Distinct regions of ATF/CREB proteins Atf1 and Pcr1 control recombination hotspot ade6-M26 and the osmotic stress response

The Atf1 protein of Schizosaccharomyces pombe contains a bZIP (DNA-binding/protein dimerization) domain characteristic of ATF/CREB proteins, but no other functional domains or clear homologs have been reported. Atf1-containing, bZIP protein dimers bind to CRE-like DNA sites, regulate numerous stress responses, and activate meiotic recombination at hotspots like ade6–M26. We defined systematically the organization of Atf1 and its heterodimer partner Pcr1, which is required for a subset of Atf1-dependent functions. Surprisingly, only the bZIP domain of Pcr1 is required for hotspot activity and tethering of Atf1 to ade6 promotes recombination in the absence of its bZIP domain and the Pcr1 protein. Therefore the recombination–activation domain of Atf1-Pcr1 heterodimer resides exclusively in Atf1, and Pcr1 confers DNA-binding site specificity in vivo. Atf1 has a modular organization in which distinct regions affect differentially the osmotic stress response (OSA) and meiotic recombination (HRA, HRR). The HRA and HRR regions are necessary and sufficient to activate and repress recombination, respectively. Moreover, Atf1 defines a family of conserved proteins with discrete sequence motifs in the functional domains (OSA, HRA, HRR, bZIP). These findings reveal the functional organization of Atf1 and Pcr1, and illustrate several mechanisms by which bZIP proteins can regulate multiple, seemingly disparate activities.     

Phosphorylation-Independent Regulation of Atf1-Promoted Meiotic Recombination by Stress-Activated,p38 Kinase Spc1 of Fission Yeast

Background

Stress-activated protein kinases regulate multiple cellular responses to a wide variety of intracellular and extracellular conditions. The conserved, multifunctional, ATF/CREB protein Atf1 (Mts1, Gad7) of fission yeast binds to CRE-like (M26) DNA sites. Atf1 is phosphorylated by the conserved, p38-family kinase Spc1 (Sty1, Phh1) and is required for many Spc1-dependent stress responses, efficient sexual differentiation, and activation of Rec12 (Spo11)-dependent meiotic recombination hotspots like ade6-M26.

Methodology/Principal Findings

We sought to define mechanisms by which Spc1 regulates Atf1 function at the ade6-M26 hotspot. The Spc1 kinase was essential for hotspot activity, but dispensable for basal recombination. Unexpectedly, a protein lacking all eleven MAPK phospho-acceptor sites and detectable phosphorylation (Atf1-11M) was fully proficient for hotspot recombination. Furthermore, tethering of Atf1 to ade6 in the chromosome by a heterologous DNA binding domain bypassed the requirement for Spc1 in promoting recombination.

Conclusions/Significance

The Spc1 protein kinase regulates the pathway of Atf1-promoted recombination at or before the point where Atf1 binds to chromosomes, and this pathway regulation is independent of the phosphorylation status of Atf1. Since basal recombination is Spc1-independent, the principal function of the Spc1 kinase in meiotic recombination is to correctly position Atf1-promoted recombination at hotspots along chromosomes. We also propose new hypotheses on regulatory mechanisms for shared (e.g., DNA binding) and distinct (e.g., osmoregulatory vs. recombinogenic) activities of multifunctional, stress-activated protein Atf1.     

14-3-3 protein interferes with the binding of RNA to the phosphorylated form of fission yeast meiotic regulator Mei2p

The switch from mitosis to meiosis is controlled by the Pat1(Ran1) kinase-Mei2p system in Schizosaccharomyces pombe. Mei2p promotes both premeiotic DNA synthesis and meiosis I, and its RNA binding ability is essential for these two processes. Mei2p forms a dot structure in the nucleus prior to meiosis I, aided by a specific RNA species named "meiRNA". Pat1 kinase phosphorylates Mei2p on two positions and downregulates its activity. Pat1 kinase undergoes inactivation under meiotic conditions, as a result of the production of a tethering pseudosubstrate Mei3p, and accumulation of the unphosphorylated form of Mei2p commits cells to meiosis. However, the mechanism of how phosphorylation of Mei2p suppresses its activity to induce meiosis remains largely unknown. Here we show that S. pombe Rad24p, a 14-3-3 protein, functions as a negative factor for meiosis by antagonizing the function of meiRNA to promote the formation of a nuclear Mei2p dot. Rad24p binds preferentially to Mei2p phosphorylated by Pat1 kinase. It inhibits association of meiRNA to the phosphorylated form of Mei2p but not to the unphosphorylated form in vitro. We speculate that Rad24p, bound tightly to the residues phosphorylated by Pat1 kinase, may mask the RNA recognition motifs on Mei2p. This model will explain, at least partly, why phosphorylation by Pat1 kinase inhibits the meiosis-inducing activity of Mei2p.     

