The Multiple Roles of Cohesin in Meiotic Chromosome Morphogenesis and Pairing

Sister chromatid cohesion, mediated by cohesin complexes, is laid down during DNA replication and is essential for the accurate segregation of chromosomes. Previous studies indicated that, in addition to their cohesion function, cohesins are essential for completion of recombination, pairing, meiotic chromosome axis formation, and assembly of the synaptonemal complex (SC). Using mutants in the cohesin subunit Rec8, in which phosphorylated residues were mutated to alanines, we show that cohesin phosphorylation is not only important for cohesin removal, but that cohesin's meiotic prophase functions are distinct from each other. We find pairing and SC formation to be dependent on Rec8, but independent of the presence of a sister chromatid and hence sister chromatid cohesion. We identified mutations in REC8 that differentially affect Rec8's cohesion, pairing, recombination, chromosome axis and SC assembly function. These findings define Rec8 as a key determinant of meiotic chromosome morphogenesis and a central player in multiple meiotic events.     

Meiotic Chromosome Pairing in Triploid and Tetraploid Saccharomyces Cerevisiae

Meiotic chromosome pairing in isogenic triploid and tetraploid strains of yeast and the consequences of polyploidy on meiotic chromosome segregation are studied. Synaptonemal complex formation at pachytene was found to be different in the triploid and in the tetraploid. In the triploid, triple-synapsis, that is, the connection of three homologues at a given site, is common. It can even extend all the way along the chromosomes. In the tetraploid, homologous chromosomes mostly come in pairs of synapsed bivalents. Multiple synapsis, that is, synapsis of more than two homologues in one and the same region, was virtually absent in the tetraploid. About five quadrivalents per cell occurred due to the switching of pairing partners. From the frequency of pairing partner switches it can be deduced that in most chromosomes synapsis is initiated primarily at one end, occasionally at both ends and rarely at an additional intercalary position. In contrast to a considerably reduced spore viability (~40%) in the triploid, spore viability is only mildly affected in the tetraploid. The good spore viability is presumably due to the low frequency of quadrivalents and to the highly regular 2:2 segregation of the few quadrivalents that do occur. Occasionally, however, quadrivalents appear to be subject to 3:1 nondisjunction that leads to spore death in the second generation.     

植物减数分裂染色体配对与染色体组分析的研究进展

简要介绍了植物减数分裂染色体配对研究.综述了减数分裂染色体配对研究在鉴定异源易位系、确定多倍体物种类型、分析物种间亲缘关系和物种的染色体组来源及探讨杂种不育的细胞遗传学机制等诸多方面的应用进展.分析了影响染色体配对的主要因素,如配对控制体系、遗传背景和外界环境条件等,并展望了染色体配对研究与其他技术结合在染色体组分析中的应用前景.     

Precocious Meiotic Centromere Separation of a Novel Yeast Chromosome

In Saccharomyces cerevisiae, a reciprocal translocation between chromosome II and a linear plasmid carrying a centromere (CEN6) has split chromosome II into two fragments: one, approximately 530 kilobase pairs (kbp) in size, has the left arm and part of the right arm of chromosome II; the other, a telocentric fragment approximately 350 kbp in size, has CEN6 and the rest of the right arm of chromosome II. A cross of this yeast strain with a strain containing a complete chromosome II exhibits a high frequency of precocious centromere separation (separation of sister chromatids during meiosis I) of the telocentric fragment. Precocious centromere separation is not due to the position of the centromere per se, since diploids that are homozygous for both fragments of chromosome II segregate the telocentric fragment with normal meiotic behavior. The precocious centromere separation described here differs from previously described examples in that pairing and synapsis of this telocentric chromosome seem to be normal. One model of how centromeres function in meiosis is that replication of the centromere is delayed until the second meiotic division. Data presented in this paper indicate that replication of the centromere is complete before the first meiotic division. The precocious separation of the centromere described here may be due to improper synapsis of sequences flanking the centromere.     

