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

Utilization of an online module bank for a research training curriculum: development, implementation, evolution, evaluation, and lessons learned

Background: Students enter Oakland University William Beaumont School of Medicine’s required research program, Embark, with variable levels of experience. Recognizing this, Embark allows for progression through the individual research project with flexibility. Since 2014, student self-directed curriculum personalization is promoted through a menu of online modules. Objective: This evaluation sought to understand student usage of the modules, identified strengths of the modules and preferred attributes of the modules. Gaining this evidence will provide information on how to best meet students’ needs in a just-in-time format. Methods: A retrospective mixed methods analysis of the module library was conducted. The library was constructed using best practices as an educational tool. The retrospective evaluation included analysis of students’ viewing patterns and answers to required course evaluations during the fall semesters of 2014 to 2017. Students’ preference for modules was determined by viewing records and conjoint analysis. Results: Students’ milestone preparation was not negatively impacted by relocation of curricular content from lecture to modules. Changes in module implementation within the course (2016) resulted in an increase of students viewing modules beyond only the minimum course requirements (71% (2016) from 10% (2014)). Data from both quantitative and qualitative evaluation questions show an increase in students’ identifying the modules as a strength to individualize the course. The identified module strengths include content individualization, just-in-time access, while identified needs included a desire for additional modules. Students preferred modules that were animated, shorter in duration and curated from an external source. Conclusions: Online modules provide students with a rich set of resources allowing for individualized learning. Lessons learned in the implementation of the online modules may be transferable to many educational topics. When implementing similar technology projects, usage rates, learner feedback, and effect on appreciation of the content are important to frequently monitor

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

Expression of SBF and MBF targets in early meiosis.

<p>Gene-normalized hierarchical clustering analysis of MBF and SBF targets. For clustering procedure, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031575#s4" target="_blank"><i>Materials and Methods</i></a>.</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

Epistasis analysis of <i>IME2</i> and <i>WHI5</i>.

<p>WT and mutant cells were induced to enter meiosis synchronously and DNA content was analyzed by flow cytometry at the indicated time points. This experiment was conducted in conjunction with the microarray experiment; therefore, sets of WT and <i>ime2Δ</i> histograms shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031575#pone-0031575-g002" target="_blank">Fig. 2</a> are reproduced here. Note that we have observed variability in the degree of DNA replication observed in <i>ime2Δ</i> cells at 24 h (<i>e.g.</i> compare with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031575#pone-0031575-g001" target="_blank">Fig. 1</a>). Strains used were DSY1089, YGB221, YGB752, and YGB753.</p

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

Sic1 steady-state levels.

<p>WT and indicated mutant cells were induced to enter meiosis in a synchronous fashion and followed through time (h = hours). DNA content was analyzed by flow cytometry to detect pre-meiotic DNA replication (2C to 4C transition). Sic1^13myc and tubulin were detected by western blotting. For each time point, Sic1^13myc level was quantified by determining the relative band intensities of Sic1^13myc (red) and tubulin (green) and normalizing the resulting Sic1^13myc/tubulin ratio to the corresponding 0 hour ratio. Results are shown in graphical form (a.u., arbitrary units). Prior to immunodetection, membranes were stained with Ponceau S for total protein content assessment; regions that include Sic1^13myc and tubulin are shown. Strains used were YGB803, YGB787, and YGB804.</p