The inherent asymmetry of female meiosis presents an opportunity for genetic material to gain an evolutionary advantage during the formation of the egg. Since centromeres mediate chromosomal segregation by forming the bridge between microtubules and chromosomes during cell division, they are loci that can drive, or selfishly evolve, during female meiosis by manipulating the process of entering the egg. Mimulus guttatus, a species of yellow monkeyflowers, has the best documented case of centromeric drive (Fishman and Saunders, 2008). Since homozygotes for drive have decreased pollen viability, lower seed counts, and poor reproductive success, CENH3 —the gene that encodes the H3 histone specific to centromeres— has evolved to suppress centromeric drive. CENH3 is duplicated in Mimulus, and the sequence variation of CENH3_A suggests that this paralog can suppress centromeric drive during female meiosis (Finseth et al. 2015). Our analysis of gene expression levels in meiotic and mitotic tissues indicates that both CENH3_A and CENH3_B are still expressed at similar levels, suggesting that the paralogs have not specialized for different roles in cell divisions. However, qPCR was only performed with nine tissue samples, so further analysis of gene expression with a larger sample set is needed to confirm whether or not the CENH3 paralogs have specialized roles in meiosis and mitosis.

Here I describe the construction and characterization of a new generation of human artificial chromosome that contains an array of DNA sequences that can be used to manipulate the chromosome in vivo and possibly in vitro. This HAC was originated in human fibrosarcoma HT1080 cells from a synthetic alphoid DNA containing an array of TetOperator sequences, cloned in a BAC-based vector. This synthetic ά-satellite DNA formed HACs that were stably maintained throughtout replication and segregation in HT1080 cells. However, I succeeded to also transfer and manipulate the alphoidtetO HAC into a HeLa-based hybrid cell line. The synthetic alphoidtetO HAC chromatin was similar to the chromatin at endogenous centromeric alphoid DNA. Importantly, the DNA sequences embedded in the synthetic HAC were accessible to targeting TetR-fused constructs in vivo. The alphoidtetO HAC could be successfully targeted with a number of TetR:fusion proteins without affecting its chromatin structure, kinetochore assembly and mitotic behaviour. However, the targeting of a transcriptional activator (tTA) inactivated the HAC synthetic alphoidtetO DNA in a fraction of transfected cells. Surprisingly, the targeting of the transcriptional repressor tTS, co-repressor KAP1 or the heterochromatin-associated protein HPIά severely inactivated the synthetic alphoidtetO kinetochore . In fact, upon targeting several inner and outer kinetochore proteins were delocalized from the alphoidtetO sequences. The dissociation of kinetochore proteins CENP-H and CENP-C appeared to precede that of CENP-A. The alphoidtetO HAC lacking inner kinetochore protein complexes showed mitotic defects including misalignment at the metaphase plate and defective anaphase segregation, ultimately being included in tiny DAPI-positive nano-nuclei in the cytoplasm. The transcriptional repressor tTS repressed the low levels of transcription from the alphoidtetO sequences. In addition, targeting of transcriptional repressors altered the HAC chromatin towards a more “closed”, heterochromatic conformation, as seen from the changes in histone tail modifications. Interestingly, the targeting of the histone methyltransferase EZH2 to the alphoidteto HAC showed a much milder inactivating activity compared to KAP1. Based on these results, I propose that the formation of HPI-type of heterochromatin or accumulation of HPIά to the centromeric regions could disrupt the association of constitutive kinetochore proteins to the underlying sequences. Centromeric alphoid sequences lacking a functional kinetochore structure then also loose the centromere-specific histone H3 variant CENP-A becoming definitively inactive. Alternatively, a basal transcriptional activity from centromeric sequences might be required for centromere functionality.

