Equal partitioning of genetic materials of the chromosomes is key to the mitotic cell cycle, as unequal segregation of chromosomes during mitosis leads to aneuploidy, a hall mark of human cancer. Accurate chromosome segregation is directed by the kinetochore, a proteinaceous structure on each sister chromosome that physically connects the chromosome to the spindle microtubules. Kinetochore assembles at the centromere, a specialized chromosome region epigenetically defined by the histone H3 variant centromere protein A (CENP-A) in higher eukaryotes including mammals. In order to maintain centromere identity against CENP-A dilution caused by S phase genome replication, new CENP-A molecules are loaded at preexisting centromeres in G1 phase of the cell cycle. Despite of the several important stages and molecular components identified in CENP-A replenishment, little is known about how new CENP-A proteins become stably incorporated into centromeric nucleosomes. Here by using quantitative imaging, pulse-chase labeling, mutant analysis, cellular fractionation and computational simulations, I have identified the cytoskeleton protein diaphanous formin mDia2 to be essential for the essential for the stable incorporation of newly synthesized CENP-A at the centromere. The novel function of mDia2 depends on its nuclear localization and its actin nucleation activity. Furthermore, mDia2 functions downstream of a small GTPase molecular switch during CENP-A loading, and is responsible for the formation of dynamic and short actin filaments observed in early G1 nuclei. Importantly, the maintenance of centromeric CENP-A levels requires a pool of polymerizable actin inside the nucleus. Single particle tracking and quantitative analysis revealed that centromere movement in early G1 nuclei is relatively confined over the time scale of initial CENP-A loading, and the subdiffusive behavior was significantly altered upon mDia2 knockdown. Finally, knocking down mDia2 results in prolonged centromere association of Holliday junction recognition protein (HJURP), a chaperone required to undergo timely turnover to allow for new CENP-A loading at the centromere. Our findings suggest that diaphanous formin mDia2 forms a link between the upstream small GTPase signaling and the downstream confined viscoelastic nuclear environment, and therefore regulates the stable assembly of new CENP-A containing nucleosomes to mark centromeres’ epigenetic identity (Chapter 2 and 3). While centromere identity is essential for kinetochore assembly, once kinetochores are assembled, fine-tuned interactions between kinetochores and microtubules become important for a fully functioning mitotic spindle during chromosome segregation. It has been previously found that another diaphanous formin protein mDia3 and its interaction with EB1, a microtubule plus-end tracking protein, are essential for accurate chromosome segregation1. In Chapter 4 of this thesis, I found that knocking down mDia3 caused a compositional change at the microtubule plus-end attached to the kinetochores, marked by a loss of EB1 and a gain of CLIP-170 and the dynein light chain protein Tctex-1. Interestingly, this compositional change does not affect the release of cytoplasmic dynein from aligned kinetochores, suggesting a population of Tctex-1 can be recruited to the kinetochores without dynein. During mitosis, Tctex-1 associates with unattached kinetochores and is required for accurate chromosome segregation. Tctex-1 knockdown in cells does not affect the localization and function of dynein at the kinetochore, but produces a prolonged mitotic arrest with a few misaligned chromosomes, which are subsequently missegregated during anaphase. This function is independent of Tctex-1’s association with dynein. The kinetochore localization of Tctex-1 is independent of the ZW10-dynein pathway, but requires the Ndc80 complex. Thus, our findings reveal a dynein independent role of Tctex-1 at the kinetochore to enhance the stability of kinetochore-microtubule attachment. Together, these work suggest novel regulatory roles of the cytoskeletal systems in the maintenance as well as subsequent functions of the centromere/kinetochore, and provide mechanistic insights into the complex control principles of accurate chromosome segregation. Our findings provide a new model in understanding the epigenetic maintenance of genome integrity, and will have implications with regard to how aberrant cell divisions underlying aneuploidy can be targeted in the treatment of cancer.

