The cell's nucleus contains DNA in the form of chromosomes, which are the hereditary content of the organism. The proper transmission of DNA from one generation to the next is critical. Along with this crucial process, cells will also need to transcribe the DNA, silence certain genes (or whole chromosomes) during development and regulate other chromosome dynamics that are still being identified. The molecular components responsible for these processes are starting to be identified. However, the regulation of these components and how they interact with each other is not well understood.The condensin complex is one component that has been identified to play a role in chromosome dynamics. Activity of the complex has been studied in vitro but in vivo activity has been difficult to measure. Similarly, understanding the regulation of the complex has been difficult given the lack of assays and that the complex is essential for cell survival. In this dissertation, I have identified and characterized a regulator of condensin II function using Drosophila melanogaster. The chromo-domain protein Mrg15 interacts with condensin II to inhibit homologous chromosome interactions.Lastly, I look at the role of condensin II in female meiosis. Meiosis involves pairing and subsequent segregation of homologous chromosomes. The process of the initial pairing has remained elusive but specialized structures have evolved to maintain this pairing. Condensin II can antagonize a basal level of homologous pairing and also removes the specialized structure that pair meiotic chromosomes. This dissertation will add to the growing knowledge of the regulation of the condensin II complex and its role in female meiosis.

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.

Within the span of a single human lifetime, we have discovered that DNA is the basis of genetic inheritance, deciphered the genetic code, and determined the entire sequence of multiple human genomes. However, we still have only a basic understanding of many of the processes that regulate DNA structure, function, and dynamics. The work presented in this dissertation describes the roles of two sets of genes that regulate the expression of genetic information and its transmission from one generation to the next.The condensin II complex has been implicated in the maintenance of genomic integrity during cell division and in transcriptional regulation during interphase. These roles stem from its ability to regulate chromosome structure though the mechanisms of this regulation are unclear. Evidence suggests that it is important for chromosome condensation and segregation during mitosis and meiosis. We have shown that this complex regulates the condensation of chromosomes during interphase. Its ability to reduce chromosome axial length provides a mechanism for the establishment of chromosome territories. We have also shown that condensin II differentially regulates interactions between homologous and heterologous DNA sequences. These findings contribute to our understanding of the overall structure of the nucleus, the regulation of chromosome structure, and the regulation of gene expression.The function of the Drosophila gene, sticky, is poorly understood. It contributes to cytokinesis by phosphorylating myosin II, but it also has a role in the regulation of chromatin structure. Mutations in sticky are associated with a wide range of developmental abnormalities. We provide evidence that this gene regulates the expression of numerous other genes which contribute to the phenotypes observed when sticky is mutated. We also show that sticky function overlaps with that of dfmr1, an ortholog of the gene associated with the most common form of human mental retardation. These findings contribute to our understanding of transcriptional regulation in chromatin and its implications in development and disease.

At the beginning of mitosis, chromosomes are condensed and segregated to facilitate correct alignment later in cytokinesis. Condensin is the pentameric enzyme responsible for this DNA compaction and is composed of two structural maintenance of chromosomes (SMC) subunits and three non-SMC subunits. Condensin mutations generate chromosomal abnormalities due to improper segregation, leading to genome instability and eventual malignant transformation of the cell. Cdc2 phosphorylation of the non-SMC subunits, CAP-G, CAP-D2, and CAP-H, has been demonstrated to be important for condensin supercoiling activity and function. While these subunits are thought to be phosphorylated by Cdc2, the exact sites have not yet been identified and characterized. The basis of this research was to determine the Cdc2 phosphorylation sites in the CAP-G subunit of the condensin enzyme and to characterize the functional significance of the sites in the regulation of condensin activity using site-directed mutagenesis and immunofluoresence microscopy. While DNA condensation represents a critical step early in mitosis, formation of the mitotic spindle represents a vital event leading to the division of a cell into two daughter cells in a process known as cytokinesis. Protein regulating cytokinesis 1 (PRC1) is a mitotic protein essential for cytokinesis that participates in formation of the mitotic spindle in a phosphorylation dependent manner. PRC1 possesses microtubule bundling properties. Loss of PRC1 leads to mis-segregation of chromosomes and abnormal cytokinesis. HSF2 is a transcription factor known to be important in development and differentiation. Previous research has determined that HSF2 plays a significant mechanistic role in the process of hsp70i gene bookmarking during mitosis. Bookmarking is an epigenetic phenomenon whereby certain gene promoters remain uncompacted, in contrast to the majoritiy of genomic DNA during mitosis. This lack of compaction allows quick reassembly to a transcriptionally competent in G1 of the cell cycle and ensures the ability of the cell to induce expression of the cytoprotective hsp70i protein. HSF2 and PRC1 were found to interact in a yeast-two hybrid screen. Given the importance of both of these proteins during mitosis, this study seeks to characterize the HSF2/PRC1 interaction and determine the potential role for PRC1 in hsp70i gene bookmarking.

