Clp1p and Mid1p form links between cell cycle progression and gene expression at cytokinesis in fission yeast

Comment on: Papadopoulou K, et al. J Cell Sci 2010; 123:4374–81 and Agarwal M, et al. J Cell Sci 2010; 123:4366–7

Cell cycle regulated gene expression in yeasts

The regulation of gene expression through the mitotic cell cycle, so that genes are transcribed at particular cell cycle times, is widespread among eukaryotes. In some cases, it appears to be important for control mechanisms, as deregulated expression results in uncontrolled cell divisions, which can cause cell death, disease, and malignancy. In this review, I describe the current understanding of such regulated gene expression in two established simple eukaryotic model organisms, the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. In these two yeasts, the global pattern of cell cycle gene expression has been well described, and most of the transcription factors that control the various waves of gene expression, and how they are in turn themselves regulated, have been characterized. As related mechanisms occur in all other eukaryotes, including humans, yeasts offer an excellent paradigm to understand this important molecular process

Pol5p, a novel binding partner to Cdc10p in fission yeast involved in rRNA production

Cdc10p is a major component of the cell cycle transcription factor complex MBF that controls G1-S phase specific gene expression in the fission yeast Schizosaccharomyces pombe. Here, we describe the identification of a new binding partner to Cdc10p and Pol5p. Pol5p was discovered through a 2-hybrid screen, with the direct interaction confirmed by in vitro "pull-down" experiments with bacterially expressed proteins. Pol5p appears to have no role in cell cycle gene expression, but is instead required for rRNA production. Pol5p is an essential gene, expressed constitutively throughout both the mitotic and meiotic life cycles, and localises to the nucleus. Over-expressing Pol5p has no phenotype, but reducing levels of Pol5p inhibits rRNA production. Pol5p is shown to bind to rDNA promoter fragments. Potentially, we have identified a mechanism by which Cdc10p controls rDNA gene expression, therefore linking the cell cycle with cellular growth

Regulation of gene expression and cell division by Polo-like kinases

Much scientific research has focused on characterising regulatory pathways and mechanisms responsible for cell integrity, growth and division. This area of study is of direct relevance to human medicine as uncontrolled growth and division underlies many diseases, most strikingly cancer. In cancer cells, normal regulatory mechanisms for growth and division are often altered, or even fail to exist. This review summarises the mechanisms that control the genes and gene products regulating cytokinesis and cell separation in the fission yeast Schizosaccharomyces pombe, as well as highlighting conserved aspects in the budding yeast Saccharomyces cerevisiae and higher eukaryotes. Particular emphasis is put on the role of gene expression, the Polo-like kinases (Plks), and the signal transduction pathways that control these processes

Positive and negative roles for cdc10 in cell cycle gene expression

In this paper we describe properties of the cdc10-C4 mutant of the fission yeast Schizosaccharomyces pombe. The cdc10<sup>+</sup> gene encodes a component of the DSC1<sup>Sp</sup>/MBF transcription complex, which is required for cell-cycle regulated expression at G1-S of several genes via cis-acting MCB (MIuI cell cycle box) elements. At permissive temperatures cdc10-C4 causes expression of MCB-regulated genes through the whole cell cycle, which in asynchronously dividing cells is manifested in overall higher expression levels. This overexpression phenotype is cold sensitive: in cdc10-C4 cells, MCB genes are expressed offprogressively higher levels at lower temperatures. In heterozygous cdc10-C4/cdc10<sup>+</sup> diploid strains, MCB-regulated genes are not overexpressed, suggesting that loss, rather than alteration, of function of the cdc10-C4 gene product is the reason for unregulated target gene expression. Consistent with this, the cdc10-C4 mutant allele is known to encode a truncated protein. We have also overexpressed the region of the cdc10 protein absent in cdc10-C4 under the control of an inducible promoter. This induces a G1 delay, and additionally causes a reduction of the overexpression of MCB genes in cdc10-C4 strains. These results suggest that DSC1<sup>Sp</sup>/MBF represses, as well as activates, MCB gene expression during the cell cycle

Cell cycle, DNA damage and heat shock regulate <i>suc22</i>⁺ expression in fission yeast

The &lt;i&gt;suc22&lt;/i&gt;&lt;sup&gt;+&lt;/sup&gt; gene of Schizosaccharomyces pombe encodes the small subunit of ribonucleotide reductase. Two transcripts that hybridise to &lt;i&gt;suc22&lt;/i&gt;&lt;sup&gt;+&lt;/sup&gt; have previously been described: a constitutive transcript of 1.5 kb, and a transcript of approximately 1.9 kb that is induced when DNA replication is blocked by hydroxyurea. In this paper we show that both transcripts derive from the suc22+ gene, are polyadenylated, and have transcription initiation sites separated by approximately 550 nucleotides. The absence of translation initiation codons and predicted intron splice sites within this 550 nucleotide region suggests strongly that both transcripts encode the same protein. Under normal growth conditions, the larger &lt;i&gt;suc22&lt;/i&gt;&lt;sup&gt;+&lt;/sup&gt; transcript is present at a very low level. This low level expression is periodic during the cell cycle, showing a pattern similar to that of other genes under regulation by MCB elements with a maximum in G1/S phase. Consistent with this, there are MCB elements upstream of the initiation site of the transcript. This pattern of expression contrasts with the continuous expression, at a much higher level, of the smaller &lt;i&gt;suc22&lt;/i&gt;&lt;sup&gt;+&lt;/sup&gt; transcript. The larger &lt;i&gt;suc22&lt;/i&gt;&lt;sup&gt;+&lt;/sup&gt; transcript is induced by exposure of cells to 4-nitroquinoline oxide (4-NQO),a UV-mimetic agent that causes DNA damage. The transcriptional response to 4-NQO is observed in cells previously arrested in G2 by a &lt;i&gt;cdc2&lt;/i&gt;&lt;sup&gt;ts&lt;/sup&gt; mutation, demonstrating that induction can occur outside S phase. We show that the rad1+ gene, part of the mitotic checkpoint, is required for induction of the large transcript. Exposure of cells to heat shock also induces the &lt;i&gt;suc22&lt;/i&gt;&lt;sup&gt;+&lt;/sup&gt; large transcript: a consensus heat shock element has been identified upstream of the large transcript start site