DNA methylation is essential for mammalian development and transcriptional repression of genes and retrotransposons during embryo development and in somatic cells. The patterns of DNA methylation are established by de novo DNA methyltransferases, which are regulated by developmental signalling and require access to chromatin. Besides DNA methyltransferases, other proteins have recently been implicated in DNA methylation, such as the ATP-dependent chromatin remodeler LSH. The absence of LSH in mouse embryos leads to defects in DNA methylation and development. In relation to this, mutations in LSH have been found to cause Immunodeficiency-Centromeric instability-Facial anomalies (ICF) syndrome. This syndrome is characterized by centromeric instability and CpG hypomethylation of centromeric satellite repeats, and is most often caused by mutations in the catalytic domain of the DNA methyltransferase DNMT3B. LSH is essential for developmentally programmed de novo DNA methylation of large chromosomal domains including promoters of protein coding genes and repetitive sequences. Importantly, fibroblasts derived from chromatin remodeling ATPase LSH-null mouse embryos, which lack DNA methylation at transposons and specific gene promoters, are capable of re-establishing normal patterns of DNA methylation and transcriptional silencing of misregulated genes upon re-expression of LSH. The ATP hydrolysis by LSH is essential for its function in gene silencing and de novo DNA methylation. However, the molecular mechanisms of LSH-dependent gene silencing and de novo DNA methylation are yet unclear. Here we use an inducible system that enables controlled expression of LSH in Lsh-null mouse embryonic fibroblasts (MEFs) to follow chromatin dynamics, transcriptional silencing and establishment of de novo DNA methylation. This conditionally reversible Lsh knockout cellular system allowed us to study the order of events occurring immediately after LSH restoration in MEF cell lines in order to elucidate the molecular mechanism of LSH-dependent gene silencing. We have demonstrated that LSH upon its restoration localises to the promoters of LSH-dependent loci leading to a mild decrease in the occupancy of H3, which reinforces the previously shown role of LSH as a chromatin remodeler. Simultaneously, there is removal of acetyl groups from H3 tails when LSH is bound to these target regions, which might be facilitated by the interaction of HDACs with LSH. The removal of H3Ac marks is followed by deposition of H3K9me2 by G9a/GLP histone methylases at the same time point when misregulated genes are silenced. This suggests that LSH creates a suitable substrate for G9a/GLP promoting gene silencing. Surprisingly, transcriptional repression occurs without acquisition of DNA methylation at the promoters of these loci. This order of events implies that LSH plays a role as a chromatin remodeler leading to changes in chromatin structure and modifications that facilitate epigenetic gene silencing without DNA methylation in the initial period when LSH is restored in MEF cell lines. Furthermore, deposition of H3K9me2 by the G9a/GLP complex is critical for silencing of specific genes, but not for repetitive elements such as IAPs. The histone modification H3K27me3 seems to play a transitory role in the silencing of IAP retrotransposons in the absence of G9a/GLP activity. In conclusion, this work has demonstrated that changes in chromatin modifications leading to a transcriptionally repressive chromatin state can be established in somatic cells by the chromatin remodeler LSH without acquisition of DNA methylation. This suggests that the primary role of LSH is to promote changes in chromatin structure and modifications that lead to gene silencing and not DNA methylation, which most likely occurs as a consequence of transcriptional silencing.

