La méiose est une étape de la différenciation germinale qui permet la formation des gamètes. Elle est composée de deux divisions successives. La ségrégation des chromosomes homologues à la première division nécessite des connexions entre homologues, mises en place par des événements de crossing-over (CO). Les CO augmentent également la diversité génétique, et leur fréquence et leur distribution sont étroitement régulées. Ils sont générés par un mécanisme de formation et réparation de cassures double brins de l'ADN (CDBs), catalysées par la protéine SPO11 et préférentiellement localisées dans des régions de 1-2 kb appelées points chauds de recombinaison méiotique. Une question majeure est de comprendre comment sont régulés ces CO, ce qui détermine leur fréquence et leur distribution, car toute altération de cette régulation peut conduire à des anomalies chromosomiques graves.Dans ce travail de thèse, pour la première fois chez les mammifères, nous avons montré que des modifications de la chromatine sont associées à l'initiation de la recombinaison méiotique (formation des CDBs par SPO11). Ces résultats ont été obtenus par des analyses d'immunoprécipitation de chromatine (ChIP) sur des spermatocytes purifiés ou non, isolés de différentes lignées de souris. Une des modifications associées à l'activité de deux points chauds testés est la triméthylation de la lysine 4 de l'histone H3 (H3K4Me3). Une analyse fonctionnelle et temporelle de cette modification a permis de montrer qu'elle ne dépend pas de SPO11 et apparaît avant la formation de CDBs. Nous avons montré ici que c'est la protéine PRDM9, récemment identifiée comme un déterminant majeur des points chauds de recombinaison chez les mammifères et possédant une activité méthyltransférase, qui appose H3K4Me3. Nous proposons un modèle où H3K4Me3 et d'autres caractéristiques inconnues constitueraient un substrat pour la machinerie d'initiation et recruteraient SPO11 en des points précis du génome, qui deviendront des points chauds. / Meiosis is a specialized cell division to produce haploid gametes from a diploid cell. It segregates parental genomes by two successive divisions. The faithful segregation of homologous chromosomes is achieved during the first unique division via formation of crossovers (COs). COs establish physical connections between homologs by the reciprocal exchange of genetic material and require the formation and subsequent repair of SPO11-dependent DNA double-strand breaks (DSBs). Studies in many organisms revealed that COs are distributed in highly localized regions (1-2Kb) of genomes called recombination hotspots. The mechanisms of COs regulation are elusive and a main question in the field is to understand how the frequency and distribution of CO are regulated, because either absence or defects of recombination can lead to aneuploidy or reduced fertility. In the present study, for the very first time in mammals, we investigate whether recombination hotspots are associated with any chromatin modifications. We performed chromatin immunoprecipitation (ChIP) on spermatocytes isolated from different mice strains harbouring either active or inactive hotspots. Comparison of hot and cold spots revealed that a specific histone modification i.e. trimethylation of the lysine 4 of histone H3 (H3K4Me3) is enriched at two tested hotspots in mice. Temporal and functional analysis show that H3K4Me3 is not dependent on SPO11 and appears before DSBs formation. Furthermore, we demonstrate here that H3K4Me3 is methylated via the histone methyltransferase activity of PRDM9, recently identified as a major determinant of recombination hotspots in mammals. We propose a model that H3K4Me3 and other unknown chromatin features may specify recruitment of SPO11 initiation machinery to initiate meiotic recombination at the hotspots.

