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Dispatch R461 Meiosis: Step-by-step through sporulation Mary J. Clancy A transcription factor, Ndt80p, has been identified that has a critical role in the pathway that controls meiosis — sporulation — in budding yeast. Ndt80p coordinately controls genes that mediate spore formation and progression through the two meiotic divisions; it may also be a target of a checkpoint control. meiosis, called the prophase to metaphase I transition; those with DNA damage arrest in meiosis at the end of prophase, at a stage called pachytene. Results discussed below suggest that this checkpoint mechanism could be acting in part through the Ndt80p transcription factor to accomplish regulation of this critical step in meiosis. Address: Department of Biological Sciences, University of New Orleans, New Orleans, Louisiana 70148, USA. Temporal organization of sporulation in S. cerevisiae Current Biology 1998, 8:R461–R463 http://biomednet.com/elecref/09609822008R0461 © Current Biology Ltd ISSN 0960-9822 Meiotic cells, whatever their source, face some common general problems. One is to ensure the integrity of the genetic material during the potentially threatening process of meiosis, which includes extensive recombination and two rounds of meiotic division. Another is to provide good homes for the haploid nuclei generated by meiosis: gamete morphogenesis must follow the meiotic divisions to form the mature sperm, pollen or ascospores, as appropriate for the species in question. Finally, these two separate processes, meiosis and gametogenesis, must be physically and temporally coupled to each other. Recent work has illuminated some of the mechanisms used by the simple budding yeast, Saccharomyces cerevisiae, to ensure the safe passage of its genome through the varied events of meiosis and ascospore formation. One of these mechanisms, involving the transcription factor Ndt80p, ensures that the meiotic divisions and morphogenesis are initiated at the same time. The targets of Ndt80p are the ‘middle’ class of sporulation-specific genes [1]. The best known of these middle genes are part of the morphogenetic pathway that carves out the ascospores after meiosis is finished (reviewed in [2]), so their activation by Ndt80p initiates the spore formation process. The more surprising, and more recently recognized, members of the middle gene class include some that activate the meiotic division pathway. It turns out that the CLB genes, which encode the B-type cyclins [3], also respond to the Ndt80p transcription factor and can be regarded as middle sporulation genes [1]. Sporulation of diploid yeast cells is initiated by a simple nutritional shift. Shortly thereafter, the cells induce the ‘early’ wave of sporulation-specific transcription units (reviewed in [2]). Guided by these early gene products, DNA replication and the pairing of homologous chromosomes then occur, and the chromosomes undergo recombination. The chromosomes are monitored throughout this first phase of meiosis by the recombination checkpoint system, which detects the presence of unresolved recombination intermediates [5]. When all recombination intermediates have been resolved, the cells are permitted to proceed into the meiotic nuclear divisions. Until that time, the cells do not start the meiotic divisions, but halt at the pachytene stage near the end of prophase. This first phase of sporulation culminates in the completion of premeiotic prophase I, when the paired and recombined homologous chromosomes are ready for segregation at meiosis I. When this has been achieved, the cells undergo the prophase to metaphase I transition. In yeast [7], as well as in metazoans, this requires ‘maturation promoting factor’ (MPF), which is composed of a catalytic subunit — encoded by CDC28 in S. cerevisiae — and a regulatory subunit, a B-type cyclin. The requisite B-type cyclins are synthesized at this time [3]. Successful negotiation of the prophase to metaphase I transition sets several processes in motion. The two sequential meiotic divisions are begun, with the separation of the spindle pole bodies to form the meiosis I spindle. At the same time, the morphogenetic pathway for ascospore formation initiates with the dramatic accumulation of the middle sporulation-specific transcripts. Meanwhile, the ‘early’ sporulation genes are shut down (reviewed in [8]). Identification and phenotypic characterization of ndt80 Another protective mechanism, known as the recombination checkpoint, forces the cells to wait at the brink of the first of the two meiotic divisions — meiosis I — until recombination events have been completed, thus safeguarding the genome from the lethal effects of unresolved recombination intermediates [4–6]. Cells that have finished recombination can pass through a key regulatory point in How is the prophase to metaphase I transition accomplished? Mutations affecting this step have identified CDC28 and NDT80 as two of the critical genes [7,9]. In an early study, Shuster and Byers [7] showed that temperature-sensitive cdc28 alleles permit entry into meiosis but cause arrest at pachytene, shortly before the prophase to metaphase I transition. More recently, ndt80 R462 Current Biology, Vol 8 No 13 strains were shown to arrest meiosis at this same stage, with a cluster of phenotypes indistinguishable from those conferred by cdc28 alleles [9]. Xu et al. [9] considered several possible