed in autosomal genes. Finally, the X chromosome general expression analyses showed that cells transduced with shH3F3B.2 and selected on HAT had increased gene expression relative to the control. In conclusion, our results suggest a decondensation of the chromatin in the Xi chromosome and therefore a probable involvement of the H3F3B gene in the maintenance of ICX.

Entre le XIème et le XVIème siècle, la Mer des Indes fut le théâtre de nombreux mouvements populationnels aux fins essentiellement commerciales ou coloniales. Madagascar se trouve à la croisée des mondes asiatiques et africains. La côte sud-est malgache a vu l'arrivée de plusieurs migrations : la dernière, probablement vers la fin du XVème siècle, serait celle des Antemoro dont une partie d'entre eux se réclame d'une origine arabe et se rattache à La Mecque. L'éthnie des Antemoro a fait l'objet de nombreuses études anthropologiques et linguistiques. Néanmoins, le débat sur l'origine des migrants fait toujours l'objet d'hypothèses contradictoires. Leurs origines génétiques pourraient ainsi être l'Arabie, l'Afrique de l'Est, l'Inde ou encore l'Asie du Sud-Est à une époque où ces régions étaient déjà islamisées. Ce travail a consisté à étudier la diversité génétique d'une population Antemoro afin d'apporter des éléments de réponse à la question de leur origine biologique. Ce projet interdisciplinaire a pour objectif de mettre en relation l'anthropologie culturelle et sociale avec l'anthropologie biologique. Le polymorphisme du chromosome Y a été étudié afin de rechercher les origines des lignées paternelles par l'analyse de 17 marqueurs microsatellites ainsi que des mutations ponctuelles de l'ADN de la partie non recombinante du chromosome Y. De même, la variabilité génétique des lignées maternelles a été analysée par séquençage des régions hypervariables I et II de l'ADN mitochondrial, et par la définition de polymorphismes bialléliques dans sa région codante. Nous avons mis en évidence la présence de deux haplogroupes du chromosome Y chez certains groupes Antemoro, qui les différencient de la diversité habituellement rencontrée dans les populations malgaches. Bien que la majeure partie des Antemoro entre dans la diversité observée en Afrique sub-Saharienne et en Asie du Sud-Est, quelques haplotypes, des lignées paternelles, les lieraient au Moyen-Orient. Les lignées maternelles, quant à elles, ne les différencient pas de celles des autres populations malgaches. L'isolat génétique formé par certaines « pseudo-castes » Antemoro confirme bien l'isolat culturel. Ce travail apporte une nouvelle vision de la diversité génétique humaine à Madagascar. / Between the 11th and 16th century, the Indian Ocean was the scene of many population movements notably for commercial and colonial purposes. Madagascar is located at the crossroads of the Asian and African continents. Several migrations have occurred in this region; the last one during the late 15th century involved the Antemoro population who claimed an Arabian origin in Mecca. Many anthropological and linguistic studies have been carried out on this ethnic group, but the origin of these migrants remains contentious. It is uncertain whether their origins were in Arabia, East Africa, India or Southeast Asia, when these regions were Islamized. In this study we assessed the genetic diversity of an Antemoro population from villages between Manakara and Vohipeno, to determine their biological origin. The aim of our interdisciplinary study was to link cultural and social anthropology with biological anthropology. Y-chromosome polymorphisms were studied by analyzing 17 microsatellites markers and some SNPs in the non-recombining region of the Y-chromosome to determine the biological origins of the paternal lineages. In addition, genetic variability of maternal lineages was analyzed by sequencing hypervariables regions I and II, and by defining bi-allelic polymorphisms in the coding region of mitochondrial DNA. We found two Y-chromosome haplogroups in some Antemoro groups that differentiated them from the typical genetic variability found in other Malagasy populations. Although most of the Antemoro showed a genetic diversity similar to that observed in sub-Saharan Africa and Southeast Asia, few haplotypes associated to paternal lineages linked them to the Middle East. Maternal lineages did not differ from those found in other Malagasy populations. The genetic isolate formed by some Antemoro groups confirmed their cultural isolation. This study provides a new view of the human genetic diversity in Madagascar.