The centromere is a specialized chromatin domain that serves as the assembly site for the mitotic kinetochore structure, thereby playing a fundamental role in facilitating the maintenance of the genetic information. A histone H3 variant termed CENP-A is specifically found at all active centromeres. Beyond this, however, little is known about how and to which extent the chromatin environment of centromeres modulates and contributes towards centromere identity and function. Here, I have employed a novel Human Artificial Chromosome (HAC), the centromere of which can be targeted by fusions to the tet repressor, to characterize the chromatin environment underlying active kinetochores, as well as to specifically probe the role of this environment in the maintenance of kinetochore structure and function. My data demonstrate that centromeric chromatin resembles the downstream regions of actively transcribed genes. This includes the previously unrecognized presence of histone H3 nucleosomes methylated at lysine 36 within the chromatin underlying functional kinetochores. Targeted manipulation of this chromatin through tethering of a heterochromatin-seeding transcriptional repressor results in the inactivation of HAC kinetochore function concomitant with a hierarchical disassembly of the structure. Through an even more selective engineering of the HAC centromere chromatin, I have provided evidence supporting a critical role for nucleosomes dimethylated at lysine 4 on histone H3 in facilitating local transcription of the underlying DNA. Tethering of different chromatin-modifying activities into the HAC kinetochore collectively reveals a critical role for both, histone H3 dimethylated on lysine 4 and low-level, non-coding transcription in the maintenance of the CENP-A chromatin domain. On one hand, repression of centromeric transcription negatively correlates with the maintenance of CENP-A and ultimately results in the loss of kinetochore function. On the other hand, increasing kinetochore-associated RNA polymerase activity to within physiological levels for euchromatin is associated with rapid loss of CENP-A from the HAC centromere. Together, my data point towards the requirement for a delicate balance of transcriptional activity that is required to shape and maintain the chromatin environment of active centromeres.

Thesis advisor: Marc-Jan Gubbels / The obligate intracellular parasite Toxoplasma gondii exhibits closed mitosis, as chromosome segregation occurs with the confines of the nuclear envelope. Distinct structural changes are absent during mitosis, as the nucleolus is maintained and condensation of chromosomes is largely restricted. Moreover, the centromeres are clustered and remain persistently associated with the centrocone (spindle pole). To elucidate the process of chromosome segregation during mitosis in the parasite, the role of kinetochores and microtubules was examined. Localization studies of the functionally conserved kinetochore proteins TgNuf2 and TgNdc80 revealed that clustered kinetochores colocalize with clustered centromeres at the centrocone throughout the cell cycle. Pharmacological disruption of microtubules resulted in partial loss of clustering, which indicates spindle microtubules are necessary, but not strictly required for this process. Furthermore, the generation of a conditional TgNuf2 knockdown revealed this kinetochore protein is essential for chromosome segregation but dispensable for clustering of centromeres, which remain associated with the centrocone. Moreover, in the absence of TgNuf2 the centrosome behaves normally, but looses its association with the centrocone. Further analysis of this phenotype revealed that the centrocone is devoid of spindle microtubules following depletion of this essential kinetochore protein. Examination of tubulin localization dynamics through parasite development showed that the initiation of spindle microtubules occurs at the basal region of the nucleus prior to centrosome duplication. Furthermore, acetylation of α-tubulin, a posttranslational modification associated with microtubule stability, was confirmed to be specifically associated with stabilization of the spindle microtubules following comigration of the centrocone and centrosome to the apical end of the nucleus. Collectively, these data demonstrate that the persistent association of clustered centromeres with the centrocone is independent of spindle microtubules. These discoveries are contributing unprecedented details to chromosome anchoring and segregation during the cell cycle in this protozoan parasite. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Chromosome alignment and orientation within the spindle in mitosis and meiosis are determined by chromosome-microtubule interaction. Evidence suggests that within the acentrosomal spindle the mechanism of chromosome positioning is different from in mitotic spindle but its molecular bases are not well understood. I investigated how chromosome-microtubule interactions position the chromosomes within the spindle using Drosophila oocytes. I addressed the role and molecular mechanisms of kinetochore and chromosome interaction with microtubules in this process. I developed new live imaging reagents to observe dynamic chromosome-microtubule interaction. Live imaging combined with inactivation of kinetochores in oocytes revealed that kinetochore-microtubule attachment is required for three-step chromosome positioning in Drosophila oocytes: de-congression, change of orientation and re-congression. Augmin, a γ-tubulin recruiting complex, has been previously shown to be important for chromosome congression specifically in oocytes. Live imaging further showed that Augmin facilitates chromosome congression particularly in early stages of spindle assembly. Study of Augmin dynamics revealed that Augmin stably associates with spindle polar regions, specifically in oocytes. This meiotic regulation of Augmin function may contribute to generation of force pushing chromosomes toward spindle equator. Sentin protein has been shown to be important for microtubule plus end dynamics in mitosis. In meiosis, sentin mutant results in reduced distance between centromeres of homologous chromosomes. However, its meiotic role is unknown. Live imaging of the sentin mutant showed that in oocytes Sentin is required for preventing premature stabilization of kinetochore-microtubule attachments. As conclusion, I have used live imaging to reveal molecular basis of the interaction between chromosomes and microtubules particularly important for oocytes.