Sporulation in Saccharomyces cerevisiae includes the processes of meiosis and spore formation. The genes involved in this developmental process are tightly regulated at the level of transcription to ensure that genes are expressed at the correct time and level. The co-ordinated expression of middle sporulation genes is mediated by a key timing promoter element called the middle sporulation element (MSE). While this element sets the timing of gene expression to middle sporulation, in some cases, the level of expression is mediated by cis-acting auxiliary promoter elements. This study has addressed the role that auxiliary transcription factors play in fine-tuning of timing and level of expression of the MSE-regulated middle sporulation genes SPS18 and SPS19 and the mid-late sporulation genes DIT1 and DIT2. The MSE*SPS18/19 was shown previously to set the timing of expression of SPS18 and SPS19 to middle sporulation. In order to achieve the full level of meiotic activation, a novel bipartite auxiliary promoter element called the MAE (MSE-associated element) was required (Dalton, 2004). This study has revealed that proteins bind to specific regions of the MAE motif during sporulation in vitro and has attempted to isolate the proteins by affinity chromatography and identify them by mass spectrometry. The timing of expression of DIT1 and DIT2 during sporulation was of particular interest since two MSE-like elements had been identified in the promoter of the these genes (Hepworth et al., 1995). If these MSEs were functional, it was thought that auxiliary elements may delay expression of these genes until mid-late sporulation. This study has shown that the MSE*NRE confers a normal middle sporulation pattern of expression on a reporter gene. The DRE (DIT repressor element) previously identified by Bogengruber et al. (1998) was further characterised as an element that alters the level of expression conferred by an MSE without altering the timing. Several proteins were shown to bind to specific regions of the DIT promoter surrounding the DRE motif in vitro, with a different set of proteins binding during vegetative growth and sporulation. Attempts to isolate and identify these proteins by affinity chromatography and mass spectrometry are discussed.

The Saccharomyces cerevisiae iron-responsive transcription factor, Aft1p, has a well established role in regulating iron homeostasis through the transcriptional induction of iron-regulon genes. However, recent studies have implicated Aft1p in other cellular processes independent of iron-regulation such as chromosome stability. In addition, chromosome spreads and two-hybrid data suggest that Aft1p interacts with and co-localizes with kinetochore proteins, however the cellular implications of this have not been established. Here, we demonstrate that Aft1p associates with the kinetochore complex through Iml3p. Furthermore, we show that Aft1p, like Iml3p, is required for the increased association of cohesin with the pericentromere and that aft1Δ cells display sister chromatid cohesion defects in both mitosis and meiosis. Our work defines a new role for Aft1p in the sister chromatid cohesion pathway.

The Saccharomyces cerevisiae iron-responsive transcription factor, Aft1p, has a well established role in regulating iron homeostasis through the transcriptional induction of iron-regulon genes. However, recent studies have implicated Aft1p in other cellular processes independent of iron-regulation such as chromosome stability. In addition, chromosome spreads and two-hybrid data suggest that Aft1p interacts with and co-localizes with kinetochore proteins, however the cellular implications of this have not been established. Here, we demonstrate that Aft1p associates with the kinetochore complex through Iml3p. Furthermore, we show that Aft1p, like Iml3p, is required for the increased association of cohesin with the pericentromere and that aft1Δ cells display sister chromatid cohesion defects in both mitosis and meiosis. Our work defines a new role for Aft1p in the sister chromatid cohesion pathway.

<p>Although the majority of the population carries <em>Candida spp</em> as normal components of their microflora, these species are important human pathogens that have the ability to cause disease under conditions of immunosuppression or altered host defenses. The spectrum of disease caused by these species ranges from cutaneous infections of the skin, mouth, esophagus and vagina, to life-threatening systemic disease. Despite increases in drug resistance, the antifungal armamentarium has changed little over the past decade. Thus increasing our understanding of the life cycles of these organisms, not only how they propagate themselves, but also how genetic diversity is created within the population is of considerable import. Additionally expanding our knowledge of key signal transduction cascades that are important for cell survival and response to stress will add in developing new antifungal therapies and strategies. </p><p>This thesis addresses both of these key areas of fungal pathogenesis. In the first chapter, we use genome comparisons between