Impact of ploidy and cell size on genome expression in fission yeast

Cells are classified based on their ploidy into haploids, containing a single chromosome set, diploids, containing two chromosome sets, polyploids, containing more than two chromosome sets, and aneuploids, containing abnormal chromosome numbers. Polyploidy is typically accompanied by increased cell size. Polyploid cells are found in most tumors and exhibit chromosomal instability that leads to aneuploidy. The effects of aberrant ploidy on genome regulation and on cell size are not well understood. I used fission yeast as a model to analyse impacts of altered ploidy and cell size on gene expression. Using aneuploids that are disomic or trisomic for a portion of chromosome III, I find that total mRNA levels scale with DNA copy numbers. Aneuploidy also affects the transcription of some genes present in monosomic areas, possibly reflecting associated regulatory genes in disomic or trisomic areas. I also analysed the effect of polyploidy on genome expression by constructing diploid and tetraploid strains. Diploids were stable with normal cell shape, while tetraploids showed irregular morphologies and often lost chromosomes. Increased ploidy resulted in increased cell size, and also in a linear increase in cellular RNA levels. Using spike-in controls and normalization, we showed that increased transcription in polyploids does not affect ratios between total RNA and mRNA. Cells kept a tight control on genome-wide transcription which generally scaled with the copy numbers of genes, a few genes were differentially regulated as a function of polyploidy and/or cell size. These genes were present in multiple copies close to telomeres and may function at the cell surface. They were also differentially regulated in haploid cell-size mutants, indicating a role of cell size, rather than ploidy, in controlling these genes. Intriguingly, deletion and overexpression of these genes in turn resulted in a significant decrease or increase in cell size, respectively, raising the possibility that the genes are involved in size control

Telomerase Regulation in Arabidopsis thaliana

Telomeres form a nucleoprotein cap at the end of eukaryotic chromosomes. The telomere protein constituents repress the DNA damage response (DDR) and facilitate maintenance of terminal sequences by a specialized ribonucleoprotein complex called telomerase. In turn, factors involved in the DDR guarantee telomerase acts only in telomere homeostasis, and not at double-strand breaks (DSBs). Thus, the three pathways surrounding telomeres display incredible overlap and are immensely complex. Here, I report a novel regulatory pathway that limits telomerase action during DNA damage. Duplication of the telomerase RNA subunit (TER) in Arabidopsis has given rise to a TER that is not required for telomere homeostasis. Indeed, this TER, termed TER2, is a competitive inhibitor of TER1 RNP complexes. Exposure to genotoxic agents results in TER2 upregulation and a subsequent inhibition of telomerase activity. Using data from the 1,001 Arabidopsis genomes project, I determine that the TER duplication and inhibitory nature of TER2 is likely derived from a transposon-like element within TER2. This element is found throughout Brassicaceae, with at least 32 members in Arabidopsis lyrata. These findings highlight the complex and diverse mechanisms by which an organism will regulate telomerase action. Here I characterize two members of the A. thaliana POT1 gene family. Contrary to POT1a, these proteins appear to have derived unique ways to perform their roles in chromosome-end protection. POT1b may protect telomeres as part of a TER2 telomerase RNP complex, as telomere defects only appear in the absence of both POT1b and TER2. POT1c is also appears to provide for chromosome end protection and appears to compete with POT1a to regulate telomerase access to the G-overhang. Together, these proteins represent part of a critical telomere capping complex distinct from CST. Additionally, I describe a means for elucidating factors that regulate telomere addition at DSBs. This incredibly detrimental process, termed de novo telomere formation (DNTF), is toxic, and thus this work describes the first in depth characterization of DNTF in multicellular eukaryotes. In summary, my work describes several novel regulatory and protective mechanisms for keeping telomeres and DSBs distinct

Program and abstracts from the 24th Fungal Genetics Conference

Abstracts of the plenary and poster sessions from the 24th Fungal Genetics Conference, March 20-25, 2007, Pacific Grove, CA

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

XXIII Fungal Genetics Conference

Program and abstracts from the 23rd Fungal Genetics Conference and Poster Abstracts at Asilomar, March 15-20, 200

Exploration of Cell Cycle-Specific Essential Gene Functions in the Microbial Plant Chlamydomonas Reinhardtii

