Causes and consequences of Aneuploidy

The goal of our research is to obtain a detailed molecular understanding of the regulatory circuits that control chromosome segregation and what happens to cells in which these mechanisms fail and hence become aneuploid. We use the budding yeast S. cerevisiae as a model system to study chromosome segregation and the effects of aneuploidy on cell physiology, and probe discoveries made in yeast in the mouse.

Control of exit from mitosis.

Exit from mitosis is the final cell cycle transition when mitotic cyclin-dependent kinases (CDKs) are inactivated, the mitotic spindle disassembles, chromosomes decondense and cytokinesis occurs. In budding yeast, the protein phosphatase Cdc14 is the central regulator of this cell cycle transition (Visintin et al., 1998). We study how this phosphatase is controlled and how it regulates the final stages of mitosis.

Cdc14 is regulated by an inhibitor in the nucleolus

The conserved protein phosphatase Cdc14 induces mitotic CDK inactivation and hence exit from mitosis. Cdc14 is regulated by an inhibitor Cfi1/Net1 that binds to and sequesters Cdc14 in the nucleolus during G1, S phase, G2 and metaphase (Shou et al., 1999; Visintin et al., 1999). During anaphase, Cdc14 is released from its inhibitor and spreads throughout the nucleus and cytoplasm, where it dephosphorylates its targets. We identified two pathways that control the association between Cdc14 and its inhibitor. The Cdc14 Early Anaphase Release Network (FEAR network) promotes Cdc14 release from the nucleolus during early anaphase (Stegmeier et al., 2002) and the Mitotic Exit Network (MEN) maintains Cdc14 in its released state during late stages of anaphase (Shou et al., 1999; Visintin et al., 1999). Currently we are determining how the individual MEN components are regulated and how their activity is integrated with other cell cycle events. We also study the role and regulation of the MEN and FEAR network during meiosis.

Figure 1: The FEAR network and the MEN regulate Cdc14 release from the nucleolus.

The FEAR network is composed of Esp1, Slk19, Clb-CDKs and Cdc5. This network initiates Cdc14 release from the inhibitor Cfi1/Net1 in the nucleolus during early anaphase. During later stages of anaphase the Mitotic Exit Network maintains Cdc14 in the released state. The MEN resembles a GTPase signaling cascade that is composed of the GTPase Tem1 and the protein kinases Cdc15 and Dbf2-Mob1.

The MEN senses spindle position:

Correctly positioning the mitotic spindle within a cell is essential for accurate chromosome segregation. A surveillance mechanism known as the spindle position checkpoint (SPOC) senses spindle position and conveys this information to the cell cycle machinery (reviewed in Lew and Burke, 2003). We found that in budding yeast the localization of Mitotic Exit Network components and their regulators is at the heart of spindle position sensing and the response to spindle mis-position.

The Mitotic Exit Network is a Ras-like GTPase signaling cascade, whose constituents localize to the cytoplasmic face of spindle pole bodies (SPBs), the budding yeast equivalent of centrosomes (Figure 2; reviewed in Stegmeier and Amon, 2004). The MEN activator Lte1 localizes to the bud, the MEN inhibitor Kin4 localizes to the mother cell (Bardin et al., 2000; D’Aquino et al., 2005) These localization patterns led to a simple hypothesis: The cell is divided into a MEN inhibitory zone in the mother cell, where Kin4 resides, and a MEN activating zone in the bud, where Lte1 resides (Figure 2). The MEN component carrying SPB functions as the sensor. Only when the MEN bearing SPB escapes the MEN inhibitor Kin4 in the mother cell and moves into the bud during anaphase where the MEN activator Lte1 resides can exit from mitosis occur.