Proper attachment of chromosomes to microtubules is important for the accurate segregation of chromosomes and genome stability. The initial engagement of chromosomes happens along the lateral wall of microtubules through a highly specialised protein structure assembled on the centromeric DNA, the kinetochore. Ultimately, kinetochores must be attached to the ends of microtubules (a geometry called end- on attachment). A series of highly dynamic steps called the end-on conversion process, converts the initial immature lateral attachments into mature end-on attachments. How this process is finely tuned by phosphorylation and dephosphorylation to achieve stable attachments is still unclear. Furthermore, what is the role of microtubule-associated proteins in the stabilisation of kinetochore-microtubule attachments is unknown. This project aimed to study the role of phosphatases in the regulation of the end-on conversion process. First, I investigated the different contribution of the two outer-kinetochore phosphatases - BubR1- recruited PP2A-B56 and KNL1-recruited PP1 - in counteracting Aurora B kinase during the end-on conversion process. I found that BubR1-recruited PP2A-B56 plays an essential role in the process, but KNL1-recruited PP1 does not. I also investigated whether the HEC1/Ndc80 N-tail is a critical substrate of Aurora B phosphorylation for the stabilisation of the end-on attachments. Using a phospho-dead mutant of the HEC1/Ndc80 N-tail, I discovered that cells are still susceptible to Aurora B activity, indicating downstream pathways independent of HEC1/Ndc80. Then, I studied the biological role of the Astrin C-terminus, where an evolutionarily conserved RVMF motif, a putative PP1 binding site, is located. My findings show C-terminal Astrin mutants fail to localise at kinetochores of both monopolar and bipolar spindles; induce defects in the end-on conversion process in monopolar spindles and prolong mitosis time with increased Mad2 levels at the outer-kinetochore. A kinase inhibitor assay showed that kinetochore-microtubule attachment defects in Astrin mutant expressing cells could be rescued when both Aurora B and Cdk1 kinases are inhibited, suggesting a role for Astrin’s C-terminus in counteracting Aurora B and Cdk1 activity. Finally, I probed the putative interaction of the Astrin C-terminus and PP1 using biochemistry, cell biology and fluorescence microscopy techniques. I discovered that artificially targeting PP1 onto the Astrin C-terminus but not on the N-terminus rescues mutants localisation defects at the kinetochore. In summary, my results indicate that Astrin and PP1 interact at the kinetochore of living cells. In conclusion, my work shows that mitotic phosphatases have distinctive contributions in the regulation of the dynamic steps of the end-on conversion process and that Astrin is a potential PP1 phosphatase recruiter at the outer-kinetochore, where is necessary for the stabilisation of end-on attachments.

When cells divide, a series of events must proceed in a timely and co-ordinated manner to ensure that all DNA is replicated and partitioned equally between the two daughter cells. A central component of this process is the kinetochore, a large proteinaceous complex (>100 proteins) found within the centromere of all chromosomes. During the dynamic process of cell division, this machinery must be able to capture microtubules, promote chromosome movements towards the spindle midzone and ensure that segregration only occurs once this alignment has been successfully completed. This requires intricate mechanical and regulatory co-ordination between components and it is therefore no surprise that the structures responsible are structurally and functionally varied. It has, however, become clear that many kinetochore proteins assemble into distinct sub-complexes and despite the fact that their specific contributions are well studied, the way the many unique sub-assemblies come together to form a fully operational kinetochore is still poorly understood. Here, chromosome isolation techniques from chicken DT40 cells combined with mass spectrometry employing Stable Isotope Labeling by Amino acids in Cell culture (SILAC), is used to compare the proteome of mitotic chromosomes from different conditional kinetochore knockout (KO) cell lines. This includes components of the inner kinetochore; CENP-C, CENP-T and CENP-W, and a sub-unit of the Ndc80 complex that is important for microtubule attachment. With these large data sets I have focused on the impact these depletions have on the architecture of the holo-kinetochore by measuring the SILAC ratios of individual proteins. From these measurements I can define whether specific components are decreased, increased or unchanged in terms of their abundance on chromosomes in response to the various deletions. I have found that proteins within the same complex typically behave in a similar manner across the different KO conditions. By integrating all of the data sets, dependency networks are revealed, as well as highlighting potential novel kinetochore proteins worthy of further study.