The cell cycle encompasses all the steps required for cell proliferation, and is normally tightly coupled to growth and division in all organisms. Much research has resulted in a well-supported model of eukaryotic cell cycle control. However, since most of this research has been carried out in yeast and animals (opisthokonts), it could in principle apply poorly to early-diverging groups of organisms, such as the green plants. Plant cell cycle research has largely followed a candidate strategy based on reverse genetics. These studies have a provided insights into plant cell cycle control, but are generally dependent upon sequence conservation between plant and opisthokont genes. This thesis presents work from an ongoing screen to identify critical components of the plant cell cycle by forward genetic methods that are independent of prior knowledge of specific mechanisms of cell cycle control. The screen was carried out in the unicellular green alga Chlamydomonas, a microbial member of the Viridiplantae, which has wellestablished experimental Mendelian genetics, and many features that might facilitate identification of loss-of-function mutations. We have developed semi-automatic techniques for isolation of temperature-sensitive lethal mutants that are capable of cell growth at a near-wild-type rate, but that exhibit first-cycle failure of cell division (div phenotype). We developed efficient methods for identification of causative mutations by next-generation sequencing of bulked segregant pools. The normal cell division cycle in Chlamydomonas is characterized by a long period of G1 growth, followed by a series of rapidly alternating rounds of S phases and mitoses (S/M phase). Analysis of more than 50 div mutants identified two main phenotypic classes. One class showed somewhat reduced growth and arrested in a G1-like state. This class included genes with diverse molecular functions based on gene annotations, including transcription, translation, and membrane biogenesis. The other class exhibited wild-type cell growth rate, and entered the S/M program on time; mutant cells then developed various S/M-specific defects. This class included genes directly involved in DNA replication and chromosome segregation. Other mutations identified genes likely involved in cell cycle control, including the cyclin-dependent kinases CDKA and CDKB, two anaphase-promoting complex subunits, and the mitotic kinases Aurora B and MPS1. The phenotype of the cdka-1 mutant suggested a specific role for CDKA in the transition from cell growth to initiation of the S/M cell division program. CDKB, in contrast, functions specifically after DNA replication, in entry into the first mitosis. Although most DIV genes had clear homologues involved in cell cycle progression in opisthokonts, some genes had clear homologues in Viridiplantae but not in opisthokonts, including the BSL1 phosphatase, which we demonstrate to have a role in mitotic entry similar to that of CDKB. The div mutants isolated in this screen provide an opportunity to study the plant cell cycle in a simple microbial setting. Since a large majority of the mutants alter genes with clear Arabidopsis sequelogues, the results also suggest targeted candidates for cell cycle experiments in Angiosperms

Abstracts from the 25th Fungal Genetics Conference

Abstracts from the 25th Fungal Genetics Conferenc

Réplication, condensation et division des chromosomes parentaux dans le zygote de drosophile

In animals, sexual reproduction requires the union between two distinct parental gametes: the spermatozoon and the oocyte. The unique nuclear conformation of the sperm, in which the chromatin is organized with sperm-specific chromosomal protein like protamines, abolishes its activity. The paternal chromatin remodeling and the maintenance of its integrity at fertilization by maternal activities are therefore essential processes for zygote formation. However, although their mechanisms are crucial, they remain poorly understood. During my thesis, I tried to better understand the processes involved during de novo paternal chromatin assembly in Drosophila through the study of a maternal embryonic lethal mutation: maternal haploid (mh). The mutant affects the incorporation of paternal chromosomes during the first zygotic division, leading to the development of gynogenetic haploid embryos. The identification of the mh gene as CG9203, and the generation of the null allele mh2 allowed me to characterize its function. In eggs led by mh mutant females, paternal chromosomes abnormally condense and fail to divide leading to the formation of chromatin bridges at the first embryonic division. Recently, its human ortholog Spartan/DVC1, has been described to be involved in translesion synthesis (TLS), a DNA damage tolerance pathway that ensures replication fork progression. Combining genetic and cytological approaches, I demonstrated that the Spartan function in TLS is conserved in Drosophila. However, I discovered that the critical function of MH during the first embryonic division, was not consistent with a canonical TLS. Alternatively, it is specifically required to maintain paternal integrity and to allow its proper replication at the first cycle. The mh phenotype characterization, led me to compare it with others phenotypes induced by the knock-down of replication factors and to study parental chromosome condensation in the zygote. Surprisingly, one of the proteins allowing the establishment of the pre-replication complex is dispensable for the proper paternal chromosome segregation contrarily to the maternal counterpart. Altogether, these works highlight the difference that exists between the two parental pronuclei and the complexity of maintaining their integrity at fertilizationChez les animaux, la conformation unique du noyau du spermatozoïde dont la chromatine est organisée avec des protéines chromosomiques spécifiques telles que les protamines le rend totalement inactif. Le remodelage de la chromatine paternelle à la fécondation par des activités d'origine maternelle sont donc des processus essentiels à la formation d'un embryon diploïde, dont les mécanismes restent très mal connus. Lors de ma thèse j'ai essayé de mieux comprendre ces processus par l'étude, chez la drosophile, d'un mutant létal embryonnaire à effet maternel : maternal haploid (mh). Ce mutant affecte l'incorporation des chromosomes paternels à la première division zygotique menant à la formation d'embryons haploïdes gynogénétiques. L'identification du gène de mh comme CG9203 m'ont permis de caractériser sa fonction. Dans les œufs mh, les chromosomes paternels se condensent anormalement et ne parviennent pas à se diviser correctement lors de la première mitose de l'embryon. Récemment, des études sur son orthologue humain, appelé Spartan/DVC1, ont montré qu'il était impliqué dans la synthèse translésionnelle (TLS), un mécanisme de tolérance aux dommages d'ADN. J'ai pu démontrer que dans les cellules somatiques, la fonction de Spartan dans le TLS est conservée chez la drosophile. Cependant, la fonction maternelle de MH ne relève pas du TLS canonique, mais permet de maintenir l'intégrité de l'ADN paternel avant la réplication. Ensemble, mes travaux soulignent la singularité du pronoyau mâle et la complexité que présente le maintien de son intégrité à la fécondatio