We showed that spindle position is indeed sensed in this manner. When Lte1 is targeted to the mother cell, cells with mis-positioned spindles inappropriately exit from mitosis (Bardin et al., 2000). When Kin4 is targeted to the bud, cells with correctly positioned spindles fail to exit from mitosis (Chan and Amon, 2010). Thus, spatial information is sensed and translated into a chemical signal by targeting activators and inhibitors of signal transduction pathways to specific cellular locations. Currently, we are determining how Lte1 controls Tem1 activity and how Kin4 localization is regulated.

Figure 2: Spindle position is sensed through MEN inhibitory and activating zones.

The Mitotic Exit Network components (MEN, blue) localize to the cytoplasmic face of spindle pole bodies (SPBs). The MEN activator Lte1 (green) localizes to the bud, the MEN inhibitor Kin4 localizes to the mother cell (red). In cells with correctly positioned anaphase spindles, Lte1 activates the MEN and induces exit from mitosis. In cells where the spindle is mis-positioned, Kin4 inhibits the MEN, in part by stimulating the MEN GAP Bub2-Bfa1 and in part by preventing MEN components from associating with SPBs.

Control of cell growth

Cell growth – the accumulation of cell mass - is an essential requirement for cell cycle progression, but whether the reciprocal relationship holds has not been established. We find that in budding yeast, the ability of cells to grow changes during the cell cycle. Cell growth is faster in cells arrested in anaphase and G1 than in other cell cycle positions (Goranov et al., 2009). We furthermore find that the establishment of a polarized actin cytoskeleton - either as a consequence of normal cell division or through activation of the mating pheromone response - potently attenuates protein synthesis and growth. We furthermore find by population and single cell analyses that growth varies during the cell cycle. Currently, we are investigating how the actin cytoskeleton affects cell growth.

Establishing a specialized cell division - meiosis.

Our insights into mitosis spawned our interest in how the molecular mechanisms that govern mitosis are modulated to bring about a specialized division, meiosis. Meiosis consists of two consecutive chromosome segregation phases. During meiosis I, separation of homologous chromosomes occurs; during meiosis II, segregation of sister chromatids takes place (reviewed in Marston and Amon, 2004). Our studies contributed significantly towards understanding the modifications necessary to transform mitosis into meiosis. We study how removal of cohesins, the proteins that hold sister chromatids together, is changed during meiosis and how kinetochore – microtubule attachments are modified during meiosis I. Finally, we determine how meiosis-specific control of CDKs establishes the meiotic chromosome segregation pattern.

Loss of cohesins during meiosis.

In mitosis cohesins are lost along the entire length of the chromosome at the onset of anaphase (Figure 3). In contrast, these protein complexes are lost in a step-wise manner during meiosis. Cohesins are removed from chromosome arms during meiosis I but are protected from removal around centromeres. These residual centromeric cohesins are removed only during meiosis II (Figure 3). Our work contributed to unraveling the mechanism of cohesin removal during meiosis.

Figure 3: Modulations necessary to bring about the meiotic chromosome segregation program.

During mitosis, cohesin complexes (shown as red bars) are lost along the entire length of the chromosomes. During meiosis cohesin complexes are lost in a stepwise manner. Cohesins are removed from chromosome arms during meiosis I and from centromeric regions during meiosis II. Sister kinetochore orientation also changes during meiosis. Sister kinetochores attach to microtubules emanating from the same pole (co-orientation) during meiosis I to facilitate co-segregation of sister-chromatid pairs. Sister kinetochores then attach to microtubules emanating from opposite poles (bi-orientation) during meiosis II, which separates sister chromatids in anaphase II.

We tested the hypothesis that it is differential phosphorylation of the meiosis-specific cohesin subunit Rec8 that brings about the step-wise loss of cohesins (Brar et al., 2006). This appears to be the case. Rec8 mutants lacking multiple phosphorylation sites exhibit a delay in the removal of cohesins from chromosomes. Furthermore, Rec8 appears to be phosphorylated on chromosome arms but not around centromeres during meiosis I.