Mitosis is the shortest phase of the cell cycle but visually the most outstanding. The key goal of mitosis is to accurately drive chromosome segregation. On one hand, DNA has to be condensed into characteristically shaped chromosomes. On the other hand, a very specialized structure needs to be built to conduct segregation, the mitotic spindle which is composed of microtubules organized into an antiparallel array between the two poles. The interaction between microtubules and chromosomes occurs at the kinetochore, a macromolecular complex assembled in mitosis at the centromere. The centromere/kinetochore monitors proper spindle microtubule attachment to each of the chromosomes, aligning them at the metaphase plate and also ensuring that chromosome segregation happens in perfect synchrony. Although centromeres are present in all eukaryotes, their basic structure and chromatin folding are still poorly understood. One of the aims of my work was to understand the function of the condensin complex specifically at the centromere during mitosis. Condensin I and II are pentameric protein complexes that are among the most abundant components of mitotic chromosomes. I have shown that condensin is important to confer stiffness to the innercentromeric chromatin once spindle microtubules interact with kinetochores in metaphase. Labile inner-centromeric regions delay mitotic progression by altering microtubule-kinetochore attachments and/or dynamics with a consequent increase in levels of Mad2 checkpoint protein bound to kinetochores. In the absence of condensin, kinetochores perform prominent “excursions” toward the poles trailing behind a thin thread of chromatin. These excursions are reversible suggesting that the centromeric chromatin behaves like an elastic polymer. During these excursions I noticed that only the inner centromeric chromatin was subjected to reversible deformations while the kinetochores (inner and outer plates) remained mostly unaltered. This suggested that the centromeric chromatin part of the inner kinetochore plate was organised differently from the subjacent chromatin. I went on to investigate how the centromeric chromatin is organised within the inner kinetochore domain. Super-resolution analyses of artificially unfolded centromeric chromatin revealed novel details of the vertebrate inner kinetochore domain. All together, the data allowed me to propose a new model for the centromeric chromatin folding: CENP-A domains are interspersed with H3 domains arranged in a linear segment that forms planar sinusoidal waves distributed in several layers. Both CENP-A and H3 arrays face the external surface, building a platform for CCAN proteins. CENP-C binds to more internal CENP-A blocks thereby crosslinking the layers. This organization of the chromatin explains the localisation and similar compliant behaviour that CENP-A and CENP-C showed when kinetochores come under tension. Other kinetochore proteins (the KMN complex) assemble in mitosis on top of the CCAN and bind microtubules. KMN binding may confer an extra degree of stability to the kinetochore by crosslinking CENP-C either directly or indirectly. My work and the testable model that I have developed for kinetochore organization provide a fundamental advance in our understanding of this specialized chromosomal substructure.

The kinetochore is a highly complex proteinaceous structure located at the primary constriction of mitotic chromosomes. Here, it performs an essential role in accurate chromosome segregation. Recently, much interest has been directed towards the Constitutive Centromere Associated Network (CCAN) components, as they participate in the formation of a scaffold involved in kinetochore assembly. It is therefore important to fully understand their role, and their distribution, at the kinetochore. Although many kinetochore proteins have already been identified, it is still unclear how centromeric chromatin folds to form the structure of the inner kinetochore. This is an interesting yet still open field of study, where the literature reports are still quite divided. In this study we take advantage of the high homologous recombination efficiency in DT40 B-lymphoma chicken cell lines, allowing the generation of conditional knockouts and deletion cell lines of several centromere proteins, subsequently engineered to stably express GFP:CENP-A. In the parental cell line the unfolding properties of the centromeric region were investigated by using TEEN buffer. Using fluorescence microscopy we were able to measure the length of many unfolded centromeric chromatin fibres, from both interphase and mitotic samples, based on the signal of GFP:CENP-A. A multi-peak analysis revealed the presence of discrete populations of fibres, recognised as peaks, in both interphase and mitotic samples. Compared with interphase, mitotic centromeres showed a greater level of compaction. Next, mutants for CCAN components, blocked in mitosis, were subjected to centromere chromatin unfolding. Results revealed that mitotic kinetochores depleted of CENP-C and CENP-S behaved similarly to the parental interphase samples, suggesting a role of those proteins in maintaining kinetochore structure. In contrast, CENP-O, CENP-H and CENP-I depletion did not seem to weaken the structure of the kinetochore. Additionally, we tested a hypothesis revealed by the multi-peak analyses, that chromatin layers exist in the inner kinetochore. Our data, when combined with published electron microscopy and crystallography measurements of centromere/kinetochore components, allowed us to assemble a robust and mathematically viable model that supports a boustrophedon organisation of the kinetochore chromatin. Finally, characterization studies of the novel kinetochore protein CENP-Z were performed. An involvement of CENP-Z in controlling the levels of di-methylation on lysine 4 of histone H3 was shown. This work represents an advance in our understanding of kinetochore structure in vertebrates.