Roles of Hop1 and Mek1 in Meiotic Chromosome Pairing and Recombination Partner Choice in Schizosaccharomyces pombe

Synaptonemal complex (SC) proteins Hop1 and Mek1 have been proposed to promote homologous recombination in meiosis of Saccharomyces cerevisiae by establishment of a barrier against sister chromatid recombination. Therefore, it is interesting to know whether the homologous proteins play a similar role in Schizosaccharomyces pombe. Unequal sister chromatid recombination (USCR) was found to be increased in hop1 and mek1 single and double deletion mutants in assays for intrachromosomal recombination (ICR). Meiotic intergenic (crossover) and intragenic (conversion) recombination between homologous chromosomes was reduced. Double-strand break (DSB) levels were also lowered. Notably, deletion of hop1 restored DSB repair in rad50S meiosis. This may indicate altered DSB repair kinetics in hop1 and mek1 deletion strains. A hypothesis is advanced proposing transient inhibition of DSB processing by Hop1 and Mek1 and thus providing more time for repair by interaction with the homologous chromosome. Loss of Hop1 and Mek1 would then result in faster repair and more interaction with the sister chromatid. Thus, in S. pombe meiosis, where an excess of sister Holliday junction over homologous Holliday junction formation has been demonstrated, Hop1 and Mek1 possibly enhance homolog interactions to ensure wild-type level of crossover formation rather than inhibiting sister chromatid interactions.Sexual reproduction in eukaryotes involves formation of haploid gametes from diploid cells by one round of DNA replication, pairing of the homologous chromosomes, and recombination and then by the two meiotic divisions (53). In fungi the gametes differentiate into haploid spores, which germinate to form vegetative cells. Crossover (CO) formation between homologous chromosomes and DNA repair processes between sister chromatids are required for spore viability (10, 55, 58).In vegetative cells homologous recombination (HR) is important for repair of DNA damage and stalled replication forks, with the sister chromatid as the preferred partner (28). Many of the enzymes involved in mitotic HR also contribute to meiotic recombination. In addition, meiosis-specific cytological structures and enzymes enhance recombination frequency (meiotic induction) and shift partner preference from sister chromatids to homologous chromosomes (3, 47, 64, 74). In detail the steps of HR vary between different types of sequence organization (allelic versus sister versus ectopic), between different types of DNA damage, between meiotic and mitotic cells, and between species (10, 55, 58).Meiotic recombination, including CO formation, is initiated by DNA double-strand breaks (DSBs). In Saccharomyces cerevisiae and other eukaryotes, DSBs are formed by Spo11. Many cofactors are required (29). The Schizosaccharomyces pombe homolog is Rec12, also requiring auxiliary factors whose elimination leads to loss of meiotic DSB formation (12). The 5′ single-strand ends at DSBs are processed by nucleases. In S. cerevisiae the MRX complex made up by the proteins Rad50, Mre11, and Xrs2 is required for this resection, as well as for DSB formation. The corresponding MRN complex of S. pombe (Rad50, Rad32, and Nbs1) is not required for DSB formation but is essential for DSB repair (43, 72). Deletion of rad50, rad32, or ctp1 (homologous to SAE2/COM1 in S. cerevisiae and CtIP in humans) leads to very low spore viability. These proteins are also essential for DSB processing (23, 24, 32, 43, 60, 62).Free DNA 3′ ends at DSBs are recruited for invasion of a sister or homologous chromatid by the strand transfer proteins Rad51 and Dmc1, again involving many accessory proteins (16). This results in the central intermediates of HR: heteroduplex DNA consisting of single strands originating from different chromatids and Holliday junctions (HJs). In S. cerevisiae HJs form preferably between homologs with a two- to sixfold excess over intersister HJs (64). Surprisingly, meiotic HJs form with about a fourfold excess between sisters in S. pombe (11). Eventually the intermediates are resolved into crossover (CO) and noncrossover (NCO) events. COs show exchange of the flanking sequences of the two chromatids involved and usually carry a patch of conversion (unilateral transfer of DNA sequences from one chromatid to its interacting partner) near the DSB site. NCOs are conversion events without associated COs (22). In S. pombe loss of core HR functions leads to very low spore viability: deletion of rad51 but not of dmc1 (20), double mutation of rad54 and rdh54 (7), inactivation of the endonuclease activity encoded by mus81 and eme1 (5, 52), and combined deletion of rad22 and rti1 (homologs of RAD52 of S. cerevisiae). But, differently from the other core functions, Rad22 and Rti1 are not required for CO and NCO (50).Early in meiotic prophase of many eukaryotes, axial elements (called lateral elements in later stages) form along sister chromatids, and pairing of homologous chromosomes is initiated, leading to juxtaposition of the homologous chromosomes along their whole length in the synaptonemal complex (SC) (54). In S. pombe no SC is formed, but linear elements (LEs), resembling axial elements of