A key question that our phosphorylation studies pose is: Who prevents cohesin phosphorylation around centromeres during meiosis I? Insight into this question came from our studies of Sgo1, a protein that prevents cohesin removal from centromeric regions during meiosis I (Kiburz et al., 2005). We found that cohesins that are protected from removal during meiosis I, co-localize with Sgo1 to a 50 kb region surrounding centromeres. Our studies furthermore showed that Sgo1 is loaded onto chromosomes at the 120 bp core centromere from where it spread to establish the 50 kb cohesin protective domain. Studies by others (Kitajima et al., 2006; Riedel et al., 2006) showed that Sgo1 recruits a protein phosphatase to centromeric regions during meiosis I, providing an explanation for the differential phosphorylation of cohesins along chromosomes.

Sister kinetochore orientation during meiosis.

Kinetochore orientation changes during meiosis (Figure 3). Sister kinetochores attach to microtubules so that they face the same spindle pole (co-orientation) during meiosis I. During meiosis II sister kinetochores attach to microtubules emanating from opposite poles (bi-orientation). We have established a key role for the Aurora B kinase and the chromosome structure complex condensin in sister-kinetochore co-orientation during meiosis I and determined the mechanism by which they do so (Monje-Casas et al., 2007; Brito et al., 2010). Condensins promote the association of the co-orientation complex, the monopolin complex, with kinetochores (Brito et al., 2010). Aurora B ensures that faulty kinetochore – microtubule attachments do not persist (Monje-Casas et al., 2007). Furthermore, we determined that association of the monopolin complex with kinetochores is sufficient to promote sister kinetochore co-orientation during mitosis. The ability to induce sister kinetochore co-orientation at will, provided a key opportunity to gain insight into how sister kinetochore co-orientation is established. The monopolin complex joins sister kinetochores independently of cohesins thereby allowing Aurora B to distinguish between sister chromatids and homologs (Monje-Casas et al., 2007). Currently, we are determining the mechanisms whereby this fusing of sister kinetochores occurs.

Role of Clb-CDKs in establishing the meiotic chromosome segregation pattern.

To study how the meiotic cell division program is established, we developed a method that creates cultures that progress through meiosis in a highly synchronized fashion (Carlile and Amon, 2008). Using this method we discovered a surprising diversity in the regulation of the CDKs known as Clb1 to 6-CDKs that govern progression through the meiotic divisions. Clb1-CDK activity is restricted to meiosis I, and Clb3-CDK activity to meiosis II. The analysis of cells inappropriately producing Clb3-CDKs during meiosis I furthermore demonstrated that meiosis-specific CDK regulation is essential for establishing the meiotic chromosome segregation pattern (Carlile and Amon, 2008). Cells expressing Clb3 during meiosis I undergo a meiosis II instead of a meiosis I-like division.

Currently, we are determining how Clb-CDKs are regulated during meiosis. These studies will not only provide critical insight into the molecular mechanisms that underlie the meiotic chromosome segregation program but also expand our understanding of CDK control. Unexpectedly, Clb3-CDK activity is restricted to meiosis II by 5’UTR-mediated translational control. Using genetic and biochemical purification strategies, we will identify the factors that mediate this translational control and determine the molecular mechanisms whereby they do so.

The relationship between aging and meiosis

Last but not least, we have initiated a new research area that addresses the question: Why does the ability to enter the meiotic program and chromosome segregation fidelity decrease with age? We previously found that aged yeast cells exhibit both these phenotypes (Boselli et al., 2009). The tools we established to study meiotic chromosome segregation will now allow us to determine the molecular basis for these defects. Because meiotic defects increase dramatically with age also in humans, it is our hope that these studies in yeast will pave the way for elucidating the age-dependent increase in germ cell defects in humans. We are also interested in determining the effects of meiosis on age in budding yeast. Specifically, we are investigating whether meiosis can reset the replicative life span in yeast.

The consequences of aneuploidy on cell physiology.