Le virus Herpes Simplex de type 1 (HSV-1) possède un mode d’infection particulier dit bimodal. Il peut soit se répliquer de manière active lors d’une phase dite lytique soit migrer dans les neurones et rester en latence. Il peut réactiver pour rétablir une infection lytique. Une protéine virale majeure dans la réactivation de HSV-1 est ICP0. C’est une protéine nucléaire à activité E3 ubiquitine ligase, qui possède la particularité d’induire la dégradation par le protéasome de plusieurs protéines centromériques constitutives, ce qui provoque une déstabilisation du centromère interphasique. Mon équipe a découvert une réponse cellulaire à l’instabilité centromérique, induite par la protéine ICP0, et nommée iCDR (pour interphase Centromere Damage Response.). L’objectif général de ma thèse est de déterminer les modifications structurales que subissent les centromères endommagés par ICP0 à l’origine de l’iCDR et probablement de la réactivation. J’ai pu démontrer qu’ICP0 affectait toute la structure protéique étroitement associée aux centromères durant l’interphase. Suite à ces résultats, j’ai pu démontrer, par des analyses de digestion de chromatine à la nucléase microccocale (MNAse), que l’occupation nucléosomique de la chromatine centromérique suite à l’activité d’ICP0 était affectée de façon significative. Une étude in vivo effectuée à partir de tissus nerveux provenant de souris infectées de manière latente, a permis de démontrer une co-localisation entre les génomes HSV-1 latents et les centromères. Cette co-localisation est associée à une répression transcriptionnelle du virus. Les résultats de ma thèse montrent donc que les effets d’ICP0 sur la déstabilisation des centromères sont en relation avec un rôle de ces centromères durant la latence. Ceci suggère fortement une implication de la déstabilisation des centromères dans le processus de réactivation contrôlé par ICP0. / The Herpes Simplex type 1 (HSV-1) virus possesses a bimodal mode of infection. It can either replicates in an active way during the lytic cycle, or it can infect neurons and stay in latency. HSV-1 reactivates from latently infected neurons for re-establishing a lytic infection. A major viral protein implicated in reactivation is ICP0. It is a nuclear E3 ubiquitin-ligase, which has the particularity to induce the proteasome-mediated degradation of several constitutive centromeric proteins. This activity severely destabilizes the interphase centromere. My team has discovered a novel cellular response triggered by the estabilization of centromeres by ICP0, called iCDR (interphase Centromere Damage Response). The general aim of my thesis is to determine the centromere structural modifications induced by ICP0 that can trigger the iCDR and probably the reactivation. I was able to demonstrate that ICP0 affected the entire proteinacious structure of interphase centromeres. Following this, I showed by micrococcal nuclease (MNase) digestion approach that the nucleosomal organization of centromeric chromatin was significantly affected by ICP0. An in vivo study in nervous tissues coming from latently infected mice enabled to show a co-localization between latent HSV-1 genomes and centromeres. This co-localisation is linked to a transcriptional repression of the virus. The results of my thesis show that the destabilization of centromere by ICP0 correlates with a role of the centromeres during latency. This strongly suggests an implication of centromere destabilization in the ICP0-controlled reactivation process.

Chez Arabidopsis, l’enveloppe nucléaire constitue un site de nucléation des microtubules à partir des complexes à gamma-tubuline. Conservées des plantes à l'Homme, les protéines GIPs/MZT1 ont été initialement découvertes comme partenaires d’AtGCP3. J’ai consacré ma thèse à la caractérisation moléculaire et fonctionnelle des AtGIPs et de leurs partenaires à l’interface nucléocytoplasmique. Mes résultats confirment l’appartenance des GIPs aux complexes à gamma-tubuline, et démontrent leur association entre elles et avec TSA1 (TonSoKu [TSK]-Associating protein 1) et l'histone centromérique CenH3. Les interactions génétiques entre les gènes GIPs, TSA1 et TSK révèlent des anomalies sévères à l'échelle de l'organisme, des cellules et des noyaux. Les mutants gip1gip2 démontrent une diminution de la cohésion des régions centromériques. L’ensemble de nos résultats suggère un rôle des AtGIPs dans un continuum nucléocytoplasmique inédit, la régulation de l'architecture nucléaire et du centromère. / In Arabidopsis, the nuclear envelope is a nucleation center where gamma-tubulin complexes initiate the polymerization of microtubules. Conserved from plants to humans, GIPs/MZT1 proteins were initially discovered as AtGCP3 interacting partners. Our investigations were devoted to the molecular and functional characterization of AtGIPs and their associated proteins at the nucleocytoplasmic interface. We confirmed that AtGIPs are integral components of gamma-tubulin complexes, and showed that they interact with each other, TSA1 (TonSoKu [TSK]-Associating protein 1) and centromeric histone H3 (CenH3). Genetic interactions between GIPs, TSA1 and TSK reveal severe defects at the organism, cellular and nuclear scales. gip1gip2 mutants exhibit a decrease of centromeric and pericentromeric cohesion. Altogether, this is the first evidence for the role of a gamma–tubulin complex component in the structural maintenance of centromeric regions, and in defining nuclear morphology and architecture.

No description available.