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Thesis advisor: Charles Hoffman / Faithful chromosome segregation is necessary for the successful completion of mitosis and meiosis. The centromere is the site of kinetochore and microtubule attachment during chromosome segregation, and it is critical that the centromere is properly formed and maintained. Many proteins contribute to centromere formation, and this process has been extensively studied during the mitotic cell cycle. However, the roles of the centromere and its associated proteins during meiosis and their contribution to the fidelity of chromosome segregation process are not as well understood. Here, I aim to elucidate a mechanism that may contribute to aneuploidy in gametes, which is a major contributing factor in human infertility. In this study, I investigate the role of Abp1, the most prominent member of the transposase-derived protein family homologous to mammalian CENP-B in the assembly of centromeric chromatin during meiosis in the fission yeast Schizosaccharomyces pombe. I reveal that in contrast to its known role as a major regulator of LTR retrotransposons during the mitotic and meiotic cell cycles, Abp1 has a specialized role at the centromere during meiosis. My results indicate that Abp1 displays dynamic localization to the centromeres during meiosis compared to the vegetative cell cycle. I show that loss of abp1 impairs pericentromeric heterochromatin and the localization of Cnp1, a CENP-A ortholog, to the centromere central cores during meiosis. Moreover, Abp1 appears to suppress formation of meiotic neocentromeres by restricting deposition of Cnp1 at certain heterochromatin loci. Loss of abp1 has a drastic effect on chromosome segregation, resulting in dramatic frequency of aneuploidy. Furthermore, the genome surveillance role for retrotransposons by Abp1 appears to encompass centromeres as the mere insertion of an LTR sequence within the centromere central cores further exacerbates incidence of meiotic aneuploidy in abp1 null cells. This study provides intriguing insights into factors controlling the assembly of centromeric chromatin and its impact on the fidelity of chromosome segregation process during meiosis with important implications for advancing our understanding of the evolutionary forces driving the evolution of eukaryotic centromeres. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Hugh P. Cam / Meiotic homologous recombination (HR) is not uniform across eukaryotic genomes, creating regions of strong recombination activity dubbed recombination hotspots, and regions of low recombination activity dubbed coldspots. Considerable attention has led to discoveries of a host of factors controlling the formation of hotspots. However, the determinants of coldspots are not as clearly defined. I have previously shown that CENP-B homologs of the fission yeast Schizosaccharomyces pombe have a genome surveillance role in regulating the nuclear organization and expression of Tf2 retrotransposons. Here, I reveal an additional role for CENP-Bs in suppressing meiotic recombination of Tf2s. I describe the development of a random sporulation assay to rapidly screen thousands of meiotic progeny for recombination across a locus in a variety of genetic backgrounds. Loss of any CENP-B family members (Abp1, Cbh1, Cbh2), results in increased HR at Tf2s. I show that Abp1, which acts as the primary determinant of HR suppression at Tf2s, is required to maintain proper recombination exchange of homologous alleles flanking a Tf2. In addition, Abp1-mediated suppression of HR at Tf2s requires all three of its domains with distinct functions in transcriptional repression and higher-order genome organization. I show that this suppression is likely mediated by Abp1 binding to specific motifs near the 3’end of flanking LTRs. I demonstrate that HR suppression of Tf2s can be robustly maintained despite disruption to chromatin factors essential for transcriptional repression and nuclear organization of Tf2s. Intriguingly, I uncover a surprising cooperation between the histone methyltransferase Set1 responsible for histone H3 lysine 4 methylation and the non-homologous end joining pathway in ensuring the suppression of HR at Tf2s. Furthermore, I identify a role for the architectural protein condensin involved in 3D chromatin organization and chromosome condensation in restricting HR at Tf2s. My study identifies a molecular pathway involving functional cooperation between a transcription factor with epigenetic regulators, DNA repair pathway, and chromosome organizers to regulate meiotic recombination at interspersed repeats. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

