Scientific Report 2006

Molecular Biology

Transcriptional and Proteolytic Control of Cell Proliferation and Adaptation to Environmental Stimuli

C. Wittenberg, M. Ashe, R. de Bruin, B.-K. Han. M. Guaderrama, T. Kalashnikova

Cellular decision making and coordination of cellular events often involves the differential regulation of the expression of genes. Recently, we have focused on the mechanisms through which cells exert control over gene expression to regulate cell proliferation and the response to changes in environmental conditions.

Regulation of Cell Proliferation

In most cells, commitment to a new round of cell division during the G₁ phase of the cell cycle is accompanied by the activation of a large family of genes that encode activities involved in the duplication and segregation of cellular components. G₁-specific genes also encode regulatory factors that promote subsequent cell-cycle events. In the budding yeast Saccharomyces cerevisiae, G₁-specific genes are regulated by 2 transcription factors: SBF and MBF. Using mass spectrometry–based multidimensional protein identification technology, we have identified novel regulators of these transcription factors.

SBF acts as a transcriptional activator and promotes expression of its targets specifically during the G₁ interval. We established that promoter-bound SBF associates with the Whi5 repressor during early G₁ phase and that Whi5 is inactivated via phosphorylation by a G₁-specific cyclin-dependent protein kinase, thereby activating transcription (Fig. 1). This regulation is analogous to the regulation of E2F by the tumor suppressor Rb in metazoans.

MBF, in conjunction with specific corepressors, acts primarily as a transcriptional repressor and limits transcription of target genes to the G₁ phase. We identified Nrm1, a novel MBF-associated corepressor. When expressed as an MBF target during late G₁ phase, Nrm1 associates with MBF at target promoters and represses expression as cells enter S phase (Fig. 1). Similarly, the Nrm1 homolog, SpNrm1, in the fission yeast Schizosaccharomyces pombe regulates its only G₁-specific transcription factor, MBF.

Fig. 1. Transcriptional circuitry regulating G1-specific gene expression. G1-specific transcription in S cerevisiae is regulated by 2 heterodimeric transcription factors: SBF and MBF. Both transcription factors are bound to promoters during G1 phase before commitment to a new cell cycle. Commitment occurs when their target genes are activated by the action of the cyclin-dependent protein kinase Cln3/CDK1. For SBF, Cln3/CDK1 activates transcription by phosphorylation and inactivation of the SBF-specific repressor Whi5. Once activated, G1-specific transcription leads to the accumulation of many proteins, including those that promote repression of G1-specific transcription. Nrm1 is an MBF-specific corepressor encoded by an MBF target gene. Together, these regulatory proteins can explain the confinement of G1-specific transcription to the G1 phase.

The G₁-specific transcriptional machinery is regulated by checkpoints that monitor the integrity of cellular structures and processes. When replication forks are stalled during S phase in the fission yeast, repression of MBF-regulated transcription is disrupted. We have shown that that response, which requires the Rad3 (ATM) and Cds1 (Chk2) checkpoint protein kinases, leads to the phosphorylation of SpNrm1 and dissociation from MBF-regulated promoters. Unexpectedly, according to the literature, derepression of MBF target genes also occurs via regulation of Nrm1 in response to activation of the DNA replication checkpoint in budding yeast. Consequently, replication stress appears to be associated with genomic instability in the absence of Nrm1.

Adaptation to Environmental Stimuli

Adaptation to environmental changes generally involves remodeling of the gene expression program. We have studied the regulation of the HXT genes, which encode hexose permeases, in response to extracellular glucose. Those genes are induced by glucose and are repressed for most other carbon sources. Extracellular glucose interacts with the Snf3 and Rgt2 receptors, initiating a signaling cascade that culminates with the activation of HXT gene transcription. We have shown that signaling leads to the phosphorylation-dependent destruction of a transcriptional corepressor, Mth1, by the E3 ubiquitin ligase SCF^Grr1. Destruction of Mth1 leads to the phosphorylation of the transcriptional repressor Rgt1 and its dissociation from HXT gene promoters. Conversely, repression of HXT gene expression requires Mth1 and is associated with Rgt1 dephosphorylation. We recently identified a type 2A protein phosphatase complex involved in Rgt1 dephosphorylation and are actively pursuing the protein kinase involved in Rgt1 phosphorylation.

Interestingly, SCF^Grr1, the E3 ubiquitin ligase required for HXT gene induction, is also important for destruction of phosphorylated G₁ cyclins, critical regulators of cell-cycle initiation. We are investigating the basis for discrimination between targets by SCF^Grr1. We found that basic residues in the leucine-rich repeat and parts of the carboxy terminus of the F-box protein Grr1 are important for recognition of phosphorylated substrates. Our recent identification of additional substrates and additional characterization of Grr1 are facilitating those studies.

Publications

de Bruin, R., Kalashnikova, T.I., Chawan, C., McDonald, W.H., Wohlschlegel, J.A., Yates, J. III, Russell, P., Wittenberg, C. Constraining G₁-specific transcription to late G₁ phase: the MBF-associated corepressor Nrm1 acts via negative feedback. Mol. Cell 23:483, 2006.