Image provided by Dr. Linda Breeden.
Cells can enter a non-dividing state called quiescence in which the metabolic activity of the cell drops dramatically, stress tolerance increases, and the lifespan of the cell is extended. The molecular mechanisms that control entry into the dividing or quiescent state are shared between humans and laboratory model organisms such as budding yeast. While much has been learned over the years about the transition into the division cyclefrom studies of budding yeast, for examplerelatively little has been uncovered about the transition out of the division cycle and into the quiescent state. The ability to enter and exit quiescence is central to tissue homeostasis and inappropriate regulation of this transition can result in uncontrolled cell division, causing cancer and cell death if left unchecked.
The transition into the dividing state, which begins when cells enter into S phase (for DNA synthesis), is controlled by a protein complex that represses S phase genes until a signal to divide is received. In their recent publication in PLOS Genetics, researchers in the Breeden Lab (Basic Sciences Division) clarify the role of several proteins in this repressive complex in the transition into quiescence. They also identify several proteins with critical roles in promoting the transition into quiescence.
Quiescent cells appear in yeast populations that have exhausted their sugar supply. Once the sugar source is limited, yeast will asymmetrically divide to produce smaller, denser quiescent (Q) cells. These cells can be purified by centrifugation because of their higher density. The Breeden Lab first found that the complexes responsible for the transition into S-phase, called SBF/MBF, also control the transition into the Q state. This led them to characterize a panel of genetic mutants that affect the length of time spent before committing to S-phase. Interestingly, they found that the length of time a particular strain of yeast spends before entering S phase correlates strongly with how many Q cells can be produced when the yeast are starved. The longer the delay of S phase for a particular genetic mutant, the more Q cells they were able to form. This indicates that the interval before S phase, referred to as G1, is the phase when cells set up for the transition to quiescence. The more time they have in G1, the more successful they are at this transition.
This led to the next question, which was what changes MBF/SBF from a complex that promotes S phase to one that prevents S phase and allows cells to enter a stable quiescent state? After finding no truly Q-specific proteins among the well-known MBF/SBF components, the scientists decided to test two lesser-known proteins called Msa1 and Msa2 that had been identified as interacting with MBF/SBF by protein-interaction studies. Intriguingly, they found that strains with both proteins mutated, called msa1msa2 double mutant, are able to enter S phase but die when sugar becomes limiting. Because they died once the sugar supply was exhausted, the msa1msa2 mutant did not produce Q cells, indicating these proteins are required for a successful transition into the Q state. The single mutants of either msa1 or msa2 alone did not appear to grow very differently from wild-type cells and were still able to enter quiescence, so it appears that either Msa1 or Msa2 protein alone can promote the transition to quiescence.
As members of the SBF/MBF complexes, Msa1 and Msa2 likely control the expression of genes required for G1-S or G1-Q transition. To investigate this in more detail, the scientists performed RNA deep sequencing on wild-type and msa1msa2 yeast to compare which genes are expressed when the cells are dividing in rich media versus when they are starved and entering Q. They found that many genes regulated by the SBF/MBF complex were differentially expressed in the msa1msa2 double mutant compared to wild-type yeast, specifically when the cells were starving. This highlights the role of Msa1 and Msa2 in a quiescence-specific SBF/MBF gene regulatory complex. Similar quiescence-specific complexes have been identified in worms, flies and mammals, indicating that this is a highly conserved strategy for controlling cell division (see Figure).
Researchers in the Breeden Lab are continuing their investigations, studying the unique properties of quiescent cells. Dr. Breeden explains, "We’ve just selected the small subset of a yeast population that can survive for a year in the non-dividing state. These are like humans that live for 100 years. Modern sequencing techniques allow us to identify the traits responsible for this longevity. We call this our Fountain of Youth project!"
Miles S, Croxford MW, Abeysinghe AP, Breeden LL. 2016. "Msa1 and Msa2 Modulate G1-Specific Transcription to Promote G1 Arrest and the Transition to Quiescence in Budding Yeast." PLOS Genetics. 12(6): e1006088.
Funding for this research was provided by the National Institute on Aging.