In order to contain many millions, or even billions of base pairs within every nucleus of a eukaryotic cell, DNA must be extensively packaged. This is achieved by association of DNA with packaging proteins, resulting in the formation of chromatin, which can lead to various degrees of compaction. The most extreme form of compaction is the highly condensed mitotic chromosome, formation of which is necessary for proper resolution and segregation of the genetic material during cell division. However, the exact nature of the structure of chromatin within the mitotic chromosome and the factors which regulate it remain subjects of debate and continued investigation. The hybrid cell line F1.1 presents a unique tool for the study of mitotic chromosome structure. This mouse cell line has been observed to present a distinct chromatin structure in mitosis assembled over a large region of DNA inserted into one of its chromosomes and originating from the fission yeast Schizosaccharomyces pombe. Direct comparison of the structure of this distinct region of chromatin with that of the adjacent endogenous chromatin could provide insight into the nature of mitotic chromosome structure as well as the properties of the chromatin which are influencing this structure. Microscopy and Hi-C analyses showed that the mitotic chromatin organising or "scaffold" proteins are not altered over the region of S. pombe chromatin, but that the amount of chromatin organised around these proteins is diminished. In accordance with the "radial-loop" model of mitotic chromosome structure, we put forward a model whereby the S. pombe chromatin is organised into smaller chromatin loops around a constant organising scaffold. Examination of the histone post-translational modifications over the region of S. pombe chromatin revealed it to be highly heterochromatic, with high levels of H3K9me3 and associated factors such as HP1α and 5meC, and low levels of activating marks. Generation of further mammalian - S. pombe fusion cell lines recapitulated both the distinct mitotic structure and the heterochromatic profile of the inserted S. pombe chromatin. However, insertion of S. pombe DNA into a mouse cell by transfection rather than fusion resulted in a large region of S. pombe DNA that lacked both a distinct structure and heterochromatin. These results suggest that H3K9me3- mediated heterochromatin may influence the structure of chromatin in mitosis, leading to an organisation into smaller chromatin loops than non-heterochromatic regions.

In the fission yeast, Schizosacchromyces pombe, blocks to DNA replication elongation trigger the intra-S phase checkpoint that leads to the activation of the Cds1 kinase. Cds1 is required to both stabilize stalled replication forks and to prevent premature entry into mitosis. Interestingly, although Cds1 is essential to maintain the viability of mutants defective in DNA replication elongation, my study shows that mutants defective in DNA replication initiation require the Chk1 kinase, rather than Cds1. This suggests that failed initiation events can lead to activation of the DNA damage checkpoint independent of the intra-S phase checkpoint. This might result from reduced origin firing that leads to an increase in replication fork stalling or replication fork collapse that activates the G2 DNA damage checkpoint. I refer to the Chk1-dependent, Cds1-independent phenotype as the rid phenotype (for replication initiation defective). The data shows that Chk1 is active in rid mutants when grown under semi-permissive conditions, and rid mutant viability is dependent on the DNA damage checkpoint, and surprisingly Mrc1, an adaptor protein required for activation of Cds1. Mutations in Mrc1 that prevent activation of Cds1 have no effect on its ability to support rid mutant viability, suggesting that Mrc1 has a checkpoint-independent role in maintaining the viability of mutants defective in DNA replication initiation. Like Mrc1, Swi1 and Swi3 have been hypothesized as a part of the replication fork protection complex (RFPC). They are required for maintaining the viability of rid mutants, but are not essential for activation of Chk1 in response to failed initiation events. This suggests that Mrc1 in conjunction with Swi1 and Swi3 function in a similar pathway to alleviate replicative stress resulting from defects in DNA replication initiation. Using flow cytometry, I demonstrate that inhibition of DNA replication initiation has no significant impact on the duration of S phase, suggesting dormant origins might be activated in response to defects in DNA replication initiation. Fission yeast Rad22 is implicated in forming nuclear foci in response to damaged DNA. By tracking YFP-labeled Rad22, I screened for potential DNA damage in rid mutants grown at semi-permissive temperatures, and the results show that DNA damage occurs as the result of defects in DNA replication initiation. I also identified camptothecin, a DNA topoisomerase I inhibitor that can at low dose (2 µM) induce the rid phenotype, suggesting our assay (Chk1-dependent, Cds1-independent) can be used to screen small molecule inhibitors that interfere with the initiation step of DNA replication.