Cheung, Wai Kuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 66-76). / Abstracts in English and Chinese. / List of Figures --- p.v / List of Tables --- p.vi / List of Abbreviations --- p.vii / Acknowledgements --- p.ix / Abstract --- p.xi / 摘要 --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Centromere and Kinetochore --- p.1 / Chapter 1.2 --- The Kinetochore Subunits: Centromeric Nucleosomes --- p.2 / Chapter 1.3 --- CenH3: The Centromere Specific Histone --- p.5 / Chapter 1.4 --- The Centromeric DNA: Tandem Repeats and Retrotransposons --- p.8 / Chapter 1.5 --- The Genetic and Epigenetic Nature of the Centromeres --- p.9 / Chapter 1.6 --- Point Centromeres and Regional Centromeres --- p.10 / Chapter 1.7 --- Holocentric Chromosomes --- p.11 / Chapter 1.8 --- Hypothesis --- p.13 / Chapter Chapter 2 --- Materials and methods --- p.15 / Chapter 2.1 --- Chemicals --- p.15 / Chapter 2.2 --- Bacterial strains in routine cloning --- p.15 / Chapter 2.3 --- Plant materials in cloning and transformation --- p.15 / Chapter 2.4 --- Construction of LnCENH3-GFP and CeHCP3-DsRED chimeric gene cassettes for rice transformation --- p.15 / Chapter 2.5 --- Cloning of CENH3 gene of Luzula spp --- p.22 / Chapter 2.6 --- Construction of full length OsCENH3 RNAi and 150bp OsCENH3 RNAi constructs for rice transformation --- p.22 / Chapter 2.7 --- Agrobacterium-mediated transformation of rice (Oryza sativa L.japonica. cv. Nipponbare) --- p.24 / Chapter 2.8 --- Gene gun transformation of rice (Oryza sativa L.japonica. cv. Nipponbare) by Biolistic PDS-1000/He´ёØ System (Bio-rad) --- p.26 / Chapter 2.9 --- Detection of transgenes expression --- p.28 / Chapter 2.10 --- Nuclear protein extraction --- p.29 / Chapter 2.11 --- Protein-DNA Binding Assay --- p.30 / Chapter 2.12 --- Protein precipitation by methanol-chloroform --- p.32 / Chapter 2.13 --- Western blot analysis of proteins from Protein-DNA binding assay --- p.33 / Chapter 2.14 --- Tubulin immunolocalization of root tips --- p.33 / Chapter 2.15 --- Bioinformatics analysis --- p.34 / Chapter Chapter 3 --- Results --- p.36 / Chapter 3.1 --- Plant transformation vectors construction --- p.36 / Chapter 3.2 --- Rice transformation --- p.38 / Chapter 3.3 --- Transgenic plants screening --- p.39 / Chapter 3.4 --- Analysis of the codon usages of CeHCP-3 gene in C. elegans and O. sativa --- p.42 / Chapter 3.5 --- In vitro Protein-DNA binding assays --- p.44 / Chapter 3.6 --- Subcellular localization study of LnCENH3 in rice --- p.46 / Chapter 3.7 --- Chromosome morphology of the transgenic rice expression LnCENH3 --- p.47 / Chapter 3.8 --- Tubulin immunolocalization of LnCENH3-GFP transgenic rice --- p.49 / Chapter 3.9 --- Cloning of CENH3s in Luzula genus --- p.51 / Chapter 3.10 --- Bioinformatics analysis of Luzula CENH3s --- p.53 / Chapter Chapter 4 --- Discussion --- p.57 / Chapter 4.1 --- Expression of fusion proteins in rice --- p.57 / Chapter 4.2 --- Incorporation of LnCENH3-GFP in nucleosomes --- p.57 / Chapter 4.3 --- Expression pattern of LnCENH3-GFP in rice --- p.58 / Chapter 4.4 --- Formation of additional kinetochores on transgenic rice chromosome --- p.60 / Chapter 4.5 --- Deletion of 8 amino acids in LeCENH3 --- p.62 / Chapter Chapter 5 --- Conclusion --- p.65 / References --- p.66 / Chapter 5.1 --- Appendix --- p.77

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.