parasexual, asexual, and sexual species of pathogenic <em>Candida</em> as a first approximation to answer the question of whether examining genome content alone can allow us to understand why species have a particular life cycle. We start by examining the structure of the mating type locus (<em>MAT</em>) of two sexual species <em>C. lusitaniae</em> and <em>C. guilliermondii</em>. Interestingly, both species are missing either one or two (respectively) canonical transcription factors suggesting that the control of sexual identity and meiosis in these organisms has been significantly rewired. Mutant analysis of the retained transcription factors is used to understand how sexual identity and sporulation are controlled in these strains. Secondly, based on the observation that these species are missing many key genes involved in mating and meiosis, we use meiotic mapping, SPO11 mutant analysis, and comparative genome hybridization to demonstrate that these species are indeed meiotic, but that the meiosis that occurs is occasional unfaithful generating aneuploid and diploid progeny. </p><p>In the second and third chapters we examine the calcineurin signaling pathway, which is crucial for mediated tolerance to cellular stresses including cations, azole antifungals, and passage through the host bloodstream. First, we show that clinical use of calcineurin inhibitors in combination with azole antifungals does not result in resistance to the combination, suggesting that if non-immunosuppressive analogs could be further developed this combinatorial strategy may have great clinical efficacy. Second, we use previous studies of the calcineurin signaling pathway in <em>S. cerevisiae</em> to direct a candidate gene approach for elucidating other components of this pathway in <em>C. albicans</em>. Specifically, we identify homologs of the <em>RCN1, MID1</em>, and <em>CCH1</em> genes, and use a combination of phenotypic assays and heterologous expression studies to understand the roles of these proteins in <em>C. albicans</em>. Although the mutant strains share some phenotypic properties with calcineurin deletion strains, none completely recapitulate a calcineurin mutant. </p><p>In the last chapter, we examine the plausibility of targeting the homoserine dehyrogenase (Hom6) protein in <em>C. albicans</em> and <em>C. glabrata</em> as a novel antifungal strategy. Studies in <em>S. cerevisiae</em> had demonstrated a synthetic lethality between hom6 and fpr1, the gene encoding FKBP12 a prolyl-isomerase that is the binding target of the immunosuppressant FK506. Thiss synthetic lethality was due to the buildup of a toxic intermediate in the methionine and threonine biosynthetic pathway as a result of deletion of hom6 and inhibition of FKBP12. We deleted <em>HOM6</em> from both <em>C. albicans</em> and the more highly drug-resistant species <em>C. glabrata</em>. Studies suggest that regulation of the threonine and methionine biosynthetic pathway in <em>C. albicans</em> has been rewired such that the synthetic lethality between hom6 and FKBP12 inhibition no longer exists. However, in <em>C. glabrata</em> preliminary analysis suggest that similarly to <em>S. cerevisiae</em> hom6 and inhibition of FKBP12 can result in cell death.</p> / Dissertation

Msh2 and Mlh1 proteins from Tetrahymena thermophla are homologues of MutS and MutL from Escherichia coli respectively. MutS and MutL are DNA mismatch repair proteins. In eukaryotes, MutS homologues recognize the replication errors and MutL homologues interact with MutS homologues and other proteins to make the repair occur. Biolistic transformation has been done to make the msh2 and mlh1 single knockouts in the macronuclei of different strains and the knockouts were verified complete. Two strains of WT crossing KO or KO crossing KO, with different mating types, were induced to conjugate. The processes were studied by microscopy using DAPI staining. For the msh2 knockouts, there were no crescent micronuclei formed throughout the conjugation of two knockout cells, and the pairing level was reduced severely. However, a knockout cell and a wild-type cell could conjugate normally at a high level pairing efficiency. Msh2 protein seems to be important to cell pairing and indispensible for the formation of the crescent micronuclei during cell conjugation. For the mlh1 knockouts, the pairing level of a knockout and a wild-type was reduced by half and the pairing level of two knockouts was reduced more than 80%; however, the paired cells in both could complete the conjugation with delay. Pms2 protein may have redundant roles in the MutL heterodimer (Mlh1-Pms2). In addition, chemical mutagens treated knockout was crossed with non-treated wild-type and the conjugation was compared with treated wild-types. Most of the treated knockout cells could not pair after starvation and mixing with non-treated wild-type cells, which means most of the cells could not enter meiotic phase. It is probable that G2/M checkpoint arrested the meiotic cell cycle and the intra-S phase was inactivated. Thus, Msh2 protein may have a role in the meiotic intra-S phase checkpoint system.

