The Ime2 protein kinase enhances the disassociation of the Sum1 repressor from middle meiotic promoters.

Meiotic development is the pathway that produces haploid gametes from diploid precursors. Following the induction of the pathway, cells duplicate the genome and then enter an elongated prophase when homolog pairing, synapsis, and genetic recombination take place. Exit from prophase is followed by two sequential rounds of chromosome segregation. In many organisms, exit from prophase is a key control point where meiotic progression is regulated. Meiotic development in the yeast Saccharomyces cerevisiae (sporulation) is tightly regulated by a transcriptional program. The transcriptional program of sporulation is characterized by the sequential expression of temporally distinct sets of genes that are induced as different steps in the program take place (5, 30). Although there are at least 12 temporally distinct subclasses of sporulation-specific genes, they can be broadly grouped into early, middle, and late classes (reviewed in reference 39). Early promoters are activated by the Ime1 transcription factor, which is expressed and activated in diploids in response to starvation signals (reviewed in references 13 and 39). Early genes are expressed throughout meiotic S phase and prophase (5, 30). Toward the end of prophase, at the pachytene stage, homologs are fully connected by synaptonemal complexes (SCs), joint molecules have been formed but crossover recombinants have not been resolved (1, 24), and the spindle pole body (centrosome) has duplicated in preparation for the first meiotic division (3, 43). Exit from pachytene, entry into the meiotic divisions, and spore formation require the induction of middle genes. Middle promoters are activated by the Ndt80 transcription factor, which specifically binds to DNA elements termed middle sporulation elements (MSEs) (6, 8). ndt80Δ mutants block meiotic development in pachytene (43). ndt80Δ-blocked cells can be held in pachytene for extended periods of time and efficiently resume mitotic growth if nutrients are replenished (43). In contrast, cells that have induced middle promoters and exited pachytene are largely unable to resume mitotic growth (33). These observations suggest that Ndt80 plays a role in the phenomenon of commitment to meiosis. Ndt80 also appears to be regulated by the recombination checkpoint (also known as the pachytene checkpoint) (6, 8, 26, 38) that blocks meiotic development in response to recombination intermediates such as double-strand breaks and defects in SC formation (see references 9 and 31 for reviews of the pachytene checkpoint). Ndt80 promotes the disassembly of SCs and crossover formation by activating the expression of the Cdc5 polo-like kinase (36). Ndt80 also promotes the nuclear divisions and spore morphogenesis by activating the expression of M-phase cyclins, cell cycle regulatory molecules, and gene products required for spore formation (5). NDT80 is itself a tightly regulated meiosis-specific gene that is expressed shortly before most other middle genes, and it has been classified as an early/middle (25) or subclass 3a (30) gene. Its expression is controlled through Ndt80-MSE interactions in its own promoter in a positive autoregulatory loop (6). Despite the critical role that NDT80 induction plays in meiotic regulation, the molecular events that trigger the NDT80 autoregulatory loop have not yet been elucidated. Sum1 is a DNA-binding protein that represses a subset of middle promoters in vegetative cells (42). Sum1 interacts with the core MSE and with adjacent bases (28). Ndt80-inducible MSEs therefore vary in their affinities for Sum1. The Sum1 and Ndt80 DNA-binding domains have been shown previously to compete for occupancy of MSE DNA in vitro. About 75 of the 150 middle promoters are Sum1 repressible, and these promoters are enriched with MSEs that are predicted to bind Sum1 (28, 40). The NDT80 promoter is controlled by a Sum1-repressible MSE, suggesting that the removal of Sum1 repression can regulate meiotic progression (25, 42). Sum1 represses transcription through the Hst1 NAD+-dependent histone deacetylase (Sir2 paralog) that is bound to Sum1 via the Rfm1 bridging protein (20). However, only a subset of Sum1-repressible promoters are derepressed in hst1Δ and rfm1Δ mutants, suggesting that Sum1 can repress transcription by an Hst1-Rfm1-independent mechanism. Consistent with the hypothesis that Sum1 regulates prophase exit, sum1Δ mutants bypass the pachytene checkpoint and carry out the nuclear divisions in the presence of recombination intermediates (broken chromosomes) to produce nonviable haploid products (15, 26). In addition to fulfilling a role in middle meiotic gene regulation, Sum1 interacts with a subset of origins of DNA replication and positively regulates S phase by an Hst1-dependent mechanism (12, 41). Sum1 is also required to repress α-specific promoters in a cells (44). Sum1 and Hst1 also regulate the expression of promoters controlling the salvage pathway for NAD+ (2). However, NAD+ salvage pathway genes and α-specific genes are not induced during meiosis (5, 30). It is unclear how Sum1's roles in S phase regulation, cell type specification, NAD+ biosynthesis, and meiosis are differentially regulated. Ime2 is a meiosis-specific cyclin-dependent kinase (CDK)-like kinase that regulates multiple steps in meiotic development (11). Ime2 has been shown previously to activate Ndt80 (34, 35). In addition, ime2Δ mutants show defects in middle meiotic gene expression that are partially suppressed by sum1Δ (25). These observations suggest that Ime2 promotes the transcription of middle promoters by positively regulating Ndt80 and by negatively regulating Sum1. Two models can explain how Sum1 repression is removed in meiotic cells. The first model posits that Sum1 is removed from chromatin by a mechanism that requires competition with Ndt80. The findings that the purified DNA-binding domain of Sum1 can be displaced from an MSE with purified Ndt80 in vitro (28) and that high-level ectopic Ndt80 expression in vegetative haploid cells can induce SMK1 and other Sum1-repressible middle genes in mitotic haploid cells (5) demonstrate that competition can occur. According to this competition model, Sum1 would function as a transcriptional damper to prevent adventitious induction of middle promoters by Ndt80 or other activators. The second model posits that Sum1 is actively removed from chromatin in response to meiotic signals in S-phase or prophase cells. According to this sequential model, the regulated removal of Sum1 repression would create a state that would permit the NDT80 autoregulatory loop to be induced. Distinguishing between the competition and sequential models for the removal of Sum1 repression has important implications for how meiotic progression is regulated. The SMK1 middle meiotic promoter provides an excellent model for the study of Sum1 regulation since it is controlled by a single, well-characterized MSE that interacts with Sum1 and Ndt80 and by an upstream activation sequence that binds the constitutive Abf1 transcriptional activator (28, 29, 42). Thus, Smk1 is not expressed when Sum1 is present, and it is expressed to a moderate level in an Abf1-dependent fashion when Sum1 has been removed. The derepressed SMK1 promoter is further activated by Ndt80 during meiotic development to yield peak levels of SMK1 expression (15). In this study, we show that Sum1 is removed from middle meiotic promoters in a pathway that does not require competition with Ndt80. We also show that the Ime2 CDK-like kinase phosphorylates Sum1 on T306 in vivo and that a form of Sum1 containing a nonphosphorylatable substitution at this position (Sum1-T306A) is not removed in ndt80Δ meiotic cells. Although the deletion of Hst1 does not derepress SMK1 in vegetative cells, it does bypass the Sum1 removal defect of a sum1(T306A) ndt80Δ strain. These findings suggest that Ime2 promotes a state that is permissive for middle-promoter expression by antagonizing Hst1. These data indicate that a regulated pathway promotes the removal of the Sum1 brake from chromatin prior to NDT80 induction and suggest that this pathway controls the expression of middle promoters and meiotic progression. Our findings also suggest that when this pathway is compromised, Sum1 can be removed in an Ndt80-dependent pathway.