Maintenance of an intact genome and proper regulation of the genes within are crucial aspects for life. The work of this dissertation has implicated the Drosophila condensin II complex in both processes. Condensin II's ability to reconfigure chromosomes into spatially separated and discrete units is necessary to ensure proper meiotic segregation. When this "individualization" activity fails in a condensin II mutant, chromosomes remain entangled, and either cosegregate or become lost during cell division. This leads to the creation of aneuploid sperm. We have also implicated condensin II as a factor necessary to individualize interphase somatic chromosomes from one another. This is relevant in Drosophila because the association of homologous chromosomes is thought to facilitate gene regulation activity in trans. We speculate that condensin II individualization spatially distances aligned chromosomes from one another and prevents this trans-communication between allelic loci. This is supported first by an increase of homologous chromosome pairing in a condensin II mutant background. Secondly, loss of condensin II leads to elevated production from alleles that are known to depend on pairing for transcriptional activation. These meiotic and interphase condensin II roles support its necessity to Drosophila genome integrity and transcriptional regulation. Given the conservation of condensin from bacteria to humans, it is likely that equivalent or related roles exist in a variety of species.

In order to equally segregate their genetic material into daughter cells during mitosis, it is essential that chromosomes undergo major restructuring to facilitate compaction. However, the process of transforming diffuse, entangled interphase chromatin into discrete, highly organised chromosomal structures is extremely complex, and currently not completely understood. The complexes involved in chromatin compaction and sister chromatid decatenation in preparation for mitosis include condensins I and II. Mutations in condensin subunits have been identified in human tumours, reflecting the importance of accurate cell division in the prevention of aneuploidy and tumour formation. Most mutations described in TCGA (The Cancer Genome Atlas) and COSMIC (Catalogue of Somatic Mutations in Cancer) are missense, and therefore likely to only partially affect condensin function. Most functional genetic studies of condensin, however, have used loss of function systems, which typically cause severe chromosome segregation defects and cell death. Mice carrying global hypomorphic mutations within the kleisin subunit of the condensin II complex develop T cell lymphomas. The Caph2nes/nes mouse model is therefore a good system for understanding how condensin dysfunction can influence tumourigenesis. However, little is known about which cellular processes are affected in mutant cells before transformation. I therefore set out to use the Caph2nes/nes mouse model to study the consequences of the condensin II deficiency on cell cycle regulation in several different hematopoietic lineages. The Caph2nes/nes mice are viable and fertile, with no obvious abnormalities other than the thymus, which is drastically reduced in size. Previous studies reported greater than a hundred-fold reduction in the number of CD4+ CD8+ thymocytes. I set out to understand why the alteration of a ubiquitously expressed protein which functions in a fundamental cellular process would result in such a cell-type specific block in development. To achieve this, I investigated the possibility that condensin II is involved in interphase processes as well as in mitosis. In addition, I studied the aspects of T cell development that may make this lineage particularly vulnerable to condensin II deficiency. Finally, I carried out a preliminary investigation into the biochemical properties of the condensin complexes. During my PhD., I found strong evidence to suggest that the Caph2nes/nes T cell-specific phenotype arises due to abnormal cell division. However, I was unable to find any evidence to support the hypothesis that the phenotype is a consequence of abnormal interphase processes. Upon systematic analysis of several stages of hematopoietic differentiation, I found that at a specific stage of T cell development, the mutation results in an increased proportion of cells with abnormal ploidy, followed by a drastic reduction in cell numbers. Erythroid cells revealed a similar increase in the frequency of hyperdiploid cells, but no reduction in cell numbers. B cells and hematopoietic precursors did not reveal an increase in hyperdiploidy, or a reduction in cell numbers in wildtype relative to mutant. Subsequently, I found preliminary evidence to suggest that the T cell-specificity may be due to more rapid progression of CD4+ CD8+ T cells from S phase to M phase, relative to other hematopoietic stages. Finally, a preliminary investigation into the biochemical properties of the condensin complex revealed apparent imbalances in the expression of condensin subunits in T, B and erythroid cells. The sedimentation profile of CAP-H2 from whole-thymus extract did not exclude the possibility that condensin subunits might be forming heavier-weight complexes with non-SMC proteins. Further work must be carried out to determine whether this sedimentation pattern is unique to T cells.

