A Switch from a Gradient to a Threshold Mode in the Regulation of a Transcriptional Cascade Promotes Robust Execution of Meiosis in Budding Yeast

Tight regulation of developmental pathways is of critical importance to all organisms, and is achieved by a transcriptional cascade ensuring the coordinated expression of sets of genes. We aimed to explore whether a strong signal is required to enter and complete a developmental pathway, by using meiosis in budding yeast as a model. We demonstrate that meiosis in budding yeast is insensitive to drastic changes in the levels of its consecutive positive regulators (Ime1, Ime2, and Ndt80). Entry into DNA replication is not correlated with the time of transcription of the early genes that regulate this event. Entry into nuclear division is directly regulated by the time of transcription of the middle genes, as premature transcription of their activator NDT80, leads to a premature entry into the first meiotic division, and loss of coordination between DNA replication and nuclear division. We demonstrate that Cdk1/Cln3 functions as a negative regulator of Ime2, and that ectopic expression of Cln3 delays entry into nuclear division as well as NDT80 transcription. Because Ime2 functions as a positive regulator for premeiotic DNA replication and NDT80 transcription, as well as a negative regulator of Cdk/Cln, we suggest that a double negative feedback loop between Ime2 and Cdk1/Cln3 promotes a bistable switch from the cell cycle to meiosis. Moreover, our results suggest a regulatory mode switch that ensures robust meiosis as the transcription of the early meiosis-specific genes responds in a graded mode to Ime1 levels, whereas that of the middle and late genes as well as initiation of DNA replication, are regulated in a threshold mode

Increase in <i>IME1</i> copy number results in an increase in the level of expression of <i>IME1</i> and all meiosis-specific genes.

<p>Isogenic diploids carrying 1 (Y1639, diamonds), 2 (Y1631, squares), 3 (Y1663, triangles) and 5 (Y1662, circles) copies of <i>IME1</i> were shifted to meiotic conditions (SPM). Samples were taken at the indicated times to isolate RNA and estimate transcript levels by qPCR. <b>A.</b> The RNA level of the indicated gene relative to RNA levels of <i>ACT1</i>. <b>B.</b> The relative RNA level is drawn in a log scale. The experiment was repeated 3 times and a representative result is shown.</p

List of strains.

<p>List of strains.</p

Meiosis is insensitive to the level of <i>IME1</i> mRNA.

<p>Isogenic diploids carrying different copy numbers of <i>IME1</i> were shifted to meiotic inducing conditions. <b>A.</b> The correlation between <i>IME1</i> copy number and its maximal level of expression. The maximal level of <i>IME1</i> RNA (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011005#pone-0011005-g003" target="_blank">Fig. 3A</a>) in comparison to the copy number of the <i>IME1</i> gene was drawn. The fitted trend line (dotted) and its formula are given. <b>B.</b> Efficiency of meiosis. The percentage of asci (line) and the number of cells that can form colonies (viability, column) at 48 hours in SPM. The results are the averages of three independent transformants and a count of ≥200 cells. Error bars represent standard deviations.</p

Ime2, a Meiosis-Specific Kinase in Yeast, Is Required for Destabilization of Its Transcriptional Activator, Ime1

In the budding yeast Saccharomyces cerevisiae, entry into meiosis and its successful completion depend on two positive regulators, Ime1 and Ime2. Ime1 is a transcriptional activator that is required for transcription of IME2, a serine/threonine protein kinase. We show that in vivo Ime2 associates with Ime1, that in vitro Ime2 phosphorylates Ime1, and that in living cells the stability of Ime1 depends on Ime2. Diploid cells with IME2 deleted show an increase in the level of Ime1, whereas haploid cells overexpressing IME2 show a decrease in the stability of Ime1. Furthermore, the level of Ime1 depends on the kinase activity of Ime2. Using a mutation in one of the ATPase subunits of the proteasome, RPT2, we demonstrate that Ime1, amino acids 270 to 360, is degraded by the 26S proteasome. We also show that Ime2 itself is an extremely unstable protein whose expression in vegetative cultures is toxic. We propose that a negative-feedback loop ensures that the activity of Ime1 will be restricted to a narrow window

Ectopic overexpresion of Cln3 increases the transcription of <i>IME1</i>, but delays initiation of premeiotic DNA replication and <i>NDT80</i> transcription.

<p>A wt strain (Y1631) carrying <i>pIME2-CLN3</i> on a 2μ plasmid (YEp3212, triangles) or the vector plasmid (squares) were shifted to meiotic conditions. At the indicated hours samples were taken to process for FACS analysis and calculate the percentage of cells with 4C DNA content (A), and to isolate RNA and determine the relative transcripts levels of <i>IME1</i> and <i>NDT80</i> by qPCR (B).</p