No description available.

La microcéphalie est une maladie neurologique du nouveau-né qui se traduit par une circonférence réduite de la tête, une déficience intellectuelle et des défauts anatomiques du cerveau. La microcéphalie peut être la conséquence d’une infection, de stress environnementaux ou de mutations génétiques.Le cerveau commence à se former dès la cinquième semaine de grossesse et est majoritairement constitué de cellules souches neuronales, cellules qui conservent une capacité a se reproduire a l’identique sans se spécialiser. Cette première phase de prolifération est importante pour générer suffisamment de cellules. Suit une phase de différenciation, durant laquelle les cellules préalablement formées se différencient en deux groupes : les neurones, qui permettent de partager l’information grâce à des influx électriques, et les cellules gliales, qui soutiennent activement les fonctions des cellules neuronales.Je m’intéresse à un gène en particulier, KNL1, muté chez certains patients microcéphales. Grace aux nouvelles techniques d’édition du génome, j’ai reproduit la mutation retrouvée chez les patients dans des cellules souches pluripotentes humaines. En utilisant un modèle tridimensionnel (mini-cerveaux en culture), à partir de cellules souches neuronales, j’ai analysé de manière quantitative les étapes-clés de développement: les phases de prolifération et de différenciation.Mes travaux de recherche ont montré que les cellules souches neuronales portant la même mutation que les patients prolifèrent moins, réduisant le nombre de cellules initiales nécessaires au développement cérébral normal. Par ailleurs, les cellules souches neuronales se différencient prématurément en neurones et cellules gliales, ce qui réduit davantage le nombre le nombre final de cellules. Cette hypothèse a été confirmée par l’utilisation du modèle tridimensionnel, ou les mini-cerveaux sont plus petits que la normale.Cette étude est essentielle non seulement pour comprendre le développement de la maladie, mais également pour comprendre les étapes clés du développement du cerveau humain, et ne pourrait pas être mener à bien sur des modèles animaux. En outre, l’utilisation de cellules souches induites nous permet de ne pas utiliser de cellules embryonnaires, si nécessaire pour raisons d’éthique. / Microcephaly is a neurological condition, resulting in patients having a small head circumference, intellectual impairment and brain anatomical defects. A pre-requisite for achieving a better understanding of the cellular events that contribute to the striking expansion of the human cerebral cortex is to elucidate cell-division mechanisms, which likely go awry in microcephaly. Most of the mutated genes identified in microcephaly patient encode centrosomal protein, KNL1 is the only gene that encodes a kinetochore protein, it plays a central role in kinetochore assembly and function during mitosis. While the involvement of centrosome functions is well established in the etiology of microcephaly, little is known about the contribution of KNL1.In an attempt to assess the role of KNL1 in brain development and its involvement in microcephaly, we generated isogenic human embryonic stem cell (hESC) lines bearing KNL1 patient mutations using CRISPR/Cas9-mediated gene targeting. We demonstrated that the point mutation leads to KNL1 reduction in neural progenitors. Moreover, mutant neural progenitors present aneuploidy, an increase in cell death and an abrogated spindle assembly checkpoint. Mutant fibroblasts, derived from hESC, do not have a reduced expression of KNL1 and do not present any defect in cell growth or karyotype, which highlight a brain-specific phenotype.The subsequent differentiation of mutant neural progenitors into two-dimensional neural culture leads to the depletion of neural progenitors in the favor of premature differentiation. We developed a three-dimensional neural spheroids model from neural progenitors and reported a reduced size of mutant neural spheroids, compare to control. Lastly, using knockdown and rescue assays, we proved that protein level of KNL1 is responsible of the premature differentiation and the reduced size.These data suggest that KNL1 has a brain-specific function during the development. Changes in its expression might contribute to the brain phenotypic divergence that appeared during human evolution.

We have used Chinese hamster ovary (CHO)-human hybrid cells containing chromosomes 16, 18, X, and 21 to test the ability of human kinetochores to successfully bind to spindle microtubules and to be distributed to the daughter cells. We have established the intrinsic rate of non-disjunction among these human chromosomes noted above and compared these rates with those in cells presented with mitotic challenges such as taxol, nocodazole, and mitosis with unreplicated genomes (MUG). Cells were grown on culture slides, fixed and processed for immunofluorescence and fluorescence in situ hybridization (FISH). Daughter pairs were identified by staining with anti-á-tubulin to identify midbodies. Human centromere DNA probes were used for FISH in order to test for the successful passage of human kinetochores to daughter cells during anaphase. Our data indicate that different human kinetochores vary in their ability to properly engage the spindle and to be successfully distributed. In addition, mitotic challenges have been shown to affect the rate of non-disjunction. The mechanism of this effect is not yet known.