In most eukaryotes methylation of histone H3 on lysine 9 (H3K9me) is the key post-translational modification required for the assembly of constitutive heterochromatin at centromeres and other chromosomal regions. H3K9me is bound by the chromodomain proteins HP1/Swi6 and the Suv39/Clr4 H3K9 methyltransferase itself suggesting that, once established, H3K9me might act as an epigenetic mark that can transmit the chromatin state independently of the initiator signal. However, it has not been demonstrated that H3K9me does indeed act as an epigenetic mark. Fission yeast represents an excellent system to address this question since S. pombe lacks DNA methylation and H3K9me is catalysed by the unique, non-essential H3K9 methyltransferase Clr4. To determine whether H3K9me carries epigenetic properties it is important to uncouple H3K9me from genomic domains that have the intrinsic ability to recruit the heterochromatin machinery. One way to solve this problem is to isolate H3K9me from its original context and investigate whether at an ectopic site H3K9me can self-propagate through cell division. To accomplish this, we tethered regulatable TetR-Clr4 fusion protein at euchromatic loci in fission yeast. This resulted in the assembly of an extensive domain of H3K9me-dependent heterochromatin that is rapidly disassembled following TetR-Clr4 release. Strikingly, the inactivation of Epe1, a putative histone demethylase, is sufficient to maintain the silent H3K9me-dependent heterochromatin at the tethering sites through mitotic and meiotic cell divisions in absence of TetR-Clr4. These results indicate that H3K9me acts as an epigenetic mark to maintain heterochromatin domains; however, a regulatory mechanism dependent on Epe1 exists to actively remove H3K9me and thus prevent heterochromatin from being transmitted when assembled at inappropriate regions of the genome.

Mes travaux de recherche portent sur le contrôle du cycle cellulaire chez la levure de fission,  Schizosaccharomyces pombe. Chez S. pombe, cette régulation est assurée principalement par une protéine kinase cycline-dépendante (CDK), nommée Cdc2. Au cours du cycle cellulaire, cette enzyme s’associe avec différentes cyclines (Cig1, Cig2, Puc1 et Cdc13), formant ainsi une variété de complexes CDK-cycline qui confèrent des activités spécifiques à chaque phase du cycle. Cependant, il a été montré que ce réseau complexe peut être simplifié et remplacé par un système minimal, qui consiste en la fusion des deux gènes cdc2 et cdc13, indépendant d’un grand nombre de régulations endogènes. Cette découverte a ainsi permis d'établir un nouveau modèle pour le contrôle du cycle cellulaire eucaryote. Dans ce travail nous avons d’une part, voulu comprendre pourquoi la régulation du cycle cellulaire s’est complexifiée au cours de l’évolution, étant donné qu’une grande partie du circuit endogène semble dispensable. Dans ce but, j’ai investigué les limites du système minimal, quand les cellules sont exposées à différents stress. De manière surprenante, nous avons découvert que la simplification du réseau des CDKs confère aux cellules une résistance au stress réplicative. Nous avons montré que ce phénotype était indépendant de la régulation de l’inhibiteur Rum1 et des points de contrôles. Il résulte plutôt du fait que le cycle cellulaire soit régulé uniquement par Cdc13. Nous avons trouvé que le programme de réplication était inchangé dans les minimale qui présentaient moins de dommage à l’ADN comparé aux cellules sauvages. Nos data suggèrent que l’activité des CDKs associée au cyclines de phase G1/S, représente un moyen alternatif de moduler la réponse au stress. D’autre part, en utilisant le même système dans lequel l’activité des CDKs peut être finement modulée par l’inhibiteur. Nous avons démontré que la transcription périodique des gènes dépendait d’une régulation quantitative par les CDKs. Par conséquence nous proposons le model, l’opposé de ce qui a été suggéré chez la S. cerevisiea. Dans notre model, la progression du cycle cellulaire ainsi que la transcription périodique des gènes sont toutes les deux sous le contrôle de l’activité des CDKs. / The cyclin-dependent protein kinases (CDKs) are at the core of cell cycle control. In fission yeast, cell proliferation is regulated by CDK1/Cdc2 in association with the four cyclins Cdc13, Cig1, Cig2 and Puc1 at different stages of the cell cycle. However, this complex endogenous system can be replaced by a minimal module consisting of a fusion between Cdc2 and Cdc13 in the absence of G1/S cyclins. Surprisingly, this minimal CDK network drives the entire cell cycle in a wild type manner. Since a number of aspects