<p>Centromeres, or sites of chromosomal spindle attachment during mitosis and meiosis, are non-randomly distributed in complex genomes and are largely associated with expansive, near-identical satellite DNA arrays. While the sequence basis of centromere identity remains a subject of considerable debate, one approach is to examine the genomic organization of satellite DNA arrays and their potential function. Current genome assembly and sequence annotation strategies, however, are dependent on robust sequence variation, and, as a result, these regions of near sequence identity remain absent from current genome reference sequences and thus are detached from explorations of centromere biology. This dissertation is designed as a foundational study for centromere genomics, providing the initial steps to characterize those sequences at endogenous centromeres, while further classifying `functional' sequences that directly interact with, or are capable of recruiting proteins involved in, centromere function. These studies build on and take advantage of the limited sequence variation in centromeric satellite DNA, providing the necessary genomic scope to promote biologically meaningful characterization of endogenous centromere sequences in both human and non-human genomes. As a result, this thesis demonstrates possible genomic standards for future studies in the emerging field of satellite biology, which is now positioned to address functional centromere sequence variation across evolutionary time.</p> / Dissertation

The centromere is an important region of the chromosome which ensures correct segregation at cell division. The DNA sequences which make up human centromeres are poorly understood. An analysis of the human Y chromosome centromere DNA has therefore been undertaken. The structures of 23 yeast artificial chromosomes (YAC) clones and 4 cosmid clones have been determined and these have contributed to a map of ~7Mb of DNA which span the centromere. The centromeric region of the human Y chromosome contains a single major block of tandemly repeating alphoid DNA which is variable in size. The 5.7kb alphoid subunits are all orientated in one direction and become diverged at the edges of the array. Flanking the alphoid DNA are small blocks of other known tandemly repeated sequences, the 5bp, 48bp and 68bp satellites. These satellites are arranged in an asymmetric manner and are interspersed with a range of low to moderate copy number repeats. Only one putative single copy sequence has been detected. Nearby lie two regions of X-Y homology: a more proximal region which contains a gene (amelogenin) and a more distal region which has previously been shown to result from a recent X-Y transposition event. These results show that the centromeric region of the human Y chromosome is a complex mosaic of tandem repeats and other repeats. Furthermore, they provide a detailed map of the region and thus provide a solid basis for functional studies of candidate centromere determining sequences.

Thesis (Ph. D.) University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on August 3, 2007) Includes bibliographical references.

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.

The centromere is a chromosomal locus required for accurate segregation of sister chromatids during cell division. They are maintained epigenetically in most eukaryotes, by incorporating the H3 variant CENP-A, and can, in rare instances, change location on the chromosome throughout generations. Centromeres are transcribed, and an active transcription chromatin signature is required for centromere maintenance. For this reason, insight into the nature of this so-called “centrochromatin” is essential for understanding a centromere’s place in the chromosome. The body of work contained in this thesis shows my efforts to understand the centromere in the context of chromatin, revealing interactions and new evidence for repressive chromatin domains with centromere activity, in two different vertebrate models: chicken DT40 cells and human HeLa cells. Centromeres are generally embedded within large domains of heterochromatic repetitive sequences in most eukaryotes, and mapping “centrochromatin” to high-resolution has proven difficult. However, chromosomes 5, 27 and Z of Gallus gallus are not located within repeat arrays, and are fully sequenced. CENP-A distribution on these centromeres has been mapped by ChIP-seq, and I have performed ChIP against selected histone modifications as part of a collaboration. While levels of heterochromatin are naturally quite low in these centromeres, I have shown that repressive polycomb chromatin instead is enriched in these non-repetitive centromeres, suggesting a replacement of one silenced chromatin state with another. Additional mapping of these centromeres showed a pattern of active chromatin marks distinct from that reported for human cells, which exhibited dynamic distribution throughout the cell cycle. Furthermore, conditionally generated neocentromeres in DT40 cells revealed that centrochromatin formation lowers, but does not eliminate, active transcription. To directly study the interaction of polycomb and heterochromatin with centrochromatin, I used a synthetic Human Artificial Chromosome (HAC), which allows for specific conditional targeting of chromatin modification enzymes, allowing manipulation of the underlying chromatin. Enrichment of the polycomb chromatin state on the HAC centromere, by EZH2 tethering, reduced its active transcriptional chromatin signature, but did not impair its actual transcription or mitotic activity. However, direct tethering of polycomb secondary silencing effector PRC1 caused centromere loss, and this effect was mimicked with homologous heterochromatin factors, indicating that centromeres can subsist within repressive chromatin domains, but are lost when direct repression is applied. To understand the contribution of the local repressive heterochromatin to centromere stability, I erased heterochromatin marks from the HAC centromere by tethering JMJD2D (an H3K9me3 demethylase): long-term (but not short-term) heterochromatin loss impaired CENP-A assembly, perturbed mitotic behaviour, and resulted in significant HAC mis-segregation. These results strongly suggest that local heterochromatin is essential to maintain normal CENP-A dynamics and centromere function. Together with previous observations, these data suggest that a repressive chromatin environment contributes to centromere stability, and that centromeres likely have natural mechanisms to maintain their transcriptional activity within such domains.