other eukaryotes, are formed. LEs do not form continuously along the chromosomes (1) but load the proteins Rec10, Hop1, and Mek1 (36, 44, 57), which are homologs of, or at least related, to the S. cerevisiae proteins Red1, Hop1, and Mek1, respectively, localizing to axial/lateral elements (2, 67). Hop1 carries a HORMA domain, also present in proteins associating with axial elements and regulating the progress of recombination in higher eukaryotes: Arabidopsis thaliana (61), Caenorhabditis elegans (9, 41), and mammals (18).In S. cerevisiae localization of Hop1 and Mek1 (meiosis-specific protein kinase) to axial elements is dependent on Red1 (2, 67). Mutation of the three S. cerevisiae genes results in reduction of DSB formation, CO and conversion frequencies, and spore viability (26, 31, 59). Direct comparison of unequal sister chromatid recombination (USCR) frequencies in an assay excluding the scoring of intrachromatid recombination (ICR) revealed no increase in the hop1 null mutant but about fourfold increases in the red1 and mek1 null mutants (69). The S. cerevisiae Hop1, Red1, and Mek1 proteins are involved in biasing meiotic DSB repair to occur between homologous chromosomes rather than between sister chromatids (47). Activated Mek1 kinase is required for the inhibition of sister chromatid-mediated DSB repair by Rad51, when the DMC1 gene is deleted and the meiotic recombination checkpoint is activated (4, 27, 38, 47). For Mek1 activation, phosphorylation of Hop1 by the Mec1/Tel1 kinases is also required (6).Less is known about the S. pombe proteins. Hop1 of S. pombe was identified as a nonsignificant hit by sequence comparison with full-length S. cerevisiae Hop1 and contains an N-terminal HORMA domain and a central zinc finger motif like Hop1 in S. cerevisiae. In addition they share a short homology block toward the C terminus (36). The Mek1 protein of S. pombe shares 34% identity and 54% similarity with its S. cerevisiae counterpart along the whole sequence. It contains an FHA domain in the N-terminal part like the other members of its family of checkpoint kinases and is involved in regulation of the meiotic cell cycle (57). Hop1 and Mek1 are strongly expressed in meiosis but not expressed or only slightly expressed in vegetative cells (42, 57). In prophase both proteins localize to LEs as defined by colocalization with the LE component Rec10 (36). Deletion of the distant RED1 homolog rec10 abolishes LE formation (36, 44) and strongly reduces meiotic recombination (17, 70). Rec10, but not Hop1 and Mek1, is required for localization of Rec7 (a distant homolog of S. cerevisiae Rec114) to meiotic chromosomes (34). Rec7 and Rec10 are required for Rec12 activity (12, 29).Obtaining information on the functions of Hop1 and Mek1 in S. pombe was the aim of the work presented here, especially on their possible roles in homolog versus sister discrimination for DSB repair. Deletion mutants have been studied with respect to spore viability and the frequencies of CO and conversion. They have also been assessed for genetic recombination events between sister chromatids in the known PS1 assay (63) and the newly developed VL1 assay (for details, see Fig. ​Fig.3).3). Physical analysis of DSB formation and repair has been performed in meiotic time course experiments. It is proposed that S. pombe Hop1 and Mek1 are promoting interactions between homologous chromosomes rather than inhibiting interactions between sister chromatids.Open in a separate windowFIG. 3.PS1 and VL1 assay systems for intrachromosomal recombination. Strains with constructs carrying repeated DNA sequences have been assayed for prototroph formation either by intrachromatid recombination (ICR, yielding prototrophs only in PS1) or by unequal sister chromatid recombination (USCR, in PS1 and VL1). Crosses of the constructs were performed with strains carrying a deletion of the ade6 gene to exclude other homologous recombination events. (A) The PS1 assay involves copies of the ade6 gene inactivated by either the hot spot mutation M26 or the mutation 469. The repeated sequences are separated by the ura4⁺ marker (63). ICR (left) or USCR (right) between the repeated sequences can lead to formation of adenine prototrophs that have lost the ura4⁺ marker by crossover (CO) or single-strand annealing (SSA) events. Adenine prototrophs maintaining the ura4⁺ marker can derive from noncrossover (NCO) events. Both types of pairing may lead to CO or NCO products. (B) The newly constructed VL1 assay (see the supplemental material) involves different truncations of the ade6 gene separated by the hyg^R marker (also called hphMX6), conferring hygromycin resistance. The left truncation carries a 3′ portion of ade6; the right truncation carries a 5′ portion of ade6. While the gray parts of the truncations are not overlapping, the white sections of 500-bp length are of almost identical sequence, allowing for homologous pairing. CO and SSA products resulting from ICR retain only the central portion of ade6 and remain auxotrophic. Adenine prototrophic CO and NCO products resulting from USCR both retain hygromycin resistance. Note that NCO events may arise through loop formation of one sister chromatid and pairing with a single block (500 bp) of the repeated ade6 sequence (39).     

