Reconstruction of the kinetochore: a prelude to meiosis

In eukaryotic organisms, chromosomes are spatially organized within the nucleus. Such nuclear architecture provides a physical framework for the genetic activities of chromosomes, and changes its functional organization as the cell moves through the phases of the cell cycle. The fission yeast Schizosaccharomyces pombe provides a striking example of nuclear reorganization during the transition from mitosis to meiosis. In this organism, centromeres remain clustered at the spindle-pole body (SPB; a centrosome-equivalent structure in fungi) during mitotic interphase. In contrast, during meiotic prophase, centromeres dissociate from the SPB and telomeres cluster to the SPB. Recent studies revealed that this repositioning of chromosomes is regulated by mating pheromone signaling. Some centromere proteins disappear from the centromere in response to mating pheromone, leading to dissociation of centromeres from the SPB. Interestingly, mating pheromone signaling is also required for monopolar orientation of the kinetochore which is crucial for proper segregation of sister chromatids during meiosis. When meiosis is induced in the absence of mating pheromone signaling, aberrant chromosome behaviors are observed: the centromere proteins remain at the centromere; the centromere remains associated with the SPB; and sister chromatids segregate precociously in the first meiotic division. These aberrant chromosome behaviors are all normalized by activating the mating pheromone signaling pathway. Thus, action of mating pheromone on the centromere is important for coherent behavior of chromosomes in meiosis. Here we discuss repositioning and reconstruction of the centromere during the transition from mitosis to meiosis, and highlight its significance for proper progression of meiosis.


Background
Eukaryotic chromosomes are spatially organized within the nucleus. While such nuclear architecture provides a physical framework for the genetic activities of chromosomes, this framework however is dynamic, able to change its functional organization during the cell cycle or developmental stages. Local chromatin structures change as chromosomes undergo processes such as replication, transcription, recombination and repair. During chromosome segregation, a specialized structure called kineto-chore is formed on the centromeric DNA. Global organization of chromosomes within the nucleus can also change in association with their activities. A prominent example of reconstruction of the nuclear and chromosomal frameworks is observed during the transition from mitosis to meiosis.
Meiosis is a process that produces haploid gametes from parental diploid germ cells in sexually reproducing organisms. In this process, a single round of chromosome rep-lication is followed by two rounds of chromosome segregation. A characteristic feature of meiosis is the behavior of sister chromatids in the first division (meiosis I). In meiosis I, homologous chromosomes segregate while sister chromatids remain held together. Sister chromatids then segregate in the second division (meiosis II). Reductional segregation of homologous chromosomes in meiosis I requires a recombination-mediated physical link between a pair of homologous chromosomes. This regulated segregation of homologous chromosomes and sister chromatids is achieved by selective use of monopolar and bipolar structures of the kinetochore, which provide a structural basis for monopolar and bipolar attachment of the spindle, respectively. Monopolar attachment of the spindle to sister kinetochores leads to movement of sister chromatids to the same pole in meiosis I. Bipolar attachment of the spindle to sister kinetochores causes segregation to the opposite poles in meiosis II (as well as in mitosis).
Meiotic reorganization of the chromosome is associated with assembly of the meiotic chromatin proteins that replace the mitotic counterparts. Cohesin proteins are among such proteins, and affect structures and segregation patterns of chromosomes [1,2]. The position of centromeres in the nucleus also changes during the transition from mitosis to meiosis. In mitotic interphase, chromosomes are organized in a polarized orientation, with centromeres confined near the centrosome and telomeres positioned at the opposite side of the nucleus (often called the Rabl orientation). In contrast, in meiotic prophase, telomeres become clustered beneath the nuclear envelope near the centrosome; this arrangement of chromosomes bundled at the telomeres is often called the bouquet arrangement because of its shape (reviewed in [3]). The fission yeast Schizosaccharomyces pombe provides a striking example of the repositioning of centromeres and telomeres upon entering meiosis. Studies on regulatory mechanisms of this phenomenon have elucidated important aspects of centromere reconstruction that may influence the progression of meiosis.