Human centromeres contain large arrays of α-satellite DNA that are thought to provide centromere function. These arrays show size and sequence variations. However, the lower limit of the sizes of these DNA arrays in normal centromeres is unknown. Using a set of chromosome-specific α-satellite probes for each of the human chromosomes, interphase Fluorescence In Situ Hybridisation (FISH) was performed in a population screening study. This study demonstrated that extreme reduction of chromosome-specific α-satellite is unusually common in chromosome 21 (screened with the αRI probe), with a prevalence of 3.70%, compared to <=.12 % for each of chromosomes 13 and 17, and 0 % for the other chromosomes. No analphoid centromere was identified in over 17,000 morphologically normal chromosomes studied. All the low-alphoid centromeres are fully functional as indicated by their mitotic stability and binding to centromere proteins including CENtromere Protein-A (CENP-A), CENtromere Protein-B (CENP-B), CENtromere Protein-C (CENP-C), and CENtromere Protein-E (CENP-E). Sensitive metaphase FISH analysis of the low-alphoid chromosome 21 centromeres established the presence of residual αRI as well as other non-αRI α-satellite DNA suggesting that centromere function may be provided by (i) the residual αRI DNA, (ii) other non-αRI a-satellite sequences, (iii) a combination of i and ii, or (iv) an activated neocentromere DNA. (For complete abstract open document)

Centromere identity is integral for proper kinetochore formation and chromosome segregation. In most species chromosomes have a centromere at a defined locus that is propagated across generations. The histone H3 variant CENP-A acts as an epigenetic mark for centromere identity in most species studied. CENP-A is absent from the inactivated centromere on dicentric chromosomes and present at neocentromeres that form on non-centromeric sequences. Thus, the canonical centromere sequence is neither necessary nor sufficient for centromere function. Nevertheless, centromeres are generally associated with particular sequences. Understanding the organisation of centromeric sequence features will provide insight into centromere function and identity. In this study I use the fission yeast Schizosaccharomyces pombe model system to address the relationship between CENP-ACnp1 and centromeric sequence features. These analyses reveal that CENP-ACnp1 nucleosomes are highly positioned within the central domain by large asymmetric AT-rich gaps. The same sequence features underlying CENP-ACnp1 positioning are conserved in the related species S. octosporus, but are not found at neocentromeres, suggesting that they are important but non-essential for centromere function. CENP-ACnp1 over-expression leads to ectopic CENP-ACnp1 incorporation primarily at sites associated with heterochromatin, including the sites where stable neocentromeres form. Ectopic CENP-ACnp1 also occupies additional sites within the central domain that are not occupied in cells with wild-type CENP-ACnp1 levels. In wild-type cells CENP-ACnp1 occupied sites are likely also occupied by H3 nucleosomes or the CENP-T/W/S/X nucleosome-like complex in a mixed population. Several candidate proteins were investigated to determine a protein residing in the large gaps between CENP-ACnp1 nucleosomes could be identified. No proteins could be localised to the AT-rich gaps between CENP-ACnp1 nucleosomes, but the origin recognition complex in a promising candidate. The results presented in this thesis demonstrate that nucleosomes within the fission yeast centromere central domain are highly positioned by sequence features in a conserved manner. This positioning also allows for another complex, possibly the origin recognition complex, to bind to DNA. Nucleosome positioning, DNA replication, and transcription could individually and collectively influence CENP-ACnp1 assembly and centromere function. Further experiments in fission yeast will continue to provide insight into the general properties of centromere function and identity.