The Saccharomyces cerevisiae iron-responsive transcription factor, Aft1p, has a well established role in regulating iron homeostasis through the transcriptional induction of iron-regulon genes. However, recent studies have implicated Aft1p in other cellular processes independent of iron-regulation such as chromosome stability. In addition, chromosome spreads and two-hybrid data suggest that Aft1p interacts with and co-localizes with kinetochore proteins, however the cellular implications of this have not been established. Here, we demonstrate that Aft1p associates with the kinetochore complex through Iml3p. Furthermore, we show that Aft1p, like Iml3p, is required for the increased association of cohesin with the pericentromere and that aft1Δ cells display sister chromatid cohesion defects in both mitosis and meiosis. Our work defines a new role for Aft1p in the sister chromatid cohesion pathway.

During development, somatic cell cues direct sex-specific differentiation of germ cells that is characterised by two distinct cell cycle states. At 12.5 days post coitum (dpc) in a testis, XY germ cells stop proliferating and enter G1/G0 arrest. In the ovary, XX germ cells bypass G1/G0 arrest and instead enter the first phase of meiosis I from 13.5 dpc. Whilst it is hypothesised that errors in cell cycle control during development precede the formation of testicular germ cell tumours, the mechanism of cell cycle control at this time has not been thoroughly investigated. This project therefore sought to explore the mechanism of XY germ cell G1/G0 arrest using several approaches. Although cell cycle regulation for somatic cells is well established, we know very little regarding germ cell control of this process. Therefore my first aim was to profile this machinery at the transcript level using a cell cycle cDNA array. Purified populations of germ cells were isolated both before and after sex differentiation and expression of 112 cell cycle related genes was assessed. From this study a comprehensive network governing apoptosis and calcium signalling that was common to both XX and XY germ cells was observed. Importantly, the retinoblastoma family and cyclin dependent kinase inhibitor p21 was implicated in the regulation of G1/G0 arrest in XY germ cells. Lastly, XX germ cells displayed a down-regulation of genes involved in both G1 and G2 phases of the cell cycle consistent with their progression past G1 phase. This study has provided a detailed analysis of cell cycle gene expression during fetal germ cell development and identified candidate factors for future investigation in order to understand cases of aberrant cell cycle control in these specialised cells. In order to investigate several candidate genes identified within the cell cycle array, I next sought to generate a germ cell-specific Cre recombinase mouse model for use in conditional knockout studies. As current Cre lines lack specificity or appropriate temporal expression, we used the germ cell-specific regions of the fragilis promoter to drive Cre expression during germ cell specification. Eleven founder lines were generated using this construct and four were analysed using a reporter line. Although we have not achieved germ cell expression from these lines to date, analysis continues in order to identify an invaluable new tool for germ cell research. Following the implication of the retinoblastoma family in XY germ cell G1/G0 arrest, I next investigated the role of RB in these cells using the Rb null mutant. RB is a known cell cycle suppressor that controls this process in many cell types and, subsequently, mice homozygous for the Rb deletion die in utero at 14.5 dpc. Using this model we analysed developing gonads from 14.5 – 16.5 dpc using ex vivo culture techniques. At 14.5 dpc when wild type germ cells have arrested, proliferating germ cells were detected in the absence of Rb using proliferation marker Ki67. This proliferation was accompanied by a slight increase in germ cell number at 14.5 dpc, however, two days later at 16.5 dpc germ cell numbers were slightly decreased in the Rb-/- testes. During this time we could also detect increased expression of other RB family members p107 and p130, suggesting that these factors may compensate for the loss of Rb in the germ line. This investigation has implicated RB in the regulation of XY germ cell G1/G0 arrest and will form the basis for future work aimed at understanding the initiation of this cell cycle state. In addition to RB, a lesser-known transcription factor was also investigated in the initiation and maintenance of XY germ cell G1/G0 arrest. The high mobility group box transcription factor 1 (HBP1) suppresses proliferation and promotes differentiation in various cell types and was recently identified within the XY germ cells at the appropriate time of sex differentiation. In my analysis two Hbp1 transcripts were identified within the XY germ cells that display different sub-cellular localisations in vitro. Next, Hbp1-LacZ reporter lines were generated to aid in understanding the germ cell-specific regulation of these transcripts and lastly, I analysed the genetrap mutation for Hbp1. Surprisingly, this model revealed no aberrations to germ cell-cell cycle control during development. In summary, I have performed the first comprehensive study of the cell cycle machinery utilised by germ cells as they undergo the first stages of sex differentiation. Using loss-of-function models I was able to implicate the cell cycle regulator RB specifically in XY germ cell G1/G0 arrest and, conversely, demonstrate that the transcription factor HBP1 is not required for this process.