of cell cycle control in fission yeast appear to be dispensable, we asked why similarly simplified circuits were not selected over the complex endogenous network during evolution. This led us to investigate the limits of such minimal systems, in particular when challenged by different stresses. Unexpectedly, we uncovered that simplification of the CDK network confers resistance to replication stress. We showed that this phenotype is independent from the CDK inhibitor Rum1 and the existing checkpoint pathways. It solely relies on operating the entire cell cycle with a single cyclin, Cdc13, and is associated with reduced genome instability when replication is challenged. However, it is not the consequence of changes in replication organisation along the chromosomes. Our data suggest that G1/S cyclin-associated Cdc2 activity may represent an alternative as yet unknown means of modulating cellular response to DNA stress. We also took advantage of a derivative of the minimal cell cycle network, in which Cdc2 is made sensitive to specific chemical inhibition. As a result, CDK activity can be externally modulated and cell cycle phases can be precisely controlled. Using this system, we re-visited the interplay between CDK and periodic transcription, a highly conserved process that is critical for proper cell proliferation. In contrast with previous studies in budding yeast, we demonstrate that periodic transcription in fission yeast is not independent from cell cycle progression. On the contrary, our work reveals that cell cycle transcriptional oscillations rely on quantitative changes in CDK activity levels. We therefore propose a new model, in which cell cycle progression and periodic transcription are intimately coupled through their common dependency on a unique input, namely CDK activity levels.

The establishment of cell polarity is a vital requirement for cellular processes such as proper cell division, growth and movement. Cell polarization relies on different internal and external cues in order to reorient the cell growth machinery along the axis of polarity. The core mechanisms involved in establishment of polarized growth are highly conserved from yeast to humans. Cells of the fission yeast Schizosaccharomyces pombe grow in a highly polarized fashion, with cell growth restricted to the cell tips, making fission yeast an excellent model system to study polarized growth. Here I describe a system for long-term live-cell imaging of fission yeast polarized growth that is stress free, physiological and accessible to media change and drug addition. I use this improved imaging system along with yeast genetics and drug perturbations to address how cell polarity is established and maintained in fission yeast. I have shown that fission yeast growth polarity depends on competition and cooperation among three distinct internal polarity cues: 1) A microtubule-based cue involving Tea1/Tea4 polarity proteins positively regulates polarized growth, initially at the “old” cell end (i.e., the end that pre-existed in the mother cell) and later at the “new” cell end (i.e. the end that is generated by septation), in order to initiate the transition from monopolar to bipolar growth (also known as New End Take-Off, or “NETO”). 2) An actin cable-based cue “clears” polarity proteins from the new end immediately after cytokinesis thereby reinforcing old-end growth. As a result perturbation of actin cable-based transport by either deleting actin cable nucleator For3 or cable-based transporter Myo52 results in premature bipolar growth. 3) A novel “memory-based” growth polarity cue helps to establish polarized growth in the absence of the microtubule-based cue. This memory-based cue is dependent on the predicted transmembrane proteins Rax1/Rax2. In the absence of both Tea1/Tea4 cue and Rax1/Rax2 cue, cells depend on septation cue and grow exclusively from the cell ends generated by septation. Furthermore, both Tea1/Tea4 and Rax1/Rax2 cue are important to maintain polarized growth under various environmental stresses. In fission yeast, during interphase, nucleus is positioned at the centre of the cell and this precise positioning of nucleus, which is important for defining the position of cytokinetic ring is thought to be exclusively MT-dependent. Here I show that MT-independent nuclear movement exists in fission yeast and this nuclear movement is mediated by actin cables and type myosin myo52. Furthermore, I show that actin cable might be important for buffering the pushing forces generated by MTs on the nucleus. In this way both microtubules and actin cables are involved in nuclear movement in fission yeast.

