Transition from growth to quiescence

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 from growth to quiescence. 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. 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. 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. We are using a combination of genetics, genomics and biochemistry to define the growth to quiescence transition in budding yeast.


Identifying factors important for longevity

Previous studies of quiescence or chronological aging in yeast have involved monitoring the long-term viability of stationary phase cultures, but these cultures are heterogeneous and the proportion of cells that attain the quiescent state varies in lab and natural yeast isolates.  We use density sedimentation to purify and characterize the quiescent population.  The longevity of quiescent cells also varies and we are exploiting this natural variation to select progeny with long life spans and then identifying the polymorphisms that are responsible for promoting longevity with genome sequencing.  We have identified mRNA binding proteins, transcription factors and chromatin remodelers that are important for longevity in the non-dividing state.


Exploring the growth to quiescence transition

When yeast naturally exhaust the glucose from their medium, they undergo one more division, which is highly asymmetric, and there is a slowing of physical growth. This results in a dramatic change in modal cell volume.  These cells arrest in G1, undergo a global 30-fold drop in transcription and fortify their cell walls. This unique program of G1 arrest, asymmetric cell division, chromatin re-programming and cell wall fortification leads to the production of distinct cell types in stationary phase cultures that can be distinguished by flow cytometry.  Using fluorescence-activated cell sorting, we showed that only one of these cell types has the properties of quiescent (Q) cells.  We have explored the timing of the log to Q transition using flow cytometry and we have used a high throughput flow cytometry screen of a deletion library to identify mutants that fail to G1 arrest and enter quiescence.


Mechanisms driving chromatin to a quiescent state

Just as in log phase cells, the Cln3 cyclin must be down-regulated to achieve G1 arrest.  The replication stress checkpoint is active during this interval, and it becomes essential for G1 arrest and viability if Cln3 is over-produced.  The transcription repressor Xbp1 is induced after the glucose is exhausted from the medium (referred to as the diauxic shift or DS), and it represses CLN3 and hundreds of other transcripts after the DS.  In the absence of Xbp1, cells undergo additional cell divisions.  The resulting dense Q cells are very small and both their longevity and their recovery are compromised. Xbp1 recruits Rpd3 to its promoter binding sites, where it deacetylates histones and is specifically responsible for the global transcriptional shut down that occurs in quiescent cells.

The genes that are most repressed in Q cells have promoters that show a large increase in histone density and a large decrease in histone acetylation. In the absence of Rpd3 deacetylase activity, the chromatin and transcript profiles are strikingly similar to that of cells just after the diauxic shift.  These results provide clear molecular evidence of an Rpd3-dependent mechanism that drives chromatin to a very distinct quiescent state, which may be conserved in higher cells.