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.

Eukaryotic centromeres are the region upon which kinetochores assemble, directing attachment of spindle microtubules and faithful segregation of chromosomes during mitosis and meiosis. Except for a transient disruption in mitosis when chromosomes are segregated, centromeres of fission yeast Schizosaccharomyces pombe remain closely associated with the nuclear periphery. Similar to multicellular eukaryotic centromeres, they also maintain unique chromatin architecture, with a central core defined by the presence of the conserved centromeric histone H3 variant CENP-A, designated Cnp1 in S. pombe, that is flanked by histone H3 containing heterochromatin. While much progress has been made in understanding chromatin-associated factors important for proper centromere function, many questions remain. In order to gain a better understanding of the factors involved in centromeric chromatin structure, we affinity purified and defined by mass spectrometry interactions among select proteins that had been implicated in proper Cnp1 localization and centromere function. These biochemical purifications revealed several proteins that may be involved in Cnp1 localization. Purification and analyses of Cnp1 also led us to the identification of the Mlp1/Tpr nucleoporin homolog Nup211. We have found that Nup211 interacts with components of the inner nuclear basket of the nuclear pore, and co-purifies with centromeric chromatin proteins. Cells lacking Nup211 have substantial chromosome segregation defects, as observed by synthetic growth assay, flow cytometric analysis, and fluorescent microscopy. A series of immunoprecipitation experiments have revealed that Nup211 associates with centromeric DNA, and that, surprisingly, cells lacking Nup211 have increased histone H3 lysine 9 methylation, a marker of heterochromatin, and a reduction in Cnp1 levels at the central core. Moreover, cells lacking Nup211 have decreased transcription at centromeric loci, disruption of the stereotypical nucleosome structure found at the central core of S. pombe, and show striking changes in the distribution of heterochromatic foci in the nucleus. By demonstrating that Nup211 is essential for the maintenance of normal central core chromatin state, these studies have shed light on a novel role for Nup211 in proper centromere structure and function in S. pombe, and suggest that Nup211 may play a role in preventing the invasion of flanking pericentric heterochromatin into the central core of centromeres.

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.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 6 uppsatser.

Thesis (Ph.D.). / Written for the Dept. of Biology. Title from title page of PDF (viewed 2008/01/12). Includes bibliographical references.

Thesis (Ph.D. in Molecular Biology) -- University of Colorado, 2006. / Typescript. Includes bibliographical references (leaves 126-147). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

CP1 is a sequence specific DNA-binding protein of the yeast Saccharomyces cerevisiae which recognizes the highly conserved centromere DNA element I (CDEI) of yeast centromeres. The gene encoding CP1, which was designated CEP1 for centromere protein 1, was cloned and sequenced. CEP1 encodes a highly acidic protein of molecular weight 39,400. CEP1 was mapped to a position 4.6 centiMorgans centromere distal to SUP4 on the right arm of chromosome X. Phenotypic analysis of cep1 mutants demonstrated that yeast strains lacking CP1 are viable but have a 35% increase in cell doubling time, a ninefold increase in the rate of mitotic chromosome loss, and are methionine auxotrophs. Detailed analysis of the mitotic chromosome-loss phenotype showed that the loss is primarily due to chromosome nondisjunction (2:0 segregation). During meiosis cep1 null mutants exhibited aberrant segregation of centromere containing plasmids, chromosome fragments, and chromosomes. The predominant missegregation event observed was precocious sister segregation. The mutants also displayed a nonrandom 20% decrease in spore viability. Missegregation of chromosomes accounted for some but not all of this decreased spore viability, the remainder of which is presumed to be related to the pleiotropic consequences of the cep1 mutation. Together with the observed mitotic missegregation phenotype the results are interpreted as suggesting that CP1 promotes sister chromatid-kinetochore adhesion. The following conclusions are based on my mutational analysis of CP1: (1) CP1 is normally present in functional excess, (2) the C-terminal 143 amino acids are sufficient for full CP1 function in chromosome segregation and methionine metabolism, and (3) while DNA binding is apparently necessary for function, DNA binding per se is not sufficient. All of the mutations which caused an observable phenotype affected both centromere function and methionine metabolism. In addition, a direct correlation was observed in the degree to which both phenotypes were affected by different mutations. None of the mutant proteins displayed trans-dominant effects in a wild type background; however, two nonfunctional DNA binding-competent mutants exerted a dominant negative effect on the ability of PHO4 to suppress cep1 methionine auxotrophy. The data are consistent with a model in which CP1 performs a similar function at centromeres and promoters.