The fission yeast meiosis: an overview
The fission yeast S. pombe is a unicellular eukaryotic organism. This yeast usually grows as haploid cells with a genome of three chromosomes. Meiotic processes of S. pombe are summarized in Figure 1. When cells are starved for nitrogen sources, they arrest their cell cycle in G1 phase and secrete mating pheromone. Cells of the opposite mating type (h + and h -) exchange mating pheromones, and subsequently conjugate to form a diploid zygote. In the diploid zygote, h + and hhaploid nuclei fuse to form a diploid nucleus. Immediately after nuclear fusion the nucleus elongates and moves back and forth between the cell poles. The elongated nucleus is often called the "horsetail" nucleus because of its shape. Meiotic DNA synthesis and recombination occur in this horsetail nucleus. After nuclear movement, the cell undergoes meiotic divisions, and the nucleus is divided into four haploid nuclei. Each nucleus is encapsulated within a spore after meiosis II.
Significantly, centromeres and telomeres switch their positions in the nucleus upon entering meiosis [4,5]. Before meiosis, centromeres are clustered at the spindlepole body (SPB, an equivalent structure to the animal centrosome), and telomeres are attached to the nuclear mem- The meiotic process in S. pombe Figure 1 The meiotic process in S. pombe. When starved of nitrogen sources, haploid cells of the h + and hmating type are arrested in G1, and secrete the mating pheromone to the opposite mating type cells. Upon sensing the pheromone, the cell elongates toward the opposite mating type cell and the cells fuse with each other (conjugation), followed by fusion of the haploid nuclei. The fused diploid nucleus then elongates (known as the horsetail nucleus), and moves back and forth between the cell ends (meiotic prophase). Meiotic DNA replication occurs at the beginning of this period, yielding a nucleus with 4C DNA contents. After movement ceases, the horsetail nucleus becomes rounded at the center of the cell, and proceeds with the first and second meiotic divisions to create four 1C nuclei. brane, showing a typical Rabl orientation. In contrast, upon entering meiosis, telomeres cluster to the SPB and centromeres become dissociated from the SPB, showing the bouquet configuration ( Figure 2). In nuclear fusion, haploid nuclei approach each other, with the telomere-SPB cluster located at the leading edge of each nucleus. After nuclear fusion, telomeres remain clustered at the SPB located at the leading edge of the elongated nucleus moving between the poles. Repositioning of centromeres and telomeres is observed in meiotic cells in many eukaryotes from yeasts to plants and animals including humans [6][7][8][9].
Telomeres are clustered in response to mating pheromone signaling While telomere clustering in meiotic prophase has been observed in a wide variety of organisms, and its role in meiosis has been inferred (reviewed in [10]), analyses in S. pombe have demonstrated that telomere clustering and nuclear movement promote homologous chromosome pairing [11,12]. In S. pombe, telomere clustering is known to take place in response to mating pheromone [13], and recently the underlying molecular mechanism has been revealed [14]. S. pombe Taz1 (an ortholog of the human TRF1 and TRF2) binds directly to telomeric DNA, and Rap1 binds to the telomere through interaction with Taz1 [15][16][17]. During the mating pheromone response, meiosis-specific proteins Bqt1 and Bqt2 are expressed and the Bqt1/Bqt2 complex recruits Sad1 (an SPB component) to Rap1 at the telomere [14]. In the presence of Bqt1 and Bqt2, Sad1, which is usually localized at the SPB, becomes accumulated at the scattered telomeres to form multiple foci on the nuclear envelope, and subsequently the Sad1 foci converge to the SPB together with telomeres. In this process, Sad1 binds to another SPB component Kms1, which interacts with the dynein motor protein [18,19], and Sad1-bound telomeres are then clustered to the SPB through interaction with the dynein motor complex along microtubules. Horsetail nuclear movement is also mediated by cytoplasmic microtubules and the dynein motor complex [19][20][21]. Thus, telomeres are clustered to the SPB via the meiosis-specific connector proteins Bqt1 and Bqt2 in response to mating pheromone signaling, and remain connected to the SPB throughout the horsetail nuclear movement.

Centromeres are disassembled in response to mating pheromone signaling
Centromeres are separated from the SPB during meiotic prophase in S. pombe. Recent studies have revealed that a group of proteins disappear from the centromere during meiotic prophase, regulated by mating pheromone signaling [22,23]. In mitotic interphase, a number of proteins assemble at the centromere and connect the centromere to the SPB; when the cell enters mitosis, they form the kinetochore as an interface between the centromere and spindle microtubules. As in other eukaryotic organisms, the S. pombe kinetochore is composed of several subcomplexes of centromere proteins: three centromere subcomplexes the meiotic monopolar kinetochore, although this remains to be proven. , probably corresponding to S-phase similarly to Rec8 ( Figure 3B). The loading of Rec8 and Moa1 does not require mating pheromone signaling [23,26]. In addition, Rec8 and Moa1 can locate to the centromere when ectopically expressed in mitotic interphase cells [31,33], while the NMS complex remains bound to the centromere. Thus, the removal of the NMS complex may be unnecessary for loading of Rec8 and Moa1 to the centromere. Monopolar sister kinetochore formation further requires mating pheromone signaling, suggesting that centromere loading of Rec8 and Moa1 is necessary, but not sufficient for formation of the monopolar kinetochore. Additional alteration of the kinetochore may occur in response to the mating pheromone signaling.

Monopolar sister kinetochore is formed in response to mating pheromone signaling
Localization of another meiosis-specific protein, Sgo1, to the centromere is regulated by mating pheromone signaling. Sgo1 protects Rec8 at the centromere to maintain cohesion between sister kinetochores until meiosis II [34]. Sgo1 appears at the centromere during late-stage horsetail nuclear movement, as does the NMS complex [23] ( Figure 3B). Importantly, when meiosis is induced by pat1 mutation in the absence of mating pheromone signaling, loading of Sgo1 to the centromere is severely diminished [23]. This suggests that loading of Sgo1 is regulated downstream of mating pheromone signaling. Rec8 is expressed in pat1 mutation-induced meiosis in the absence of mating pheromone signaling as well as normal meiosis in wild type cells. Because Sgo1 is capable of localizing to centromeres in proliferating cells when ectopically coexpressed with Rec8 [34], diminished localization of Sgo1 in the pat1 mutant might be due to insufficient expression. Details of the mechanism of Sgo1 regulation by mating pheromone signaling are yet to be elucidated.

