Gene Expression Analysis of Yeast Strains with a Nonsense Mutation in the eRF3-Coding Gene Highlights Possible Mechanisms of Adaptation.

In yeast Saccharomyces cerevisiae, there are two translation termination factors, eRF1 (Sup45) and eRF3 (Sup35), which are essential for viability. Previous studies have revealed that presence of nonsense mutations in these genes leads to amplification of mutant alleles (sup35-n and sup45-n), which appears to be necessary for the viability of such cells. However, the mechanism of this phenomenon remained unclear. In this study, we used RNA-Seq and proteome analysis to reveal the complete set of gene expression changes that occur during cellular adaptation to the introduction of the sup35-218 nonsense allele. Our analysis demonstrated significant changes in the transcription of genes that control the cell cycle: decreases in the expression of genes of the anaphase promoting complex APC/C (APC9, CDC23) and their activator CDC20, and increases in the expression of the transcription factor FKH1, the main cell cycle kinase CDC28, and cyclins that induce DNA biosynthesis. We propose a model according to which yeast adaptation to nonsense mutations in the translation termination factor genes occurs as a result of a delayed cell cycle progression beyond the G2-M stage, which leads to an extension of the S and G2 phases and an increase in the number of copies of the mutant sup35-n allele.

Forkhead transcription factor Fkh1: insights into functional regulatory domains crucial for recruitment of Sin3 histone deacetylase complex.

Transcription factors are inextricably linked with histone deacetylases leading to compact chromatin. The Forkhead transcription factor Fkh1 is mainly a negative transcriptional regulator which affects cell cycle control, silencing of mating-type cassettes and induction of pseudohyphal growth in the yeast Saccharomyces cerevisiae. Markedly, Fkh1 impinges chromatin architecture by recruiting large regulatory complexes. Implication of Fkh1 with transcriptional corepressor complexes remains largely unexplored. In this work we show that Fkh1 directly recruits corepressors Sin3 and Tup1 (but not Cyc8), providing evidence for its influence on epigenetic regulation. We also identified the specific domain of Fkh1 mediating Sin3 recruitment and substantiated that amino acids 51-125 of Fkh1 bind PAH2 of Sin3. Importantly, this part of Fkh1 overlaps with its Forkhead-associated domain (FHA). To analyse this domain in more detail, selected amino acids were replaced by alanine, revealing that hydrophobic amino acids L74 and I78 are important for Fkh1-Sin3 binding. In addition, we could prove Fkh1 recruitment to promoters of cell cycle genes CLB2 and SWI5. Notably, Sin3 is also recruited to these promoters but only in the presence of functional Fkh1. Our results disclose that recruitment of Sin3 to Fkh1 requires precisely positioned Fkh1/Sin3 binding sites which provide an extended view on the genetic control of cell cycle genes CLB2 and SWI5 and the mechanism of transcriptional repression by modulation of chromatin architecture at the G2/M transition.

Dbf4 Zn-Finger Motif Is Specifically Required for Stimulation of Ctf19-Activated Origins in Saccharomyces cerevisiae.

Eukaryotic genomes are replicated in spatiotemporal patterns that are stereotypical for individual genomes and developmental profiles. In the model system Saccharomyces cerevisiae, two primary mechanisms determine the preferential activation of replication origins during early S phase, thereby largely defining the consequent replication profiles of these cells. Both mechanisms are thought to act through specific recruitment of a rate-limiting initiation factor, Dbf4-dependent kinase (DDK), to a subset of licensed replication origins. Fkh1/2 is responsible for stimulation of most early-firing origins, except for centromere (CEN)-proximal origins that recruit DDK via the kinetochore protein Ctf19, which is required for their early firing. The C-terminus of Dbf4 has been implicated in its recruitment to origins via both the Fkh1/2 and Ctf19 mechanisms. Here, we show that the Zn-finger motif within the C-terminus is specifically required for Dbf4 recruitment to CENs to stimulate CEN-proximal/Ctf19-dependent origins, whereas stimulation of origins via the Fkh1/2 pathway remains largely intact. These findings re-open the question of exactly how Fkh1/2 and DDK act together to stimulate replication origin initiation.

Mitotic degradation of yeast Fkh1 by the Anaphase Promoting Complex is required for normal longevity, genomic stability and stress resistance.

The Saccharomyces cerevisiae Forkhead Box (Fox) orthologs, Forkheads (Fkh) 1 and 2, are conserved transcription factors required for stress response, cell cycle progression and longevity. These yeast proteins play a key role in mitotic progression through activation of the ubiquitin E3 ligase Anaphase Promoting Complex (APC) via transcriptional control. Here, we used genetic and molecular analyses to demonstrate that the APC E3 activity is necessary for mitotic Fkh1 protein degradation and subsequent cell cycle progression. We report that Fkh1 protein degradation occurs specifically during mitosis, requires APCCdc20 and proteasome activity, and that a stable Fkh1 mutant reduces normal chronological lifespan, increases genomic instability, and increases sensitivity to stress. Our data supports a model whereby cell cycle progression through mitosis and G1 requires the targeted degradation of Fkh1 by the APC. This is significant to many fields as these results impact our understanding of the mechanisms underpinning the control of aging and cancer.