Two Types of Sites Required for Meiotic Chromosome Pairing in Caenorhabditis Elegans

Previous studies have shown that isolated portions of Caenorhabditis elegans chromosomes are not equally capable of meiotic exchange. These results led to the proposal that a homolog recognition region (HRR), defined as the region containing those sequences enabling homologous chromosomes to pair and recombine, is localized near one end of each chromosome. Using translocations and duplications we have localized the chromosome I HRR to the right end. Whereas the other half of chromosome I did not confer any ability for homologs to pair and recombine, deficiencies in this region dominantly suppressed recombination to the middle of the chromosome. These deletions may have disrupted pairing mechanisms that are secondary to and require an HRR. Thus, the processes of pairing and recombination appear to utilize at least two chromosomal elements, the HRR and other pairing sites. For example, terminal sequences from other chromosomes increase the ability of free duplications to recombine with their normal homologs, suggesting that telomere-associated sequences, homologous or nonhomologous, play a role in facilitating meiotic exchange. Recombination can also initiate at internal sites separated from the HRR by chromosome rearrangement, such as deletions of the unc-54 region of chromosome I. When crossing over was suppressed in a region of chromosome I, compensatory increases were observed in other regions. Thus, the presence of the HRR enabled recombination to occur but did not determine the distribution of the crossover events. It seems most likely that there are multiple initiation sites for recombination once homolog recognition has been achieved.     

Meiotic Crossing over between Nonhomologous Chromosomes Affects Chromosome Segregation in Yeast

Meiotic recombination between artificial repeats positioned on nonhomologous chromosomes occurs efficiently in the yeast Saccharomyces cerevisiae. Both gene conversion and crossover events have been observed, with crossovers yielding reciprocal translocations. In the current study, 5.5-kb ura3 repeats positioned on chromosomes V and XV were used to examine the effect of ectopic recombination on meiotic chromosome segregation. Ura(+) random spores were selected and gene conversion vs. crossover events were distinguished by Southern blot analysis. Approximately 15% of the crossover events between chromosomes V and XV were associated with missegregation of one of these chromosomes. The missegregation was manifest as hyperploid spores containing either both translocations plus a normal chromosome, or both normal chromosomes plus one of the translocations. In those cases where it could be analyzed, missegregation occurred at the first meiotic division. These data are discussed in terms of a model in which ectopic crossovers compete efficiently with normal allelic crossovers in directing meiotic chromosome segregation.     