Le variant d’histone CENP-A marque épigénétiquement le centromère. La présence de CENP-A au centromère permet le recrutement de protéines centromériques qui constituent la plateforme pour l’assemblage de kinétochores fonctionnels.Dans les cellules humaines, l'extrémité amino-terminale de CENP-A ainsi que la phosphorylation de la sérine 7, ont été signalées comme étant cruciales pour la progression de la mitose. Cependant, aucune phosphorylation de CENP-A dans d'autres espèces de métazoaires n'a été décrite. Ici, nous montrons que le domaine NH2-terminal CENP-A, mais pas sa séquence primaire, est nécessaire pour la mitose dans les fibroblastes embryonnaires de souris (MEFs). Nos données montrent que les défauts mitotiques résultant de la déplétion de CENP-A endogène peuvent être restaurés lorsque les MEFs expriment un mutant GFP-CENP-A dont l'extrémité NH2-terminal de CENP-A a été échangée par la queue phosphorylable de l'histone canonique H3. Inversement, dans ce même mutant, lorsque l’on remplace les deux serines phosphorylables par des résidus alanines, les défauts mitotiques persistent. En outre, le mutant de fusion non- phosphorylable de CENP-A, où les sept serines du domaine NH2-terminal ont été remplacées par des résidus alanines, a été également incapable de restaurer le phénotype mitotique des cellules déplétées en CENP-A endogène.Nous avons également identifié les trois premières sérines de la queue de CENP-A comme sites potentiels de phosphorylation. De plus, nos résultats montrent que l’absence de phosphorylation du domaine amino-terminal conduit à la délocalisation de la protéine centromérique CENP-C. Ces résultats suggèrent que la phosphorylation mitotique de CENP-A est un événement potentiellement fréquent chez les métazoaires et essentiel à la progression mitotique.Dans la seconde partie de ce travail, nous avons voulu lier sans ambiguïté la fonction du domaine NH2-terminal du CENP-A à la mitose. Nous avons conçu une nouvelle méthode, appelée approche Hara-kiri, pour pouvoir éliminer le domaine NH2- terminal seulement pendant la mitose. Ceci afin de répondre à la question ci-dessus dans les cellules humaines. L'élimination du domaine NH2-terminal du CENP-A en utilisant l'approche Hara-kiri en début de mitose a conduit à une augmentation des défauts mitotiques dans les cellules. Prises collectivement, ces données montrent que le domaine NH2-terminal CENP-A est nécessaire pendant la mitose afin d’assurer le bon déroulement de la division cellulaire. / The histone variant CENP-A epigenetically marks the centromere. The presence of CENP-A at the centromeres allows the recruitment of centromeric proteins that constitute the platform for functional kinetochores.In human cells, the NH2-terminus of CENP-A and its phosphorylation at serine 7 in mitosis has been reported to be crucial for the progression of mitosis. However, no phosphorylation of CENP-A in other metazoan species has been described. Here, we show that the NH2-terminus of CENP-A, but not its primary sequence, is required for mitosis in mouse embryonic cells (MEFs). Our data show that the mitotic defects resulting from the depletion of the endogenous CENP-A can be rescued when MEFs expressing a GFP- CENP-A mutant where the NH2-terminus of CENP-A was swapped with the phosphorylatable tail of conventional histone H3. Conversely, no rescue was observed when the two phosphorylatable serines in the H3 tail mutant were replaced with alanines. Furthermore, a non-phosphorylatable fusion mutant of CENP-A where all seven serines in the amino-tail were replaced with alanines, was also unable to rescue the mitotic phenotype of CENP-A depleted cells.We also identified that the first three serines of the tail of CENP-A as potential sites for phosphorylation. Additionally, we were able to link the phosphorylation of CENP-A amino-tail to the proper localization of the key centromeric protein CENP-C. These results suggest that mitotic CENP-A phosphorylation is a potentially common event in metazoans essential for mitotic progression.In the second par of this work we wanted to unambiguously tie the NH2-terminus function of CENP-A to mitosis. To achieve this, we wanted to remove the CENP-A amino-tail only during mitosis and we devised a new method called the Hara-kiri approach in order to answer the above question in human cells. The removal of the NH2-terminal domain of CENP-A using the Hara-kiri approach at the onset of mitosis led to increased mitotic defects in cells. Taken collectively these data show that the CENP-A NH2- terminus is required during mitosis to assure proper cell division.

