To survive, organisms must resist stress. Quiescence (or G0) is an adaptive response in which cells resist stress, enter a reversible dormancy and remain viable for long periods of time. How cells enter, survive and exit G0 is a critical question in basic biology, which is currently poorly understood. This is because for decades G0 was often deemed as an `uneventful' state in the cell cycle; however, recent discoveries have challenged this dogma and underscored the importance of G0 regulation in several human pathologies, including cancers. Therapeutic intervention requires a basic understanding of how cells transition to and from G0 and the molecular switches that govern these processes. Remarkably, these remain largely open questions in biology. G0 entry requires the establishment of a unique transcriptional state, which imparts distinct properties to G0 cells. It is also concomitant with redistribution of heterochromatic marks, suggesting that heterochromatin proteins contribute to G0 establishment. To address this knowledge gap, we recently modeled G0 in the fission yeast cells. G0 in S. pombe bears many similarities to that in mammalian cells including inducing signals (nutrient depletion), long life, resistance to genotoxic agents, lower metabolism, and use the same biological pathways (e.g. autophagy) and protein complexes (e.g. proteasome). The major advantage of using fission yeast as a G0 model is that upon nitrogen starvation (or glucose deprivation) all cells enter quiescence uniformly, creating pure, synchronous and isogenic populations of G0 cells. We exploited these features and developed a time-course G0 assays to track cell viability, chromatin, transcriptional and small RNA (sRNA) changes temporally. We found that as cells enter G0 a new class of Argonaute 1(Ago1)-associated small RNAs (G0 sRNAs) emerges, which deploys constitutive heterochromatin proteins to euchromatic parts of the genome. We showed that RNAi- and heterochromatin proteins are essential for survival and establishment of the G0 program. Overall, we discovered a novel role for constitutive heterochromatin proteins (global regulation of transcription in G0) and nuclear Ago1-associated sRNAs in adaptation to long-term stress. Based on these, we proposed a general model for Quiescent-induced Transcriptional Silencing (QuieTS) in eukaryotes, in which early accumulation of sequence specificity factors (e.g. G0 sRNAs) target the global deployment of regulatory proteins, under persistent stress. In this proposal, we will determine how G0 sRNA are formed (Aim 1), and identify the complexes and the mechanisms which mediate QuieTS (Aim 2). We will also test the generality of this mechanism as an adaptive response to long term stress and delineate the core and stress-specific targets of QuieTS (Aim 3). Overall, the proposed studies will greatly impact our understanding of the mechanisms by which G0 transcriptional programs are established and may reshape the current models of stress-induced transcriptional regulation in eukaryotes. We predict that these studies also may expose novel therapeutic targets for G0 pathologies.
Dormancy is an old but poorly understood concept in cell biology. Cells enter dormancy in response to stress. Dormant cells stop dividing, survive for long periods of time, but retain their ability to grow again once the environment improves. This is a major problem in cancer because long-lived dormant cancer cells are resistant to radiation and chemotherapy and seed cancer recurrence decades after therapy. To address this knowledge gap, we created an experimentally malleable model of dormancy in yeast. This allowed us to discover a critical switch, which helps cells survive in dormancy. In this grant using cutting-edge technologies, we propose to dissect this pathway molecularly, to hopefully identify drug targets for killing dormant cancer cells.