Compact heterochromatin is essential for genome stability and hence cell survival. Studies in many organisms including humans underline the importance of pericentromeric heterochromatin in centromere function. Fission yeast centromeres share a common structural organisation with those of their metazoan counterparts. The fission yeast model has been pivotal in understanding many key events in the pathway leading to the assembly of pericentromeric heterochromatin. In particular, studies in this system have revealed that the RNA interference (RNAi) pathway connects with the chromatin modification machinery to impart proper heterochromatin formation. Transcription of the pericentromeres by RNA polymerase II (Pol II) produces double stranded RNA (ds RNA) which is processed by Dicer(Dcr1) into small interfering RNAs (siRNAs). These siRNAs are loaded onto the Argonaute protein Ago1, and target the Ago1- containing RITS (RNA-Induced Transcriptional Silencing) complex to the pericentromeres via complementary base-pairing of the siRNA to the nascent centromeric transcript. RITS then recruits the sole Histone H3-K9-methyl transferase, Clr4, as part of the Clr4-complex, CLRC. The resulting H3K9-methyl marks further result in the recruitment of downstream chromatin binding proteins including the HP1- homolgue Swi6 which plays a key role in cohesin retention. Additionally, the H3K9- methyl marks are required for stabilising the association of CLRC and RITS, thereby promoting a reinforcing loop within the RNAi-mediated heterochromatin pathway. Thus crosstalk between RITS and CLRC is important in establishing and maintaining silent chromatin at the pericentromeres. Stc1 has been proposed to act as a critical link that connects the RITS and CLRC complexes. Stc1 is required for heterochromatin establishment and maintenance at the pericentromere and association of RITS with CLRC is lost in the absence of Stc1. Moreover, Stc1 directly interacts with Ago1 and is essential for siRNA production. These and other previous observations (Bayne et al. 2010) highlight the key role played by Stc1 in the RNAi-mediated heterochromatin pathway. To understand how Stc1 mediates the specific cross-talk between RNAi and chromatin modification, I have investigated the nature of Stc1 interactions with the RNAi and chromatin modification machineries. Using in-vitro binding assays, I found that Stc1 directly interacts with the CLRC subunits Dos2 and Clr4. I also identified the RITS subunit Tas3 as a potential interactor of Stc1, in addition to Ago1. A collaborating research group elucidated the structure of Stc1 using NMR (He et al. 2013) and my study provides evidence for interactions via the distinct domains of Stc1. Stc1 utilises its disordered C-terminus to bind to Dos2 while the N-terminus, which contains a tandem zinc finger domain, acts as a multi-protein interaction interface binding the CLRC subunit Clr4 and RITS subunits Ago1 and Tas3, opening up possibilities for Stc1-containing distinct-complexes. My work provides new insights into the role of Stc1 and opens up future avenues of research key to understanding how heterochromatin domains are defined and maintained.