Fkh1 and Fkh2 associate with Sir2 to control CLB2 transcription under normal and oxidative stress conditions.

The Forkhead (Fkh) box family of transcription factors is evolutionary conserved from yeast to higher eukaryotes and its members are involved in many physiological processes including metabolism, DNA repair, cell cycle, stress resistance, apoptosis, and aging. In budding yeast, four Fkh transcription factors were identified, namely Fkh1, Fkh2, Fhl1, and Hcm1, which are implicated in chromatin silencing, cell cycle regulation, and stress response. These factors impinge transcriptional regulation during cell cycle progression, and histone deacetylases (HDACs) play an essential role in this process, e.g., the nuclear localization of Hcm1 depends on Sir2 activity, whereas Sin3/Rpd3 silence cell cycle specific gene transcription in G2/M phase. However, a direct involvement of Sir2 in Fkh1/Fkh2-dependent regulation of target genes is at present unknown. Here, we show that Fkh1 and Fkh2 associate with Sir2 in G1 and M phase, and that Fkh1/Fkh2-mediated activation of reporter genes is antagonized by Sir2. We further report that Sir2 overexpression strongly affects cell growth in an Fkh1/Fkh2-dependent manner. In addition, Sir2 regulates the expression of the mitotic cyclin Clb2 through Fkh1/Fkh2-mediated binding to the CLB2 promoter in G1 and M phase. We finally demonstrate that Sir2 is also enriched at the CLB2 promoter under stress conditions, and that the nuclear localization of Sir2 is dependent on Fkh1 and Fkh2. Taken together, our results show a functional interplay between Fkh1/Fkh2 and Sir2 suggesting a novel mechanism of cell cycle repression. Thus, in budding yeast, not only the regulation of G2/M gene expression but also the protective response against stress could be directly coordinated by Fkh1 and Fkh2.

Mechanistic insights into aging, cell-cycle progression, and stress response.

The longevity of an organism depends on the health of its cells. Throughout life cells are exposed to numerous intrinsic and extrinsic stresses, such as free radicals, generated through mitochondrial electron transport, and ultraviolet irradiation. The cell has evolved numerous mechanisms to scavenge free radicals and repair damage induced by these insults. One mechanism employed by the yeast Saccharomycescerevisiae to combat stress utilizes the Anaphase Promoting Complex (APC), an essential multi-subunit ubiquitin-protein ligase structurally and functionally conserved from yeast to humans that controls progression through mitosis and G1. We have observed that yeast cells expressing compromised APC subunits are sensitive to multiple stresses and have shorter replicative and chronological lifespans. In a pathway that runs parallel to that regulated by the APC, members of the Forkhead box (Fox) transcription factor family also regulate stress responses. The yeast Fox orthologs Fkh1 and Fkh2 appear to drive the transcription of stress response factors and slow early G1 progression, while the APC seems to regulate chromatin structure, chromosome segregation, and resetting of the transcriptome in early G1. In contrast, under non-stress conditions, the Fkhs play a complex role in cell-cycle progression, partially through activation of the APC. Direct and indirect interactions between the APC and the yeast Fkhs appear to be pivotal for lifespan determination. Here we explore the potential for these interactions to be evolutionarily conserved as a mechanism to balance cell-cycle regulation with stress responses.

The reverse, but coordinated, roles of Tor2 (TORC1) and Tor1 (TORC2) kinases for growth, cell cycle and separase-mediated mitosis in Schizosaccharomyces pombe.

Target of rapamycin complexes (TORCs), which are vital for nutrient utilization, contain a catalytic subunit with the phosphatidyl inositol kinase-related kinase (PIKK) motif. TORC1 is required for cell growth, while the functions of TORC2 are less well understood. We show here that the fission yeast Schizosaccharomyces pombe TORC2 has a cell cycle role through determining the proper timing of Cdc2 Tyr15 dephosphorylation and the cell size under limited glucose, whereas TORC1 restrains mitosis and opposes securin-separase, which are essential for chromosome segregation. These results were obtained using the previously isolated TORC1 mutant tor2-L2048S in the phosphatidyl inositol kinase (PIK) domain and a new TORC2 mutant tor1-L2045D, which harbours a mutation in the same site. While mutated TORC1 and TORC2 displayed diminished kinase activity and FKBP12/Fkh1-dependent rapamycin sensitivity, their phenotypes were nearly opposite in mitosis. Premature mitosis and the G2-M delay occurred in TORC1 and TORC2 mutants, respectively. Surprisingly, separase/cut1-securin/cut2 mutants were rescued by TORC1/tor2-L2048S mutation or rapamycin addition or even Fkh1 deletion, whereas these mutants showed synthetic defect with TORC2/tor1-L2045D. TORC1 and TORC2 coordinate growth, mitosis and cell size control, such as Wee1 and Cdc25 do for the entry into mitosis.