👤 Francisco Ferrezuelo

The G1-to-S transition of the cell cycle in the yeast Saccharomyces cerevisiae involves an extensive transcriptional program driven by transcription factors SBF (Swi4-Swi6) and MBF (Mbp1-Swi6). Activation of these factors ultimately depends on the G1 cyclin Cln3. To determine the transcriptional targets of Cln3 and their dependence on SBF or MBF, we first have used DNA microarrays to interrogate gene expression upon Cln3 overexpression in synchronized cultures of strains lacking components of SBF and/or MBF. Secondly, we have integrated this expression dataset together with other heterogeneous data sources into a single probabilistic model based on Bayesian statistics. Our analysis has produced more than 200 transcription factor-target assignments, validated by ChIP assays and by functional enrichment. Our predictions show higher internal coherence and predictive power than previous classifications. Our results support a model whereby SBF and MBF may be differentially activated by Cln3. Integration of heterogeneous genome-wide datasets is key to building accurate transcriptional networks. By such integration, we provide here a reliable transcriptional network at the G1-to-S transition in the budding yeast cell cycle. Our results suggest that to improve the reliability of predictions we need to feed our models with more informative experimental data. Show less

The Whi3 protein is associated with the endoplasmic reticulum, interacts with Cdc28, the budding-yeast Cdk, binds the mRNA of cyclin CLN3 and prevents accumulation of the Cdc28-Cln3 in the nucleus until late G(1). Besides its function as a cell size regulator, Whi3 is strictly required for filamentous growth. Here we show that emerging buds in Whi3-deficient cells are considerably rounder than in wild-type cells, indicating that Whi3 is required to maintain apical growth during S phase. This defect was not suppressed by deletion of CLB2, which is involved in switching from polar to isotropic bud growth, indicating that the observed phenotype is not the result of Whi3 acting solely as a negative regulator of cyclin Clb2. However, Cdc28 did not properly accumulate at the bud tip during S phase in whi3Delta cells, and their elongation defects were suppressed by CLN2 overexpression, suggesting a positive function for Whi3 in a Cdk-cyclin-dependent step required for apical growth. Additionally, the actin cytoskeleton was perturbed in Whi3-deficient cells, and WHI3 showed genetic interactions with actin patch components. Our results point to Whi3 as a key modulator of apical growth effectors to coordinate cell cycle events and morphogenesis. We propose that Whi3 is required for the apical localization of Cdc28-Cln1,2 complexes during bud growth and thereby, to promote the activation of Cdc42 and its effectors in the bud apex. Show less

During the cell division cycle of the yeast Saccharomyces cerevisiae, the G1-to-S transition depends upon the activation of two transcription factors (SBF and MBF), which are responsible for the cell cycle-regulated expression of more than 200 genes. Bck2 becomes essential in the absence of Cln3, the most upstream activator of this transcriptional program. Here we have used a genome-wide approach to elucidate the targets of Bck2. Our data indicate that Bck2 activates a selection of cell cycle-regulated genes from all cell cycle stages. In contrast, Cln3 activates only G1/S phase genes. Furthermore, Bck2 activates many genes independently of Swi6, the common component of SBF and MBF. Comparison of Bck2 targets with those of other transcription factors suggests that, in addition to SBF and MBF, Bck2 may elicit gene expression via Ste12 and Mcm1. We propose that Bck2 activates its targets by a mechanism fundamentally different from that of Cln3, and that it may be a necessary cofactor for the full expression of a subset of cell cycle-regulated genes. Show less

Whi3 is an RNA-binding protein associated with the endoplasmic reticulum (ER) that binds the CLN3 mRNA and plays a key role in the efficient retention of cyclin Cln3 at the ER. In the present work, we have identified new Whi3-associated mRNAs by a genomic approach. A large and significant number of these Whi3 targets encode for membrane and exocytic proteins involved in processes such as transport and cell wall biogenesis. Consistent with the genomic data, we have observed that cell wall integrity is compromised in Whi3-deficient cells and found strong genetic interactions between WHI3 and the cell integrity pathway. Whi3-associated mRNAs are enriched in clusters of the tetranucleotide GCAU, and mutation of the GCAU clusters in the CLN3 mRNA caused a reduction in its association to Whi3, suggesting that these sequences may act as cis-determinants for binding. Our data suggest that Whi3 is involved in the regulation and/or localization of a large subset of mRNAs functionally related to the ER and, since it is important for different molecular processes such as cytoplasmic retention or exocytic traffic of proteins, we propose that Whi3 is a general modulator of protein fate in budding yeast. Show less