Yeast IME2 Functions Early in Meiosis Upstream of Cell Cycle-Regulated SBF and MBF Targets

BACKGROUND: In Saccharomyces cerevisiae, the G1 cyclin/cyclin-dependent kinase (CDK) complexes Cln1,-2,-3/Cdk1 promote S phase entry during the mitotic cell cycle but do not function during meiosis. It has been proposed that the meiosis-specific protein kinase Ime2, which is required for normal timing of pre-meiotic DNA replication, is equivalent to Cln1,-2/Cdk1. These two CDK complexes directly catalyze phosphorylation of the B-type cyclin/CDK inhibitor Sic1 during the cell cycle to enable its destruction. As a result, Clb5,-6/Cdk1 become activated and facilitate initiation of DNA replication. While Ime2 is required for Sic1 destruction during meiosis, evidence now suggests that Ime2 does not directly catalyze Sic1 phosphorylation to target it for destabilization as Cln1,-2/Cdk1 do during the cell cycle. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated that Sic1 is eventually degraded in meiotic cells lacking the IME2 gene (ime2Δ), supporting an indirect role of Ime2 in Sic1 destruction. We further examined global RNA expression comparing wild type and ime2Δ cells. Analysis of these expression data has provided evidence that Ime2 is required early in meiosis for normal transcription of many genes that are also periodically expressed during late G1 of the cell cycle. CONCLUSIONS/SIGNIFICANCE: Our results place Ime2 at a position in the early meiotic pathway that lies upstream of the position occupied by Cln1,-2/Cdk1 in the analogous cell cycle pathway. Thus, Ime2 may functionally resemble Cln3/Cdk1 in promoting S phase entry, or it could play a role even further upstream in the corresponding meiotic cascade

Prevention of DNA Rereplication Through a Meiotic Recombination Checkpoint Response

In the budding yeast Saccharomyces cerevisiae, unnatural stabilization of the cyclin-dependent kinase inhibitor Sic1 during meiosis can trigger extra rounds of DNA replication. When programmed DNA double-strand breaks (DSBs) are generated but not repaired due to absence of DMC1, a pathway involving the checkpoint gene RAD17 prevents this DNA rereplication. Further genetic analysis has now revealed that prevention of DNA rereplication also requires MEC1, which encodes a protein kinase that serves as a central checkpoint regulator in several pathways including the meiotic recombination checkpoint response. Downstream of MEC1, MEK1 is required through its function to inhibit repair between sister chromatids. By contrast, meiotic recombination checkpoint effectors that regulate gene expression and cyclin-dependent kinase activity are not necessary. Phosphorylation of histone H2A, which is catalyzed by Mec1 and the related Tel1 protein kinase in response to DSBs, and can help coordinate activation of the Rad53 checkpoint protein kinase in the mitotic cell cycle, is required for the full checkpoint response. Phosphorylation sites that are targeted by Rad53 in a mitotic S phase checkpoint response are also involved, based on the behavior of cells containing mutations in the DBF4 and SLD3 DNA replication genes. However, RAD53 does not appear to be required, nor does RAD9, which encodes a mediator of Rad53, consistent with their lack of function in the recombination checkpoint pathway that prevents meiotic progression. While this response is similar to a checkpoint mechanism that inhibits initiation of DNA replication in the mitotic cell cycle, the evidence points to a new variation on DNA replication control

Model of early Ime2 function.

<p>Pathways are shown leading from Ime1 induction early in meiosis to DNA replication. Factors relevant to the discussion in the text are included. Ime1 activates early gene expression through de-repression at the URS1 element <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031575#pone.0031575-Mallory1" target="_blank">[45]</a>, which is found in many meiosis-specific promoters including that of <i>IME2 </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031575#pone.0031575-Mitchell2" target="_blank">[43]</a>. Ime2 in turn upregulates gene expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031575#pone.0031575-Mitchell2" target="_blank">[43]</a>, and negatively affects Ime1 expression transcriptionally and post- transcriptionally <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031575#pone.0031575-Mitchell1" target="_blank">[30]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031575#pone.0031575-GuttmannRaviv1" target="_blank">[44]</a>. We hypothesize that Ime2 activation of the meiotic (mei) SBF/MBF transcriptional cascade leads to activation of a protein kinase with CDK specificity that enables Sic1 destruction, Clb5,-6/Cdk1 activation, and initiation of DNA replication.</p

Examination of SBF and MBF targets at 2 hours.

<p><i>A</i>, Distributions of log₂ (2 h/0 h) ratios for SBF and MBF targets in WT and <i>ime2Δ</i> cells. <i>B</i>, Direct comparison of WT and <i>ime2Δ</i> log₂ (2 h/0 h) ratios for each SBF and MBF target.</p

T-profiler analysis of consensus motifs for WT <i>v. ime2Δ</i> at 2 hours.

<p>T-profiler analysis of consensus motifs for WT <i>v. ime2Δ</i> at 2 hours.</p

Analysis of consensus motif gene sets.

<p><i>A</i>, Gene expression data from our time course were analyzed by T-profiler for average expression of gene groups defined by consensus promoter motifs. Results for selected gene groups characterized by the indicated sequences are shown (R = A or G; W = A or T). Comparisons were made between expression levels at 2, 4, and 6 h <i>v.</i> expression levels at 0 h. Asterisks indicate statistically significant values (E<0.05). <i>B</i>, Distributions of log₂ (2 h/0 h) ratios for the CRCGAAA (left) and ACGCGT (right) gene sets are shown for WT and <i>ime2Δ</i> cells.</p

Yeast strains used in this study.

1<p>Strains are congenic with SK1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031575#pone.0031575-Kane1" target="_blank">[42]</a> and are listed in the order that they appear in the text.</p