Body mass varies considerably between different mammals and this variation is largely accounted for by a difference in total cell number rather than individual cell size. Insights into mechanisms regulating growth can therefore be gained by understanding what governs total cell number at any one point. In addition, control of cell proliferation and programmed cell death is fundamental to other areas of research such as cancer and stem cell research. Microcephalic Primordial Dwarfism (MPD) is a group of rare Mendelian human disorders in which there is an extreme global failure of growth with affected individuals often only reaching a height of around one metre in adulthood. To date, all identified disease genes follow an autosomal recessive mode of inheritance and encode key regulators of the cell cycle, where mutations impact on overall cell number and result in a substantially reduced body size. MPD therefore provides a valuable model for examining genetic and cellular mechanisms that impact on growth. The overall aims of this thesis were to identify novel disease causing genes, as well as provide further characterisation of known disease causing genes, through the analysis of whole exome sequencing (WES) within a large cohort of MPD patients. Following the design and implementation of an analytical bioinformatics pipeline, deleterious mutations were identified in multiple disease genes including LIG4 and XRCC4. Both genes encode components of the non-homologous end joining machinery, a DNA repair mechanism not previously implicated in MPD. Additionally, the pathogenicity of novel mutations in subunits of a protein complex involved in chromosome segregation was assessed using patient-derived cells. These findings demonstrate WES can be successfully implemented to identify known and novel disease causing genes within a large heterogeneous cohort of patients, expanding the phenotype of known disorders and improving diagnosis as well as providing novel insights into intrinsic cellular mechanisms critical to growth.

ADN subit une série de transformations structurelles complexes au cours de la division cellulaire, ce qui entraîne dans son compactage chromosomes mitotiques par un processus appelé la condensation des chromosomes. Le complexe de condensine pentamérique est fortement impliqué comme un effecteur majeur de ce phénomène. Il s'agit d'un complexe protéine de sous-unités multiples avec deux sous-unités catalytiques [SMC- Structural Maintenance of Chromosomes] et de trois sous-unités de régulation, hautement conservés de la levure à l'homme. Le complexe de condensine dans Saccharomyces cerevisiae est constitué de deux sous-unités de SMC [Smc2 et Smc4] et trois protéines non réglementaires [Brn1, Ycs4, Ycg1]. Malgré son importance, le mécanisme d'action de condensine reste largement inconnu. Par conséquent, l'objectif de cette recherche est de comprendre le mécanisme d'action de condensine et comment elle est affectée par l'interaction entre ses sous-unités réglementaires et non-réglementaires. Cette thèse identifie quatre morphologies dépendants du cycle cellulaire distincts du locus d'ADNr. Cette transformation du phénotype ADNr de G1 à la mitose dépend condensine. Afin de déterminer le rôle de l'interaction entre les sous-unités catalytiques et réglementaires de condensine dans la régulation du complexe condensine, nous avons identifié six résidus positifs sur l'extrémité C-terminale de BRN1 qui affectent la formation du complexe condensine, l'activité de la condensation et l'interaction avec tubuline, ce qui suggère que ces résidus ont un rôle dans la régulation de condensine. Ensemble, nos résultats suggèrent un modèle de règlement du condensine par l'interaction entre les sous-unités de condensine. / DNA undergoes a series of complex structural transformations during cell division, resulting in its compaction into intact mitotic chromosomes called chromosome condensation. The pentameric condensin complex has been strongly implicated as a major effector of this phenomenon. It is a multi-subunit protein complex with two catalytic “Structural maintenance of chromosome” [SMC] subunits and three regulatory subunits, highly conserved from yeast to humans. The condensin complex in Saccharomyces cerevisiae is made up of two SMC subunits [Smc2 and Smc4] and three regulatory non-SMC proteins [Brn1, Ycs4, Ycg1]. Despite its importance, the mechanism of action of condensin remains largely unknown. Hence, the objective of this research is to understand the mechanism of action of condensin and how it is affected by interaction between its regulatory and non-regulatory sub-units. This thesis identifies four distinct cell cycle dependent morphologies of the rDNA locus. The transformation of the rDNA phenotype from G1 to mitosis is condensin dependent. In order to determine the role of the interaction between the catalytic and regulatory subunits of condensin in the regulation of the condensin complex, we have identified six positive residues on the C-terminus of Brn1 which affect complex formation, condensation activity and interaction with tubulin, suggesting that these residues have a role in condensin regulation. Together, our results suggest a model for condensin regulation by interaction between condensin subunits.

