Gametogenesis is the process used by sexually reproducing organisms to produce haploid cells that are specialized for sexual fusion. During gametogenesis, precursor cells duplicate the genome, undergo crossing over, and then complete two rounds of chromosome segregation. The formation of haploids is coupled to differentiation programs that produce gametes. In the yeast Saccharomyces cerevisiae, the formation of haploids is coupled to spore formation. Spores can germinate and mate to regenerate diploids.
Sporulation in yeast is tightly regulated by a transcriptional cascade that controls the sequential induction of about 1,000 genes that can be broadly classified as early, middle, and late (5
). Early promoters are activated by the Ime1 transcription factor, which is produced in diploid cells in response to nutritional deprivation (the inducing signal for meiosis) (16
). Early genes are expressed during premeiotic S phase and during prophase, when homologous chromosomes pair, synapse, and undergo reciprocal recombination. The expression of many early genes continues until pachytene, when homologous pairs of chromosomes are connected end to end by synaptonemal complexes (SCs).
Middle sporulation genes (MSGs) are activated by the Ndt80 transcription factor, which specifically recognizes DNA elements termed middle sporulation elements (MSEs) (6
expression is IME1
dependent, and this may connect the early and middle phases of the transcriptional cascade. The NDT80
promoter is also activated in a positive autoregulatory loop by its own protein product. Thus, NDT80
is transcriptionally induced as a middle gene just prior to when most middle promoters are induced (23
). The Ndt80-inducible gene product that promotes pachytene exit has been identified as the Cdc5 Polo-like kinase (32
). Other Ndt80-inducible gene products include cyclins that promote the meiotic divisions and molecules such as Smk1 that control spore morphogenesis (5
). Thus, the Ndt80 regulon coordinately promotes pachytene exit, the meiotic divisions, and spore formation.
Exit from pachytene is a key decision point in meiotic development. In yeast, this transition is closely associated with the commitment point, after which the inducing signal (starvation) is no longer needed to complete the program (31
). In addition, a checkpoint pathway that monitors recombination intermediates and defects in SC formation (the pachytene checkpoint) controls pachytene exit (13
). The pachytene checkpoint inhibits Ndt80 (6
), and ndt80
Δ cells block meiotic development at pachytene (40
). These findings demonstrate that Ndt80 is a central component of the regulatory system that controls pachytene exit and commitment to meiotic development.
MSGs are repressed during vegetative growth by Sum1, a DNA-binding protein that specifically recognizes a subset of MSEs (39
). Sum1 represses middle promoters by recruiting an NAD+
-dependent histone deacetylase (Sir2 paralog), named Hst1, through a tethering factor, named Rfm1 (18
). Sum1 also represses transcription in an Hst1-independent fashion. Sum1 represses NDT80
, and it has been proposed that a regulated competition between the Sum1 repressor and the Ndt80 activator triggers the NDT80
positive autoregulatory loop (23
). The DNA-binding domain of Ndt80 can displace the DNA-binding domain of Sum1 from MSE DNA (24
). In addition, ectopic expression of Ndt80 can activate Sum1-repressible genes in vegetative cells (5
). It is therefore likely that Ndt80 can competitively displace Sum1 from DNA in meiotic cells. However, Sum1 is displaced from DNA in ndt80
Δ cells that have been transferred to sporulation medium (1
). Thus, Sum1 can be removed from DNA in meiotic cells by a mechanism that does not require Ndt80 competition.
Ime2 is a meiosis-specific cyclin-dependent kinase (CDK)-like kinase that regulates multiple steps in meiotic development, including induction of the program, S phase, the nuclear divisions, and spore formation (15
). It has been proposed that Ime2 collaborates with Cdk1 to regulate meiosis. One way that Ime2 regulates meiotic processes is to function in place of Cdk1. Thus, Ime2 is required for destruction of the Sic1 inhibitor of S phase in meiotic cells, while Cdk1 complexed with G1
cyclins (which inhibit meiotic development) targets Sic1 for destruction in mitotic cells (7
). Another well-characterized Ime2 target is the Cdh1 substrate-bridging protein for the anaphase-promoting complex that is phosphorylated by both Ime2 and Cdk1 as meiosis is taking place (14
). In this case, the differential sensitivities of the Ime2 and Cdk1 phosphoacceptor sites to phosphatases have been proposed to play a role in preventing S phase from taking place between meiosis I (MI) and MII.
It has previously been shown that Ime2 phosphorylates Sum1 on residue T306 and that a nonphosphorylatable Sum1-T306A protein (Sum1-i) is not removed from DNA in ndt80
Δ cells (1
). However, in otherwise wild-type (NDT80
) cells, Sum1-i does not block meiosis or spore formation. These findings suggest that while Ime2 promotes the Ndt80-independent removal of Sum1 from DNA, the removal of Sum1 is regulated through an additional mechanism.
In this report, we show that Cdk1 negatively regulates Sum1 specifically in meiotic cells. We describe a Sum1 mutant that is insensitive to Cdk1 and Ime2 (sum1-ci) and show that this mutant blocks meiosis in prophase with a phenotype that is indistinguishable from that of ndt80Δ. Ectopic expression of NDT80 or mutation of an MSE in the NDT80 promoter that has previously been reported to interact with Sum1 bypasses the sum1-ci block. Moreover, hst1Δ and rfm1Δ mutants also bypass the block. These findings demonstrate that Cdk1 and Ime2 inhibit Sum1 at the NDT80 promoter in a pathway that involves Rfm1/Hst1. The data demonstrate that Sum1 is a key regulatory brake that must be inhibited before cells can exit from prophase and complete meiotic development.