MCB-mediated regulation of cell cycle-specific cdc22(+) transcription in fission yeast

The cdc22+ gene of the fission yeast, Schizosaccharomyces pombe , encodes the large subunit of ribonucleotide reductase, and is periodically expressed during the mitotic cell cycle, transcript abundance reaching a maximum at the G1-S boundary. This regulation of expression is controlled by a transcription factor complex called DSC1, which binds to MCB motifs (ACGCGT) present in the promoter of cdc22+. cdc22+ has a complex pattern of MCBs, including two clusters of four motifs each, one of which is located within the transcribed region. We show that both clusters of MCBs contribute to the regulation of cdc22+ expression during the cell cycle, each having a different role. The MCB cluster within the transcribed region has the major role in regulating cdc22+, as its removal results in loss of transcription. The upstream cluster, instead, controls cell cycle-specific transcription through a negative function, as its removal results in expression of cdc22+ throughout the cell cycle. Both MCB clusters bind DSC1. We show that the interaction of DSC1 with the MCB cluster within the transcribed region has a high "on-off" rate, suggesting a mechanism by which DSC1 could activate expression, and still allow RNA polymerase to pass during transcription. Finally, we show that both clusters are orientation-dependent in their function. The significance of these results, in the context of MCB-mediated regulation of G1-S expression in fission yeast, is discussed

The role of DSC1 components cdc10⁺, rep1⁺ and rep2⁺ in MCB gene transcription at the mitotic G1-S boundary in fission yeast

In this paper, we describe the transcription profile of a group of genes at the G1-S boundary of fission yeast in synchronously dividing mitotic cells, under a variety of different conditions. This transcription profile is unaffected in cells where either cdc10&lt;sup&gt;+&lt;/sup&gt; or cdc10-C4 are constitutively overexpressed. In contrast, overexpression of either rep1&lt;sup&gt;+&lt;/sup&gt; or rep2&lt;sup&gt;+&lt;/sup&gt; results in constitutive expression of MCB-regulated genes, suggesting that these polypeptides have important regulatory properties in controlling MCB transcription. Finally, we examine the pattern of MCB-regulated transcription in cells where the G1 period is extended. Surprisingly, we find that the wee1-50 mutation causes MCB transcription throughout the cell cycle, whereas cells re-fed after nitrogen starvation have normal expression patterns. The implications of these observations for understanding MCB-regulated transcription are discussed

A novel Mcm1-dependent element in the SWI₄, CLN₃, CDC₆, and CDC₄₇ promoters activates M/G1-specific transcription

We have identified a novel promoter element that confers M/G1-specific transcription in Saccharomyces cerevisiae. This element, which we call an ECB (early cell cycle box), was first identified in the SWI&lt;sub&gt;4&lt;/sub&gt; promoter, but it is also present in the promoter of a G1 cyclin CLN&lt;sub&gt;3&lt;/sub&gt;, as well as in the promoters of three DNA replication genes: CDC&lt;sub&gt;6&lt;/sub&gt;, CDC&lt;sub&gt;47&lt;/sub&gt;, and CDC&lt;sub&gt;46&lt;/sub&gt;. Transcripts from all five of these genes oscillate during the cell cycle and peak at the M/G1 boundary, as do isolated ECB elements in reporter constructs. The ECB element contains an Mcm1 binding site to which Mcm1 binds in vitro, and an Mcm1-VP16 fusion, which places a constitutive activator on Mcm1-binding sites in vivo, can deregulate ECB-containing promoters. Mcm1 is a transcription factor that is also required for minichromosome maintenance. We provide evidence that the replication defect of mcm1 mutants can be suppressed by ectopic CDC6 transcription. Periodic expression of SWI4 and CLN3 may be important for cell cycle progression, as we find that these genes are both haploinsufficient and rate limiting for G1 progression. We suggest that ECB-regulated gene products play critical roles in promoting the initiation of S-phase, both by regulating CLN1 and CLN2 transcription and as components of the initiation complex on origins of replication

Cyclin-dependent kinase 8 regulates mitotic commitment in fission yeast

Temporal changes in transcription programs are coupled to control of cell growth and division. We here report that Mediator, a conserved coregulator of eukaryotic transcription, is part of a regulatory pathway that controls mitotic entry in fission yeast. The Mediator subunit cyclin-dependent kinase 8 (Cdk8) phosphorylates the forkhead 2 (Fkh2) protein in a periodic manner that coincides with gene activation during mitosis. Phosphorylation prevents degradation of the Fkh2 transcription factor by the proteasome, thus ensuring cell cycle-dependent variations in Fkh2 levels. Interestingly, Cdk8-dependent phosphorylation of Fkh2 controls mitotic entry, and mitotic entry is delayed by inactivation of the Cdk8 kinase activity or mutations replacing the phosphorylated serine residues of Fkh2. In addition, mutations in Fkh2, which mimic protein phosphorylation, lead to premature mitotic entry. Therefore, Fkh2 regulates not only the onset of mitotic transcription but also the correct timing of mitotic entry via effects on the Wee1 kinase. Our findings thus establish a new pathway linking the Mediator complex to control of mitotic transcription and regulation of mitotic entry in fission yeast