Centromere Pairing in Early Meiotic Prophase Requires Active Centromeres and Precedes Installation of the Synaptonemal Complex in Maize

Pairing of homologous chromosomes in meiosis is critical for their segregation to daughter cells. In most eukaryotes, clustering of telomeres precedes and facilitates chromosome pairing. In several species, centromeres also form pairwise associations, known as coupling, before the onset of pairing. We found that, in maize (Zea mays), centromere association begins at the leptotene stage and occurs earlier than the formation of the telomere bouquet. We established that centromere pairing requires centromere activity and the sole presence of centromeric repeats is not sufficient for pairing. In several species, homologs of the ZIP1 protein, which forms the central element of the synaptonemal complex in budding yeast (Saccharomyces cerevisiae), play essential roles in centromere coupling. However, we found that the maize ZIP1 homolog ZYP1 installs in the centromeric regions of chromosomes after centromeres form associations. Instead, we found that maize STRUCTURAL MAINTENANCE OF CHROMOSOMES6 homolog forms a central element of the synaptonemal complex, which is required for centromere associations. These data shed light on the poorly understood mechanism of centromere interactions and suggest that this mechanism may vary somewhat in different species.     

Multifunctional Centromere Binding Factor 1 Is Essential for Chromosome Segregation in the Human Pathogenic Yeast Candida glabrata

The CBF1 (centromere binding factor 1) gene of Candida glabrata was cloned by functional complementation of the methionine biosynthesis defect of a Saccharomyces cerevisiae cbf1 deletion mutant. The C. glabrata-coded protein, CgCbf1, contains a basic-helix-loop-helix leucine zipper domain and has features similar to those of other budding yeast Cbf1 proteins. CgCbf1p binds in vitro to the centromere DNA element I (CDEI) sequence GTCACATG with high affinity (0.9 x 10(9) M(-1)). Bandshift experiments revealed a pattern of protein-DNA complexes on CgCEN DNA different from that known for S. cerevisiae. We examined the effect of altering the CDEI binding site on CEN plasmid segregation, using a newly developed colony-sectoring assay. Internal deletion of the CDEI binding site led only to a fivefold increase in rates of plasmid loss, indicating that direct binding of Cbf1p to the centromere DNA is not required for full function. Additional deletion of sequences to the left of CDEI, however, led to a 70-fold increase in plasmid loss rates. Deletion of the CBF1 gene proved to be lethal in C. glabrata. C. glabrata cells containing the CBF1 gene under the influence of a shutdown promoter (tetO-ScHOP) arrested their growth after 5 h of cultivation in the presence of the reactive drug doxycycline. DAPI (4',6'-diamidino-2-phenylindole) staining of the arrested cells revealed a significant increase in the number of large-budded cells with single nuclei, 2C DNA content, and short spindles, indicating a defect in the G(2)/M transition of the cell cycle. Thus, we conclude that Cbf1p is required for chromosome segregation in C. glabrata.     

The Role of Topoisomerase II in Meiotic Chromosome Condensation and Segregation in Schizosaccharomyces pombe