During development, somatic cell cues direct sex-specific differentiation of germ cells that is characterised by two distinct cell cycle states. At 12.5 days post coitum (dpc) in a testis, XY germ cells stop proliferating and enter G1/G0 arrest. In the ovary, XX germ cells bypass G1/G0 arrest and instead enter the first phase of meiosis I from 13.5 dpc. Whilst it is hypothesised that errors in cell cycle control during development precede the formation of testicular germ cell tumours, the mechanism of cell cycle control at this time has not been thoroughly investigated. This project therefore sought to explore the mechanism of XY germ cell G1/G0 arrest using several approaches. Although cell cycle regulation for somatic cells is well established, we know very little regarding germ cell control of this process. Therefore my first aim was to profile this machinery at the transcript level using a cell cycle cDNA array. Purified populations of germ cells were isolated both before and after sex differentiation and expression of 112 cell cycle related genes was assessed. From this study a comprehensive network governing apoptosis and calcium signalling that was common to both XX and XY germ cells was observed. Importantly, the retinoblastoma family and cyclin dependent kinase inhibitor p21 was implicated in the regulation of G1/G0 arrest in XY germ cells. Lastly, XX germ cells displayed a down-regulation of genes involved in both G1 and G2 phases of the cell cycle consistent with their progression past G1 phase. This study has provided a detailed analysis of cell cycle gene expression during fetal germ cell development and identified candidate factors for future investigation in order to understand cases of aberrant cell cycle control in these specialised cells. In order to investigate several candidate genes identified within the cell cycle array, I next sought to generate a germ cell-specific Cre recombinase mouse model for use in conditional knockout studies. As current Cre lines lack specificity or appropriate temporal expression, we used the germ cell-specific regions of the fragilis promoter to drive Cre expression during germ cell specification. Eleven founder lines were generated using this construct and four were analysed using a reporter line. Although we have not achieved germ cell expression from these lines to date, analysis continues in order to identify an invaluable new tool for germ cell research. Following the implication of the retinoblastoma family in XY germ cell G1/G0 arrest, I next investigated the role of RB in these cells using the Rb null mutant. RB is a known cell cycle suppressor that controls this process in many cell types and, subsequently, mice homozygous for the Rb deletion die in utero at 14.5 dpc. Using this model we analysed developing gonads from 14.5 – 16.5 dpc using ex vivo culture techniques. At 14.5 dpc when wild type germ cells have arrested, proliferating germ cells were detected in the absence of Rb using proliferation marker Ki67. This proliferation was accompanied by a slight increase in germ cell number at 14.5 dpc, however, two days later at 16.5 dpc germ cell numbers were slightly decreased in the Rb-/- testes. During this time we could also detect increased expression of other RB family members p107 and p130, suggesting that these factors may compensate for the loss of Rb in the germ line. This investigation has implicated RB in the regulation of XY germ cell G1/G0 arrest and will form the basis for future work aimed at understanding the initiation of this cell cycle state. In addition to RB, a lesser-known transcription factor was also investigated in the initiation and maintenance of XY germ cell G1/G0 arrest. The high mobility group box transcription factor 1 (HBP1) suppresses proliferation and promotes differentiation in various cell types and was recently identified within the XY germ cells at the appropriate time of sex differentiation. In my analysis two Hbp1 transcripts were identified within the XY germ cells that display different sub-cellular localisations in vitro. Next, Hbp1-LacZ reporter lines were generated to aid in understanding the germ cell-specific regulation of these transcripts and lastly, I analysed the genetrap mutation for Hbp1. Surprisingly, this model revealed no aberrations to germ cell-cell cycle control during development. In summary, I have performed the first comprehensive study of the cell cycle machinery utilised by germ cells as they undergo the first stages of sex differentiation. Using loss-of-function models I was able to implicate the cell cycle regulator RB specifically in XY germ cell G1/G0 arrest and, conversely, demonstrate that the transcription factor HBP1 is not required for this process.