Our studies on the mechanisms of chromosome segregation have provided insights into the processes that prevent aneuploidy due to chromosome mis-segregation. We also began to determine what happens to yeast cells that, defying the mitotic quality controls, acquired extra chromosomes and hence are aneuploid. We discovered unanticipated commonalities among many different aneuploid yeast strains that we created. This encouraged us to extended our analysis to aneuploid primary mouse cells.

Effects of aneuploidy on cell physiology

Aneuploidy leads to death and/or developmental abnormalities in all organisms analyzed to date. Yet, at the cellular level, aneuploidy is associated with cancer, a disease characterized by high proliferative potential. These findings raise an interesting conundrum. How is it possible that a single extra chromosome causes developmental defects characterized by growth restriction, yet in the context of cancer severe karyotypic abnormalities exist in cells with high proliferative potential? To address this question we began to analyze the effects of aneuploidy on normal cell physiology in yeast and in the mouse.

The exploration of the commonalities among cells that are aneuploid involved the creation of 20 yeast strains carrying one or two additional chromosomes and, subsequently, of primary mouse embryonic fibroblasts (MEFs) carrying four different trisomies (trisomy 1, 13, 16 or 19; Torres et al., 2007; Williams et al., 2008). Their analysis revealed that aneuploidy is deleterious at the cellular level, causing cell proliferation defects in both yeast and mouse. Perhaps most exiting was our discovery that aneuploid yeast and mouse cells share a number of phenotypes that are indicative of proteotoxic and energy stress. Our studies further showed that the genes located on the additional chromosomes are expressed and that the phenotypes shared by aneuploid strains are due to the proteins that are being produced from the additional chromosomes. Currently, we are focusing our characterization of aneuploidy in the mouse. We investigate how aneuploidy impacts proliferation and development in the context of the whole animal by creating mosaic mice.

The aneuploidy stress response hypothesis

From our studies on aneuploid yeast and mouse cells we developed the following working hypothesis: aneuploidy leads to a cellular response, which we term the “aneuploidy stress response” (reviewed in Torres et al., 2008; Williams et al., 2009). In the aneuploidy stress response cells engage protein folding and degradation pathways in an attempt to correct protein stoichiometry imbalances caused by aneuploidy. Although the proteins that engage the protein degradation and folding machineries will be different for each additional chromosome, the necessity to degrade and fold excess protein, compromises the cell’s ability to fold and degrade proteins whose excess presence in the cell interferes with essential cellular processes. Thus, aneuploidy leads to an increased burden on the protein quality control pathways, an increased need for energy and contributes to the cell proliferation defect seen in aneuploids.

This hypothesis focuses our attention on the effects of aneuploidy on the protein quality control pathways of the cell, such as chaperone-mediated folding pathways and proteasomal degradation. Indeed we found that compromising proteasome function is deleterious in aneuploid cells (Torres et al., 2010).

Identification of genes that enhance or suppress the adverse effects of aneuploidy

Our results demonstrate that aneuploidy causes a decrease in cellular fitness, yet cancer cells show high proliferative potential despite sever karyotypic abnormalities. We hypothesize that genetic alterations exist that allow cancer cells to tolerate the adverse effects of aneuploidy. To probe this question, we identified aneuploid yeast strains with improved proliferative abilities. Their molecular characterization revealed strain-specific genetic alterations as well as mutations shared between different aneuploid strains. Among the latter, a loss of function mutation in the gene encoding the deubiquitinating enzyme UBP6 improves growth rates in four different aneuploid yeast strains by attenuating the changes in intracellular protein composition caused by aneuploidy. Our results demonstrate the existence of aneuploidy-tolerating mutations that improve the fitness of multiple different aneuploidies and highlight the importance of ubiquitin-proteasomal degradation in suppressing the adverse effects of aneuploidy (Torres et al., 2010). Currently, we are investigating how the other genetic alterations that we identified cause growth improvement in aneuploid cells. We also seek genetic alterations that cause synthetic lethality with the aneuploid state by probing the yeast deletion collection.

References

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