Topoisomerase II is able to break and rejoin double-strand DNA. It controls the topological state and forms and resolves knots and catenanes. Not much is known about the relation between the chromosome segregation and condensation defects as found in yeast top2 mutants and the role of topoisomerase II in meiosis. We studied meiosis in a heat-sensitive top2 mutant of Schizosaccharomyces pombe. Topoisomerase II is not required until shortly before meiosis I. The enzyme is necessary for condensation shortly before the first meiotic division but not for early meiotic prophase condensation. DNA replication, prophase morphology, and dynamics of the linear elements are normal in the top2 mutant. The top2 cells are not able to perform meiosis I. Arrested cells have four spindle pole bodies and two spindles but only one nucleus, suggesting that the arrest is nonregulatory. Finally, we show that the arrest is partly solved in a top2 rec7 double mutant, indicating that topoisomerase II functions in the segregation of recombined chromosomes. We suggest that the inability to decatenate the replicated DNA is the primary defect in top2. This leads to a loss of chromatin condensation shortly before meiosis I, failure of sister chromatid separation, and a nonregulatory arrest.     

Arabidopsis Cell Division Cycle 20.1 Is Required for Normal Meiotic Spindle Assembly and Chromosome Segregation

Cell division requires proper spindle assembly; a surveillance pathway, the spindle assembly checkpoint (SAC), monitors whether the spindle is normal and correctly attached to kinetochores. The SAC proteins regulate mitotic chromosome segregation by affecting CDC20 (Cell Division Cycle 20) function. However, it is unclear whether CDC20 regulates meiotic spindle assembly and proper homolog segregation. Here, we show that the Arabidopsis thaliana CDC20.1 gene is indispensable for meiosis and male fertility. We demonstrate that cdc20.1 meiotic chromosomes align asynchronously and segregate unequally and the metaphase I spindle has aberrant morphology. Comparison of the distribution of meiotic stages at different time points between the wild type and cdc20.1 reveals a delay of meiotic progression from diakinesis to anaphase I. Furthermore, cdc20.1 meiocytes exhibit an abnormal distribution of a histone H3 phosphorylation mark mediated by the Aurora kinase, providing evidence that CDC20.1 regulates Aurora localization for meiotic chromosome segregation. Further evidence that CDC20.1 and Aurora are functionally related was provided by meiosis-specific knockdown of At-Aurora1 expression, resulting in meiotic chromosome segregation defects similar to those of cdc20.1. Taken together, these results suggest a critical role for CDC20.1 in SAC-dependent meiotic chromosome segregation.     

A Role for the CAL1-Partner Modulo in Centromere Integrity and Accurate Chromosome Segregation in Drosophila

The relationship between the nucleolus and the centromere, although documented, remains one of the most elusive aspects of centromere assembly and maintenance. Here we identify the nucleolar protein, Modulo, in complex with CAL1, a factor essential for the centromeric deposition of the centromere-specific histone H3 variant, CID, in Drosophila. Notably, CAL1 localizes to both centromeres and the nucleolus. Depletion of Modulo, by RNAi, results in defective recruitment of newly-synthesized CAL1 at the centromere. Furthermore, depletion of Modulo negatively affects levels of CID at the centromere and results in chromosome missegregation. Interestingly, examination of Modulo localization during mitosis reveals it localizes to the chromosome periphery but not the centromere. Combined, the data suggest that rather than a direct regulatory role at the centromere, it is the nucleolar function of modulo which is regulating the assembly of the centromere by directing the localization of CAL1. We propose that a functional link between the nucleolus and centromere assembly exists in Drosophila, which is regulated by Modulo.     

X-4 Translocations and Meiotic Drive in Drosophila melanogaster Males: Role of Sex Chromosome Pairing