causes of meiotic arrest in the ndt80 mutant strain. One idea was that the checkpoint system could be preventing the cells from proceeding further. Mutations such as dmc1, for example, that cause defects in recombination or synaptonemal-complex structure, lead to the accumulation of unresolved recombination intermediates, which persist as the cells transit prophase [4]. Another idea was that ndt80 could be like cdc28, and cause arrest in pachytene because the wild-type gene product is required directly for the transition out of this stage. A simple genetic experiment distinguishes these possibilities. Mutations such as dmc1 and others, which cause ‘intermediate-block’ phenotypes, are bypassed by ‘earlyblock’ mutations such as spo11, whereas cdc28 and mutations like it do not show this interaction. The reason for the bypass is that the ‘early’ mutations prevent entry into the recombination pathway, so that the damage that triggers the checkpoint never occurs. Cells that are mutant for both spo11 and dmc1 can thus complete meiosis, even though dmc1 alone would arrest it. Conversely, mutations like cdc28 still cause arrest, because the wild-type function is required directly for meiotic progression. The results of this test showed that ndt80 was like cdc28, in that arrest still occurred in the presence of an early-block mutation, suggesting that NDT80 is also directly involved in the prophase-to-metaphase transition. A transcription factor for middle sporulation genes In wild-type S. cerevisiae cells, exit from prophase is accompanied by a major shift in gene expression. The new transcripts correspond to the middle class of sporulation-specific genes, which are expressed only in meiotic cells, and certain of the B-type cyclins, which are expressed during both meiosis and mitosis [2,3]. In ndt80 cells, by contrast, this transcriptional shift fails to occur. Chu and Herskowitz [1] demonstrated that representative middle and mid/late genes — SPS1, SMK1 and DIT1 — as well as the six CLB genes are expressed very poorly in the ndt80 mutant strain, whereas the early gene DMC1 is expressed normally. These results imply that wild-type Ndt80p is required, directly or indirectly, for expression of the later classes of sporulation genes. Might this connection be direct? If so, the Ndt80p target would likely involve a sequence called the ‘mid-sporulation element’ (MSE), which is known to be required for activation of middle genes during sporulation. The MSE was first described by Hepworth et al. [10] as a 15 base pair sequence, derived from the 5′ flanking region of the middle gene SPS4, that conferred sporulation-specific expression on a reporter construct. Activity of this minimal 15 base pair sequence was strongly augmented by an adjacent stretch of nucleotides, so that a 29 base pair sequence induced the reporter gene more than 200-fold during sporulation. Hepworth et al. [10] also noted similarity of the 15 base pair sequence to a sequence critical for SPR2 expression (D. A. Primerano, personal communication) and suggested that it might occur generally upstream of middle genes. Ozsarac et al. [11] confirmed this by identifying a related sequence within SPR3 and formulating a 9 base pair consensus sequence from this and the sequences of other known middle genes. Could Ndt80p, then, be a component of a transcription factor that activates genes via the MSE? Chu and Herskowitz [1] reported compelling evidence that this is so. They demonstrated that induced expression of Ndt80p could drive activation of an MSE–CYC1–lacZ reporter construct in mitotically-growing cells, outside the context of sporulation. Expression of the reporter gene required the invariant nucleotides of the MSE. Moreover, bacterially expressed Ndt80p formed DNA–protein complexes on MSE-containing oligonucleotides. The interaction is specific, as oligonucleotides containing wild-type MSE competed with the probe, whereas those with altered forms of the MSE did not. The B-type cyclins are middle sporulation genes Identification of Ndt80p as a transcription factor for the MSE-containing genes suggested the exciting possibility that it might also activate the CLB genes, which also depend on its activity. Indeed, MSE-like elements are present in the 5′ flanking regions of five of the six B-type cyclin genes. The only exception is CLB2, which is not induced during meiosis. Moreover, ectopic expression of Ndt80p was found to induce expression of all the CLB genes except CLB2 in mitotic cells [1]. These results provide a very satisfying explanation for the synchronous appearance of the B-type cyclins during meiosis and, moreover, contribute to a mechanism for producing them in the correct proportions. Dahmann and Futcher [12] have shown that the various B-type cyclins differ in their relative importance in meiosis and mitosis, with Clb1 and Clb4 being the most important for meiosis, and Clb2 the least, the reverse of the order for mitosis. Furthermore, the results reveal an interesting and potentially general way of recruiting mitotic functions to the meiotic pathway: a common genetic control element, the MSE, which transcriptionally recruits B-type cyclins by its presence in the 5′ flanking regions of the genes that encode them. How is NDT80 regulated? Interestingly, NDT80 is regulated in a complex way, befitting its pivotal role in the regulation of meiosis. The Dispatch gene does not fit neatly into any of the recognized