Regulatory pathways downstream of the mating pheromone
The previous sections describe the role of mating pheromone signaling in regulation of centromeres during meiosis. Moreover, ectopic activation of mating pheromone signaling can induce the normal process of meiosis. The mating pheromone signal is transmitted through a MAPK cascade, which consists of Byr2 (MAPKKK), Byr1   [14,44,45]. A more comprehensive search of factors downstream of mating pheromone signaling will lead to further understanding of meiotic regulation of the centromere.

Regulation of centromeres in other organisms
Several pieces of evidence for centromere-telomere repositioning and regulation of centromere proteins in the transition from mitosis to meiosis have been reported in other eukaryotes. In the budding yeast Saccharomyces cerevisiae, repositioning of chromosomes takes place during meiosis, although the telomere clustering is less prominent and shorter in time than that in S. pombe [8,46]. At the time of repositioning, one of the Ndc80 complex proteins, Nuf2, disappears from the centromere, suggesting that the repositioning probably involves reorganization of centromere proteins as in S. pombe [22]. In S. cerevisiae, proteins crucial for monopolar attachment of sister kinetochores to the spindle have been identified [47-53], but the correlation with spatial regulation of centromeres has not been examined. It is unlikely that the mating pheromone response regulates monopolar sister kinetochore formation because although the mating pheromone induces cell fusion, it does not induce meiosis directly in this organism.
In the nematode Caenorhabditis elegans, spatial reorganization of the nucleus occurs at the onset of meiotic prophase. Chromosomes asymmetrically cluster to one side of the nucleus, which in turn becomes crescentshaped [54], although it is unclear whether reorganization of chromosomes involves repositioning of centromeres and telomeres. In C. elegans, several centromere proteins such as Cenp-C, Him-10 and KNL-1 are localized only during chromosome segregation both in mitosis and meiosis [55][56][57][58][59]. Remarkably, histone H3 variant, Cenp-A, loses the localization selectively during meiotic prophase, while it remains at the centromere throughout the mitotic cell cycle [55,60]. It is not known, however, whether the disappearance of these proteins is related to meiotic reorganization of the nucleus and monopolar kinetochore formation in C. elegans.
In higher eukaryotes, reorganization of the nucleus occurs during meiosis [6]. A large number of kinetochore proteins, including homologues of S. pombe proteins, has been reported. However, it is largely unknown how the kinetochore proteins behave during meiosis in higher eukaryotes.

Conclusion
Regulation of meiosis is diverse among species, but the establishment of the monopolar sister kinetochore is a common and essential process in meiosis, as is the repositioning of chromosomes in meiotic prophase. Repositioning of chromosomes leading the bouquet formation promotes homologous chromosome pairing by bundling telomeres together. Although several studies indicate that repositioning of centromeres and the construction of monopolar sister kinetochores are correlated, these processes can be independently regulated in response to mating pheromone signaling ( Figure 5). Although a signal pathway may be different in yeasts and higher eukaryotes, formation of the monopolar sister kinetochore is obviously crucial for meiotic chromosome segregation. How meiosis-specific proteins interact with other centromere Schematic model of nuclear organization in S. pombe meiosis Figure 5 Schematic model of nuclear organization in S. pombe meiosis. (A) The nuclear organization induced by the pat1 mutation. When meiosis is induced by Pat1 inactivation in the absence of the mating pheromone signaling, telomeres (green) cluster at the SPB (red); however, centromeres (blue) remain associated with the SPB, and the sister kinetochores are formed in a bipolar orientation. In these cells, the NMS complex (yellow) remains at the centromere attached to the SPB. (B) Nuclear organization induced by mating pheromone signal activation. Pat1 is inactivated downstream in the mating pheromone response, leading to telomere clustering. Centromere dissociation from the SPB and formation of the monopolar sister kinetochores are regulated, probably by independent pathways, downstream of mating pheromone signaling. It remains unknown whether centromere reconstruction and/or repositioning contributes to monopolar kinetochore formation (arrow with question mark). proteins to establish monopolar sister kinetochores, and how these proteins are regulated remain to be elucidated. To address these questions, S. pombe provides a useful experimental system, as sister kinetochore formation can be manipulated in the presence or absence of mating pheromone signaling. Given that kinetochore components are highly conserved from yeasts to humans, we expect that analysis of the kinetochore and chromatin structures for monopolar attachment to the spindle will provide fundamental understanding of the mechanisms for ensuring the regulated segregation of homologous chromosomes and sister chromatids during meiosis.