Males carrying certain X-4 translocations exhibit strongly skewed sperm recovery ratios. The X^P4^D half of the translocation disjoins regularly from the Y chromosome and the 4^PX^D half disjoins regularly from the normal 4. Yet the smaller member of each bivalent is recovered in excess of its pairing partner, apparently due to differential gametic lethality. Chromosome recovery probabilities are multiplicative; the viability of each genotype is the product of the recovery probability of its component chromosomes. Meiotic drive can also be caused by deficiency for X heterochromatin. In(1)sc^4Lsc^8R males show the same size dependent chromosome recoveries and multiplicative recovery probabilities found in T(1;4)B^S males. Meiotic drive in In(1)sc^4Lsc^8R males has been shown to be due to X-Y pairing failure. Although pairing is regular in the T(X;4) males, the striking phenotypic parallels suggest a common explanation. The experiments described below show that the two phenomena are, in fact, one and the same. X-4 translocations are shown to have the same effect on recovery of independently assorting chromosomes as does In(1)sc^4Lsc^8R. Addition of pairing sites to the 4^PX^D half of the translocation eliminates drive. A common explanation—failure of the distal euchromatic portion of the X chromosome to participate in X:Y meiotic pairing—is suggested as the cause for drive. The effect of X chromosome breakpoint on X-4 translocation induced meiotic drive is investigated. It is found that translocations with breakpoints distal to 13C on the salivary map do not cause drive while translocations broken proximal to 13C cause drive. The level of drive is related to the position of the breakpoint—the more proximal the breakpoint the greater the drive.     

Histone Variant H2A.Z Regulates Centromere Silencing and Chromosome Segregation in Fission Yeast

The incorporation of histone variant H2A.Z into nucleosomes plays essential roles in regulating chromatin structure and gene expression. A multisubunit complex containing chromatin remodeling protein Swr1 is responsible for the deposition of H2A.Z in budding yeast and mammals. Here, we show that the JmjC domain protein Msc1 is a novel component of the fission yeast Swr1 complex and is required for Swr1-mediated incorporation of H2A.Z into nucleosomes at gene promoters. Loss of Msc1, Swr1, or H2A.Z results in loss of silencing at centromeres and defective chromosome segregation, although centromeric levels of CENP-A, a centromere-specific histone H3 variant that is required for setting up the chromatin structure at centromeres, remain unchanged. Intriguingly, H2A.Z is required for the expression of another centromere protein, CENP-C, and overexpression of CENP-C rescues centromere silencing defects associated with H2A.Z loss. These results demonstrate the importance of H2A.Z and CENP-C in maintaining a silenced chromatin state at centromeres.     

Variation in Crossover Frequencies Perturb Crossover Assurance Without Affecting Meiotic Chromosome Segregation in Saccharomyces cerevisiae

The segregation of homologous chromosomes during the Meiosis I division requires an obligate crossover per homolog pair (crossover assurance). In Saccharomyces cerevisiae and mammals, Msh4 and Msh5 proteins stabilize Holliday junctions and its progenitors to facilitate crossing over. S. cerevisiae msh4/5 hypomorphs that reduce crossover levels up to twofold at specific loci on chromosomes VII, VIII, and XV without affecting homolog segregation were identified recently. We use the msh4–R676W hypomorph to ask if the obligate crossover is insulated from variation in crossover frequencies, using a S. cerevisiae S288c/YJM789 hybrid to map recombination genome-wide. The msh4–R676W hypomorph made on average 64 crossovers per meiosis compared to 94 made in wild type and 49 in the msh4Δ mutant confirming the defect seen at individual loci on a genome-wide scale. Crossover reductions in msh4–R676W and msh4Δ were significant across chromosomes regardless of size, unlike previous observations made at specific loci. The msh4–R676W hypomorph showed reduced crossover interference. Although crossover reduction in msh4–R676W is modest, 42% of the four viable spore tetrads showed nonexchange chromosomes. These results, along with modeling of crossover distribution, suggest the significant reduction in crossovers across chromosomes and the loss of interference compromises the obligate crossover in the msh4 hypomorph. The high spore viability of the msh4 hypomorph is maintained by efficient segregation of the natural nonexchange chromosomes. Our results suggest that variation in crossover frequencies can compromise the obligate crossover and also support a mechanistic role for interference in obligate crossover formation.     