transcriptional classes of sporulation genes, being expressed fully two hours later than the early genes, but somewhat earlier than the middle ones [1,8,9]. Indeed, NDT80 shows features of both early and middle forms of gene regulation. On the one hand, NDT80 is a middle gene; it has an MSE in its 5′ region, so that Ndt80p actually promotes its own synthesis. This was demonstrated by tagging Ndt80p with green fluorescent protein (GFP), expressing the fusion protein under the control of the NDT80 promoter. Expression of this reporter gene was found to be Ndt80p-inducible in mitotic cells [1]. On the other hand, NDT80 behaves in some respects like an early gene. These genes share the property of being repressed during mitotic growth by the Ume6 protein, and consequently they are expressed at low levels during mitosis in ume6 mutant strains (reviewed in [2]). Chu and Herskowitz [1] found that NDT80 is also expressed under these condition, unlike the other middle genes. The authors suggest that NDT80 might be considered as a ‘delayed early’ gene. Ndt80p sits at a critical juncture in sporulation, when cells must sense that the time is right to start the meiosis I division. The connection between Ndt80p activity and Btype cyclin expression suggests that Ndt80p might provide one link between the checkpoint system, which prevents division from starting prematurely, and the cellcycle machinery, which starts the division at the correct time. It was therefore of interest to know whether NDT80 or its product, Ndt80p, are regulated by the checkpoint system. Chu and Herskowitz [1] found that dmc1 cells, which arrest at pachytene, still express the NDT80 transcript at wild-type levels, implying that NDT80 transcription is not regulated by the checkpoint system. They also observed, however, that CLB1, which is normally stimulated by Ndt80p, was not expressed in these cells. CLB1 expression could be restored to the dmc1 cells by the rad17 mutation, which inactivates the checkpoint control [13]. These results suggest that Ndt80p may actually respond to the checkpoint, and raise the possibility that the prophase to metaphase I transition is controlled in part by this mechanism. Of spores and sperm Meiotic cells of all varieties face the same fundamental biological challenges. The genome must be protected from the damaging insults of recombination, and meiosis must be successfully coordinated with gametogenesis so that the haploid meiotic products can survive. It is interesting to note the striking parallels between sporulation in yeast and spermatogenesis in the fruitfly Drosophila. In Drosophila, the aly gene product, like Ndt80p, stimulates cell-cycle progession as well as morphogenesis by regulating the two separate classes of target genes [14]. In mice, as in yeast, there are numerous mutations that block R463 meiosis at the pachytene stage [15]. Given the generality of the problem, it should perhaps not be surprising that there are common solutions to the problems of meiosis among evolutionarily diverse species. References 1. Chu S, Herskowitz I: Gametogenesis in yeast is regulated by a transcriptional cascade dependent on Ndt80. Mol Cell 1998, 1:685-696. 2. Kupiec M, Byers B, Esposito R, Mitchell A: Meiosis and sporulation in Saccharomyces cerevisiae. In The Molecular and Cellular Biology of the Yeast Saccharomyces, Volume 3. Cell Cycle and Cell Biology. Edited by Pringle J, Broach J, Jones E. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 1997:889-1036. 3. Grandin N, Reed, S: Differential function and expression of Saccharomyces cerevisiae B-type cyclins in mitosis and meiosis. Mol Cell Biol 1993, 13:2113-2125. 4. Bishop D, Park D, Xu L, Kleckner N: DMC1: a meiosis-specific homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression. Cell 1992, 69:439-456. 5. Xu L, Weiner B, Kleckner N: Meiotic cells monitor the status of the interhomolog recombination complex. Genes Dev 1997, 11:106-118. 6. Roeder GS: Meiotic chromosomes: it takes two to tango. Genes Dev 1997, 11:2600-2621. 7. Shuster E, Byers B: Pachytene arrest and other meiotic effects of the start mutations in Saccharomyces cerevisiae. Genetics 1989, 123:29-43. 8. Mitchell A: Control of meiotic gene expression in Saccharomyces cerevisiae. Microbiol Rev 1994, 58:56-70. 9. Xu L, Ajimura M, Padmore R, Klein C, Kleckner N: NDT80, a meiosisspecific gene required for exit from pachytene in Saccharomyces cerevisiae. Mol Cell Biol 1995, 15:6572-6581. 10. Hepworth S, Ebisuzaki L, Segall J: A 15 base pair element activates the SPS4 gene midway through sporulation in Saccharomyces cerevisiae. Mol Cell Biol 1995, 15:3934-3944. 11. Ozsarac N, Straffon M, Dalton H, Dawes I: Regulation of gene expression during meiosis in Saccharomyces cerevisiae: SPR3 is controlled by both ABFI and a new sporulation control element. Mol Cell Biol 1997, 17:1152-1159. 12. Dahmann C, Futcher B: Specialization of B-type cyclins for mitosis or meiosis in S. cerevisiae. Genetics 1995, 140:957-963. 13. Lydall D, Nikolsky Y, Bishop D, Weinert T: A meiotic recombination checkpoint controlled by mitotic checkpoint genes. Nature 1996, 383:840-843. 14. Lin T-Y, Viswanathan S, Wood C, Wilson P, Wolf N: Coordinate developmental control of the meiotic cell cycle and spermatid differentiation in Drosophila males. Development 1996, 122:1331-1341. 15. Sassone-Corsi P: Transcriptional checkpoints determining the fate of male germ cells. Cell 1997, 88:163-166.