Linda L. Breeden

Cell division control in budding yeast.

Our focus is to understand how the commitment to the mitotic cell cycle is regulated in response to environmental and internal cues. The critical transitions in the eukaryotic cell cycle are controlled by cyclin-dependent kinases (CDKs). In budding yeast, as in all higher eukaryotes, the decision to commit to another division cycle occurs in G1. Nine cyclins have been identified that bind and activate a single CDK, and three of these cyclins (Cln1,2 and 3) play critical roles in modulating the decision to enter the cell cycle. We are studying what controls the activity of these three G1 cyclins and other critical regulators of the transition into S phase.

Much of the work of the last decade has involved studying the cell cycle in rapidly growing cells with abundant nutrients. We have begun to investigate a far more common transition, which is the transition of quiescent, non-dividing yeast cells back into the cell cycle. All cells appear to be capable of entering a resting or quiescent state, and most spend the bulk of their life in this state. It follows that entry into, maintenance of, and recovery from quiescence is extremely important for the viability of the species. As such, it has been under constant evolutionary pressure and it is reasonable to assume that all cells have elaborated specific mechanisms to efficiently switch between mitotic growth and quiescence when conditions warrant such a switch. Interestingly, quiescent yeast and mammalian cells share several characteristics. They exit the mitotic cycle from G1 with unreplicated DNA. All quiescent cells condense their chromosomes, drastically reduce transcription and translation rates and increase rates of autophagy.

To accomplish and maintain the quiescent state, the mitotic cycle must be stably repressed without compromising viability. When conditions change and the cue to resume mitotic growth is received, this repressed state must be reversed. Releasing cells from quiescence or preventing them from entering this state are hallmarks of oncogenesis. Pathways controlling the decision to remain in the cell cycle or to exit from it are defective in most if not all human tumors {Hanahan, 2000 #1744}. The quiescent state of budding yeast is unlikely to be identical to that of metazoan cells, but the strategies for arresting and releasing cells from this non-dividing state may share important features, just as the framework of the mitotic cycle is shared. The metabolic needs of a resting cell are probably also quite similar across all phyla. As such, the strategies for maintaining this non-dividing state are also likely to share some features. Recent technical advances from the Werner-Washburne lab make it possible to examine the quiescent state and the recovery from it in pure populations of quiescent cells. We have implemented these methods to identify the genetic determinants that affect the establishment of this state and that regulate re-entry into the mitotic cycle.

Our long term goal has been to understand how the commitment to the mitotic cell cycle is regulated in response to environmental and internal cues. Most of this work has been done with rapidly growing cells and with cells subjected to DNA damage. Our most recent accomplishments are summarized below.

There are two consecutive waves of transcription that occur during G1. The first occurs at the M/G1 boundary and we have defined the promoter element, the ECB, which activates transcription of CLN3 and other key cell cycle regulators at this time (SWI4, CDC6 and MCM2-7). ECB activity is regulated by Mcm1 and a pair of homeobox proteins (Yox1 and Yhp1). The latter serve as cell cycle specific repressors of ECB activity. Using microarrays and new computational approaches to identify periodic transcripts and promoter elements, we have identified a group of ECB-regulated and M/G1-specific transcripts. We have also shown that the nascent transcription of Mcm2-7 is important for their cell cycle regulated nuclear import, which is a prerequisite for formation of pre-initiation complexes on the origins of DNA replication.

The second wave of transcription is conferred by at least two other promoter elements (SCBs and MCBs), which are activated in late G1 to induce the expression of CLN1, CLN2 and dozens of other genes that are required for DNA replication and cell wall synthesis. Among these target genes is Hcm1, which we have shown to be an S phase-specific transcription factor that activates transcription of genes primarily involved in chromosome segregation and budding. The timely expression of these Hcm1 targets promotes efficient spindle assembly. In its absence, the spindle checkpoint proteins that delay the cell cycle until chromosomes are properly aligned on the spindle become essential for viability.

During the course of these studies, we have generated microarray data across the cell cycle. We have used these data to identify and characterize yeast genes with periodic transcription. We have also collaborated with bioinformaticists to identify genes whose transcription is conserved from yeast to humans. There are about 100 of these transcripts and their gene products in yeast are eight times more likely to be phosphorylated by cyclin-dependent kinases than are random proteins. These findings support the view that there is a conserved core of proteins that are regulated at multiple levels during the cell cycle. One-third of these genes are also among the putative targets of Hcm1 in budding yeast. Hcm1 is a member of the large and conserved fork head family of transcription factors.