A Centromere DNA-binding Protein from Fission Yeast Affects Chromosome Segregation and Has Homology to Human CENP-B

Genetic and biochemical strategies have been used to identify Schizosaccharomyces pombe proteins with roles in centromere function. One protein, identified by both approaches, shows significant homology to the human centromere DNA-binding protein, CENP-B, and is identical to Abp1p (autonomously replicating sequence-binding protein 1) (Murakami, Y., J.A. Huberman, and J. Hurwitz. 1996. Proc. Natl. Acad. Sci. USA. 93:502–507). Abp1p binds in vitro specifically to at least three sites in centromeric central core DNA of S. pombe chromosome II (cc2). Overexpression of abp1 affects mitotic chromosome stability in S. pombe. Although inactivation of the abp1 gene is not lethal, the abp1 null strain displays marked mitotic chromosome instability and a pronounced meiotic defect. The identification of a CENP-B–related centromere DNA-binding protein in S. pombe strongly supports the hypothesis that fission yeast centromeres are structurally and functionally related to the centromeres of higher eukaryotes.     

Temporal Characterization of Homology-Independent Centromere Coupling in Meiotic Prophase

Background

Over the past thirty years several reports of the pairing or association of non-homologous centromeres during meiotic prophase have appeared in the literature. Recently, the homology-independent pairwise association of centromeres, termed centromere coupling, was also reported in budding yeast. It seems paradoxical that centromeres would pair with non-homologous partners during a process intended to align homologous chromosomes, yet the conservation of this phenomenon across a wide range of species suggests it may play an important role in meiosis.

Principal Findings

To better define the role of this phenomenon in budding yeast, experiments were preformed to place centromere coupling within the context of landmark meiotic events. Soon after the initiation of the meiotic program, centromeres were found to re-organize from a single cluster into non-homologous couples. Centromere coupling is detected as soon as chromosome replication is finished and persists while the recombination protein Dmc1 is loaded onto the chromosomes, suggesting that centromere coupling persists through the time of double strand break formation. In the absence of the synaptonemal complex component, Zip1, centromere coupling was undetectable, at all times examined, confirming the essential role of this protein on this process. Finally, the timely release of centromere coupling depends on the recombination-initiating enzyme, Spo11, suggesting a connection between events in homologous pairing/recombination and the regulation of centromere coupling.

Conclusions

Based on our results we propose a role for centromere coupling in blocking interactions between homologous centromeres as recombination initiation is taking place.     

Rejuvenation of Meiotic Cohesion in Oocytes during Prophase I Is Required for Chiasma Maintenance and Accurate Chromosome Segregation

Chromosome segregation errors in human oocytes are the leading cause of birth defects, and the risk of aneuploid pregnancy increases dramatically as women age. Accurate segregation demands that sister chromatid cohesion remain intact for decades in human oocytes, and gradual loss of the original cohesive linkages established in fetal oocytes is proposed to be a major cause of age-dependent segregation errors. Here we demonstrate that maintenance of meiotic cohesion in Drosophila oocytes during prophase I requires an active rejuvenation program, and provide mechanistic insight into the molecular events that underlie rejuvenation. Gal4/UAS inducible knockdown of the cohesion establishment factor Eco after meiotic S phase, but before oocyte maturation, causes premature loss of meiotic cohesion, resulting in destabilization of chiasmata and subsequent missegregation of recombinant homologs. Reduction of individual cohesin subunits or the cohesin loader Nipped B during prophase I leads to similar defects. These data indicate that loading of newly synthesized replacement cohesin rings by Nipped B and establishment of new cohesive linkages by the acetyltransferase Eco must occur during prophase I to maintain cohesion in oocytes. Moreover, we show that rejuvenation of meiotic cohesion does not depend on the programmed induction of meiotic double strand breaks that occurs during early prophase I, and is therefore mechanistically distinct from the DNA damage cohesion re-establishment pathway identified in G2 vegetative yeast cells. Our work provides the first evidence that new cohesive linkages are established in Drosophila oocytes after meiotic S phase, and that these are required for accurate chromosome segregation. If such a pathway also operates in human oocytes, meiotic cohesion defects may become pronounced in a woman''s thirties, not because the original cohesive linkages finally give out, but because the rejuvenation program can no longer supply new cohesive linkages at the same rate at which they are lost.