I was watching TV last night and serendipitously caught the opening scenes of “Austin Powers: International Man of Mystery”. The year is 1967, and Dr. Evil is growing increasingly frustrated as his plots for world domination continue to be stymied by the eponymous super spy. Failing in his last-ditch attempt to kill Mr. Powers, Dr. Evil retreats to a hidden cryogenic chamber to bide his time and await a distant future free of his nemesis and ripe for domination. “And so Dr. Evil escaped…”, spoke the narrator, “to return at a time when free love no longer reigned and greed and corruption ruled again.” The best movie villains engender a mix of loathing and sympathy, and in this instance, I felt for Dr. Evil. Would that we all, in our times of despair, could power down, wait out our adversity, and return when the world has become more amenable to our needs and goals. While this may seem a distant dream to you or me, for some organisms, and even some cells within our bodies, it is already a natural part of life.
Quiescence – a state of cellular dormancy – “allows cells to remain dormant for long periods of time to survive harsh conditions or to prevent over-proliferation,” writes Dr. Christine Cucinotta, a postdoctoral fellow in the lab of Dr. Toshi Tsukiyama in the Basic Sciences Division at Fred Hutch and aficionado of the quiescent state. In this condition, cells stop dividing, shut down transcription, and can survive for long periods. Many cells in our bodies rely on quiescence to ensure they’re ready and available when we require them – oocyte precursors, born in the embryo, await their reactivation after puberty and sexual maturation; tissue-resident stem cells occupy specialized long-term niches, ready to jump into action and help rebuild if the tissue is ever damaged; “memory” immune cells lie dormant until the infectious agent to which they are attuned reappears and must once more be fought off (much like the strategy Austin Powers employs to await Dr. Evil’s eventual return). So, too, can some organisms enter a quiescent state. Drs. Cucinotta and Tsukiyama study this process in budding yeast, which can shut down when resources are scarce and reawaken when their environment is better suited to support their growth and proliferation.
Shutting down cellular activity is one thing. But getting things back up and running is another challenge altogether. “Reversibility is a conserved hallmark of quiescent cells,” write the authors. In a new paper published in eLife, Drs. Cucinotta and Tsukiyama examined how yeast prepare their genomes for robust reactivation upon their reawakening. By feeding cells to induce quiescence exit and performing carefully timed RNA-seq to identify nascent transcripts, “we find that half of the yeast genome is significantly transcribed within 5 minutes of quiescence exit”, said Dr. Cucinotta. “This was surprising to us because in quiescence, chromatin is highly repressive and very little transcription occurs.” How, they wondered, could the DNA, which like a January (or March) string of Christmas lights had been tightly wound up and packed away within the nucleus, have been so rapidly unpacked and made accessible to the transcriptional machinery? The chromatin must, they thought, be primed in some way to reactivate transcription at a moment’s notice. Perhaps the RNA polymerase (Pol II) was already sitting on the DNA, poised and ready for a signal that quiescence was over, and it was time to get to work. But ChIP-Seq analysis showed that there was little Pol II associated with DNA in quiescent cells. Perhaps instead, they then mused, it had to do with the way the DNA was packaged. In our cells, DNA is wrapped around histone proteins into a series of bundles called nucleosomes, which are packed side by side into chromatin. Tightly wrapped chromatin, like that observed in quiescent cells, restricts access to proteins such as Pol II and blocks transcription. The authors hypothesized that the nucleosomes containing the DNA that Pol II would need to access – the promoter regions – were “fragile”, and thus primed to be quickly knocked off at the end of quiescence. To test this, they performed a weak enzyme digestion to only disrupt the most fragile nucleosomes, and indeed found that this exposed many of the regions that were rapidly reactivated.
Having found the quiescent chromatin’s sensitive spots, the group then sought to identify the protein responsible for rapidly remodeling these regions. For this, they focused on RSC, a chromatin remodeling complex that is known to be able to remove fragile nucleosomes. Using ChIP-Seq to identify RSC binding sites in quiescent cells, the authors found that this complex is bound near most of the gene promoters that get rapidly activated. As Dr. Cucinotta described, “[The RSC binding pattern] indicates it is sitting in an inactive state, essentially poised for rapid activity in exit… [RSC] sits in promoters…and is ready to open them (via moving nucleosomes) for RNA Pol II to transcribe.” To further test this concept, they depleted RSC from quiescent cells and asked whether they experienced defects with quiescent exit. “Without RSC, Pol II basically wreaks havoc on the transcriptome [during quiescent exit],” Dr. Cucinotta explained. “Genes that should be fully transcribed aren’t, transcripts start at the wrong places, and transcription occurs in the complete wrong direction. Furthermore, without RSC, cells cannot re-enter the cell cycle after quiescence, highlighting how important RSC is to quiescence exit.” These findings reveal the quiescent chromatin in a state of poised slumber – neatly inactivated and packed away but carefully organized to allow for a rapid resumption of activity when the need arises.
Looking to the future, Dr. Cucinotta is excited about the new questions her research has sparked. “There are many new directions we can go,” she says. These include studying how RSC is regulated: “One question we are asking is what mechanism sequesters RSC to [these sites] in quiescence entry”; the roles of other regulators of transcription: “One of the ‘lesser gems’ we found in this study was that histone acetylation, a mark associated with active chromatin, is delayed during [quiescence exit]; and the extent to which these principles apply outside of yeast: “Since RSC is conserved in yeast and humans, an exciting direction is to find out if RSC is required for exiting from quiescence in mammalian cells as well.”
Cucinotta C.E., Dell R.H., Braceros K.C.A, Tsukiyama T. (2021) RSC primes the quiescent genome for hypertranscription upon cell-cycle re-entry. eLife 10:e67033
This work was supported by the National Institutes of Health.
Fred Hutch/UW Cancer Consortium member Toshio Tsukiyama contributed to this work.