3D proteomics combines chemical cross-linking with mass spectrometry to study the structure of protein assemblies and protein-protein interactions both in vitro and in vivo by providing distance constraints that indicate which residues are in close spatial proximity. I addressed the main bottleneck of this technology: the reliable identification of cross-linked peptides. Reporter ion signatures for cross-linked peptides were developed, by fragmenting model compounds containing two lysine residues joined by a cross-linker backbone or a lysine residue modified with a hydrolysed cross-linker. The reporter ion signatures showed 97% specificity at 90% sensitivity and segregated cross-linked from modified and linear peptides. They decreased the false discovery rate of the identification of cross-linked peptides from 5% to 1% in a large dataset. The signatures permit data sorting during and after mass spectrometry acquisition. The advanced 3D proteomics workflow was applied to study the protein-protein interactions in Mycoplasma pneumoniae cells. In lysates of the bacterium we identified 128 protein-protein interactions (of which 24 are novel) and obtained in vivo topological data on 208 proteins, even for cases where high-resolution structures are not yet available. We showed that our data are in excellent agreement with crystal structures of proteins and complexes where available. We defined a network of ribosomal and RNA polymerase proteins that reveals an intricate link between transcription and translation in bacteria. We demonstrated that the method is suitable for identification of homomultimeric protein complexes by exploiting peptide pairs of identical amino acid sequence. The technology has the potential to provide a complete protein interaction network map after the selective enrichment of cross-lined peptides is achieved. The method was next applied to investigate the structure of condensin and cohesin complexes, which play a crucial role in stabilization of chromosome structure during mitosis. The complexes were purified, cross-linked and their linkage map created. The condensin coiled coil cross-linked on the entire length was modeled. The information was used to direct the analysis of in situ cross-linked condensin in intact chromosomes. I found two high confidence linkages between SMC2 and SMC4 coiled coils and identified H2A as a potential condensin receptor on chromosomes.

Le complexe condensine est le moteur de la condensation mitotique des chromosomes, un processus essentiel à la stabilité du génome au cours de la division cellulaire. De nombreuses données publiées indiquent qu’il existe des liens fonctionnels étroits entre le processus de transcription des gènes et le processus d’organisation des chromosomes par condensine. Ces données sont toutefois souvent contradictoires et aucun modèle ne fait actuellement consensus pour expliquer les liens entre transcription et condensine. Au cours de cette thèse, nous avons montré chez la levure Schizosaccharomyces pombe qu’en l’absence de l’hélicase à ADN/ARN Sen1, condensine s’accumule spécifiquement à proximité des gènes transcrits par l’ARN Polymérase III. Nous avons utilisé ces observations pour mieux comprendre les liens entre transcription par l’ARN polymérase III et le positionnement de condensine. Nos données montrent que Sen1 est un cofacteur de l’ARN Polymérase III impliqué dans la terminaison de la transcription. Ce résultat est important car il démontre que les modèles existants qui affirment que l’ARN polymérase III termine de transcrire de façon autonome sont erronés. Nous avons ensuite démontré que les défauts de terminaison de l’ARN polymérase III observés en l’absence de Sen1 suffisent entièrement à expliquer l’accumulation de condensine en ces sites. Cette observation importante démontre que le contrôle de qualité de la transcription est directement impliqué dans le positionnement de condensine sur les chromosomes en mitose. Nos résultats nous permettent de proposer qu’au-delà d’un certain seuil, la densité en ARN polymérases est un obstacle à la translocation de condensine sur les chromosomes. / The condensin complex is a key driver of chromosome condensation in mitosis. The condensin-dependent assembly of highly compacted chromosomes is essential for the faithful transmission of the genome during cell division. Many independent studies have established that gene transcription impacts the association of condensin with chromosomes, but the molecular mechanisms involved are still unclear. This is especially true as a number of sometimes contradictory mechanisms have been proposed so far. Here, we show in Schizosaccharomyces pombe that condensin accumulates specifically in the vicinity of a subset of RNA polymerase III-transcribed genes in the absence of the conserved DNA/RNA helicase Sen1. We demonstrate that Sen1 is a cofactor of RNA polymerase III (RNAPIII) required for efficient transcription termination. These results are important because they fundamentally challenge the pre-existing view that RNAPIII terminates transcription autonomously. Strikingly, we show that the RNAPIII transcription termination defects are directly responsible for the accumulation of condensin in the absence of Sen1. This indicates that the quality control of transcription impacts the distribution of condensin on mitotic chromosomes. We propose that above a certain density threshold, the accumulation of RNAPIII constitutes a barrier for the translocation of condensin on chromosomes.