Acetate regulation of spore formation is under the control of the Ras/cyclic AMP/protein kinase A pathway and carbon dioxide in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the Ras/cyclic AMP (cAMP)/protein kinase A (PKA) pathway is a nutrient-sensitive signaling cascade that regulates vegetative growth, carbohydrate metabolism, and entry into meiosis. How this pathway controls later steps of meiotic development is largely unknown. Here, we have analyzed the role of the Ras/cAMP/PKA pathway in spore formation by the meiosis-specific manipulation of Ras and PKA or by the disturbance of cAMP production. We found that the regulation of spore formation by acetate takes place after commitment to meiosis and depends on PKA and appropriate A kinase activation by Ras/Cyr1 adenylyl cyclase but not by activation through the Gpa2/Gpr1 branch. We further discovered that spore formation is regulated by carbon dioxide/bicarbonate, and an analysis of mutants defective in acetate transport (ady2Δ) or carbonic anhydrase (nce103Δ) provided evidence that these metabolites are involved in connecting the nutritional state of the meiotic cell to spore number control. Finally, we observed that the potential PKA target Ady1 is required for the proper localization of the meiotic plaque proteins Mpc70 and Spo74 at spindle pole bodies and for the ability of these proteins to initiate spore formation. Overall, our investigation suggests that the Ras/cAMP/PKA pathway plays a crucial role in the regulation of spore formation by acetate and indicates that the control of meiotic development by this signaling cascade takes places at several steps and is more complex than previously anticipated.     

Dot1-Dependent Histone H3K79 Methylation Promotes Activation of the Mek1 Meiotic Checkpoint Effector Kinase by Regulating the Hop1 Adaptor

During meiosis, accurate chromosome segregation relies on the proper interaction between homologous chromosomes, including synapsis and recombination. The meiotic recombination checkpoint is a quality control mechanism that monitors those crucial events. In response to defects in synapsis and/or recombination, this checkpoint blocks or delays progression of meiosis, preventing the formation of aberrant gametes. Meiotic recombination occurs in the context of chromatin and histone modifications, which play crucial roles in the maintenance of genomic integrity. Here, we unveil the role of Dot1-dependent histone H3 methylation at lysine 79 (H3K79me) in this meiotic surveillance mechanism. We demonstrate that the meiotic checkpoint function of Dot1 relies on H3K79me because, like the dot1 deletion, H3-K79A or H3-K79R mutations suppress the checkpoint-imposed meiotic delay of a synapsis-defective zip1 mutant. Moreover, by genetically manipulating Dot1 catalytic activity, we find that the status of H3K79me modulates the meiotic checkpoint response. We also define the phosphorylation events involving activation of the meiotic checkpoint effector Mek1 kinase. Dot1 is required for Mek1 autophosphorylation, but not for its Mec1/Tel1-dependent phosphorylation. Dot1-dependent H3K79me also promotes Hop1 activation and its proper distribution along zip1 meiotic chromosomes, at least in part, by regulating Pch2 localization. Furthermore, HOP1 overexpression bypasses the Dot1 requirement for checkpoint activation. We propose that chromatin remodeling resulting from unrepaired meiotic DSBs and/or faulty interhomolog interactions allows Dot1-mediated H3K79-me to exclude Pch2 from the chromosomes, thus driving localization of Hop1 along chromosome axes and enabling Mek1 full activation to trigger downstream responses, such as meiotic arrest.     

The meiotic recombination checkpoint suppresses NHK-1 kinase to prevent reorganisation of the oocyte nucleus in Drosophila

The meiotic recombination checkpoint is a signalling pathway that blocks meiotic progression when the repair of DNA breaks formed during recombination is delayed. In comparison to the signalling pathway itself, however, the molecular targets of the checkpoint that control meiotic progression are not well understood in metazoans. In Drosophila, activation of the meiotic checkpoint is known to prevent formation of the karyosome, a meiosis-specific organisation of chromosomes, but the molecular pathway by which this occurs remains to be identified. Here we show that the conserved kinase NHK-1 (Drosophila Vrk-1) is a crucial meiotic regulator controlled by the meiotic checkpoint. An nhk-1 mutation, whilst resulting in karyosome defects, does so independent of meiotic checkpoint activation. Rather, we find unrepaired DNA breaks formed during recombination suppress NHK-1 activity (inferred from the phosphorylation level of one of its substrates) through the meiotic checkpoint. Additionally DNA breaks induced by X-rays in cultured cells also suppress NHK-1 kinase activity. Unrepaired DNA breaks in oocytes also delay other NHK-1 dependent nuclear events, such as synaptonemal complex disassembly and condensin loading onto chromosomes. Therefore we propose that NHK-1 is a crucial regulator of meiosis and that the meiotic checkpoint suppresses NHK-1 activity to prevent oocyte nuclear reorganisation until DNA breaks are repaired.     

Elevated Rad53 kinase activity influences formation and interhomolog repair of meiotic DNA double-strand breaks in budding yeast

Meiotic cells generate physiological programmed DNA double-strand breaks (DSBs) to initiate meiotic recombination. Interhomolog repair of the programmed DSBs by meiotic recombination is vital to ensure accurate chromosome segregation at meiosis I to produce normal gametes. In budding yeast, the DNA damage checkpoint kinase Rad53 is activated by DSBs which accidentally occur as DNA lesions in mitosis and meiosis; however, meiotic programmed DSBs which occur at ∼160 loci per genome fail to activate the kinase. Thus, Rad53 activation appears to be silenced in response to meiotic programmed DSBs. In this study, to address the biological significance of Rad53’s insensitivity to meiotic DSBs, we examined the effects of Rad53 overexpression on meiotic processes. The overexpression led to partial activation of Rad53, uncovering that the negative impacts of Rad53 kinase activation on meiotic progression, and formation and interhomolog repair of meiotic programmed DSBs.