Linda L. Breeden

Cell division control in yeast.

Our focus is to understand how the commitment to the mitotic cell cycle is regulated in response to environmental and internal cues, as well as adversities such as DNA damage. 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 exit from G1 and begin another cell cycle. We are studying what controls the expression of these three G1 cyclins and other critical cell cycle regulators that are specifically expressed during G1.

There are two consecutive waves of transcription that occur during G1. The first occurs at the M/G1 boundary and we have defined a new 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.

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. Our goal is to understand how internal and external signals modulate the activity of these transcription complexes and control the timing of the transition to S phase and determine cell size. A complex of Swi4 and Swi6 (referred to as SBF) plays an essential and rate limiting role in the transcription of the late G1 transcripts. As noted above, Swi4 is transcriptionally regulated by ECB elements, and Swi6 activity is regulated, in part, by cell cycle specific phosphorylation which controls its nuclear localization. We continue to investigate the regulation of these transcription complexes during the mitotic cycle. We have also begun to utilize a new purification method for investigating the cell cycle in quiescent yeast cells undergoing the G0 to S transition.

With the explosion of transcript data available through microarray technology, there is a need for new methods for analyzing and managing this data. We have collaborated with UW and FHCRC bioinformaticists to develop statistical methods that enable investigators to identify transcripts which oscillate through the cell cycle and the transcription factors responsible. Using these methods we have recently identified an S phase-specific transcription factor that activates transcription of genes primarily involved in chromosome segregation and budding.

We have also used our cell cycle microarray data to identify genes whose periodic 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.