During development, somatic cell cues direct sex-specific differentiation of germ cells that is characterised by two distinct cell cycle states. At 12.5 days post coitum (dpc) in a testis, XY germ cells stop proliferating and enter G1/G0 arrest. In the ovary, XX germ cells bypass G1/G0 arrest and instead enter the first phase of meiosis I from 13.5 dpc. Whilst it is hypothesised that errors in cell cycle control during development precede the formation of testicular germ cell tumours, the mechanism of cell cycle control at this time has not been thoroughly investigated. This project therefore sought to explore the mechanism of XY germ cell G1/G0 arrest using several approaches. Although cell cycle regulation for somatic cells is well established, we know very little regarding germ cell control of this process. Therefore my first aim was to profile this machinery at the transcript level using a cell cycle cDNA array. Purified populations of germ cells were isolated both before and after sex differentiation and expression of 112 cell cycle related genes was assessed. From this study a comprehensive network governing apoptosis and calcium signalling that was common to both XX and XY germ cells was observed. Importantly, the retinoblastoma family and cyclin dependent kinase inhibitor p21 was implicated in the regulation of G1/G0 arrest in XY germ cells. Lastly, XX germ cells displayed a down-regulation of genes involved in both G1 and G2 phases of the cell cycle consistent with their progression past G1 phase. This study has provided a detailed analysis of cell cycle gene expression during fetal germ cell development and identified candidate factors for future investigation in order to understand cases of aberrant cell cycle control in these specialised cells. In order to investigate several candidate genes identified within the cell cycle array, I next sought to generate a germ cell-specific Cre recombinase mouse model for use in conditional knockout studies. As current Cre lines lack specificity or appropriate temporal expression, we used the germ cell-specific regions of the fragilis promoter to drive Cre expression during germ cell specification. Eleven founder lines were generated using this construct and four were analysed using a reporter line. Although we have not achieved germ cell expression from these lines to date, analysis continues in order to identify an invaluable new tool for germ cell research. Following the implication of the retinoblastoma family in XY germ cell G1/G0 arrest, I next investigated the role of RB in these cells using the Rb null mutant. RB is a known cell cycle suppressor that controls this process in many cell types and, subsequently, mice homozygous for the Rb deletion die in utero at 14.5 dpc. Using this model we analysed developing gonads from 14.5 – 16.5 dpc using ex vivo culture techniques. At 14.5 dpc when wild type germ cells have arrested, proliferating germ cells were detected in the absence of Rb using proliferation marker Ki67. This proliferation was accompanied by a slight increase in germ cell number at 14.5 dpc, however, two days later at 16.5 dpc germ cell numbers were slightly decreased in the Rb-/- testes. During this time we could also detect increased expression of other RB family members p107 and p130, suggesting that these factors may compensate for the loss of Rb in the germ line. This investigation has implicated RB in the regulation of XY germ cell G1/G0 arrest and will form the basis for future work aimed at understanding the initiation of this cell cycle state. In addition to RB, a lesser-known transcription factor was also investigated in the initiation and maintenance of XY germ cell G1/G0 arrest. The high mobility group box transcription factor 1 (HBP1) suppresses proliferation and promotes differentiation in various cell types and was recently identified within the XY germ cells at the appropriate time of sex differentiation. In my analysis two Hbp1 transcripts were identified within the XY germ cells that display different sub-cellular localisations in vitro. Next, Hbp1-LacZ reporter lines were generated to aid in understanding the germ cell-specific regulation of these transcripts and lastly, I analysed the genetrap mutation for Hbp1. Surprisingly, this model revealed no aberrations to germ cell-cell cycle control during development. In summary, I have performed the first comprehensive study of the cell cycle machinery utilised by germ cells as they undergo the first stages of sex differentiation. Using loss-of-function models I was able to implicate the cell cycle regulator RB specifically in XY germ cell G1/G0 arrest and, conversely, demonstrate that the transcription factor HBP1 is not required for this process.