CP1 (encoded by the gene CEP1) is a sequence-specific DNA-binding protein of Saccharomyces cerevisiae that recognizes a sequence element (CDEI) found in both yeast centromeres and gene promoters. Strains lacking CP1 are viable but exhibit defects in growth, chromosome segregation, and methionine biosynthesis. To investigate the basis of the methionine requirement, a YEp24-based yeast genomic DNA library was screened for plasmids which suppressed the methionine auxotrophy of a cep1 null mutant. The suppressing plasmids contained either CEP1 or DNA derived from the PHO4 locus. PHO4 encodes a factor which positively regulates transcription of genes involved in phosphate metabolism via an interaction with CDEI-like elements within the promoters of these genes. Subcloning experiments confirmed that suppression correlated with increased dosage of PHO4. PHO4c, pho80, and pho84 mutations, all of which lead to constitutive activation of the PHO4 transcription factor, also suppressed cep1 methionine auxotrophy. The suppression appeared to be a direct effect of PHO4, not a secondary effect of PHO regulon derepression, and was dependent on a second transcriptional regulatory protein encoded by PHO2. Spontaneously arising extragenic suppressors of the cep1 methionine auxotrophy were also isolated; approximately one-third of the them were alleles of pho80. While PHO4 overexpression suppressed the methionine auxotrophy of a cep1 mutant, CEP1 overexpression failed to suppress the phenotype of a pho4 mutant; however, a cep1 null mutation suppressed the low-Pi growth deficiency of a pho84 mutant. The results suggest that CP1 functions as a transcriptional regulator of MET genes, and that activation of PHO4 restores expression to those genes transcriptionally-disabled by the cep1mutation. The results also suggest the existence of a network that cross-regulates transcription of genes involved in methionine biosynthesis and phosphate metabolism. A direct molecular approach to investigate CP1's role in MET gene expression was also taken. CDEI sites are associated with the promoter regions of most MET genes, but only MET16, the gene encoding PAPS reductase, has been shown to require CP1 for expression; both PAPS reductase activity, and MET16 mRNA are absent in cep1 mutants. Results of the present study demonstrate that CP1 participates in two systems which regulate expression of MET16, one triggered by methionine starvation and requiring the transactivator MET4 (pathway-specific control), and the other triggered by starvation for many different amino acids and requiring GCN4 (general control). CP1 was shown to mediate its regulatory function through the upstream CDEI site, and to act directly or indirectly to modulate the chromatin structure of the MET16 promoter. In addition, the pho80 mutation was found to partially restore MET16 expression to the cep1 strain, confirming the proposed nature of PHO4 suppression. A second methionine biosynthetic gene MET25, was also analyzed. Like MET16, MET25 was found to be regulated by both pathway-specific and general control mechanisms, but in contrast to MET16, CP1 only participated in the pathway-specific response of this gene. The results demonstrate that CP1, possibly by modulating changes in chromatin structure, assists the regulatory proteins MET4 and GCN4 in activating transcription of MET genes.