RSC primes the quiescent genome for hypertranscription upon cell-cycle re-entry
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Evaluation Summary:
Cucinotta et al. describe mechanisms that support an intense burst of transcription from many genes within minutes of nutrient repletion as Saccharomyces cerevisiae cells emerge from quiescence. They focus primarily on the role of the nucleosome remodeler RSC in managing chromatin architecture over promoters during quiescence and as cells re-enter the cell cycle using a broad range of genome-wide measurements that strongly support the conclusions. This important process of cell cycle re-entry from quiescence is understudied but impacts areas as diverse as development and carcinogenesis in multicellular organisms to long-term survival and adaptation of microorganisms to environmental cues, so the results will be of interest to a broad audience.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Quiescence is a reversible G 0 state essential for differentiation, regeneration, stem-cell renewal, and immune cell activation. Necessary for long-term survival, quiescent chromatin is compact, hypoacetylated, and transcriptionally inactive. How transcription activates upon cell-cycle re-entry is undefined. Here we report robust, widespread transcription within the first minutes of quiescence exit. During quiescence, the chromatin-remodeling enzyme RSC was already bound to the genes induced upon quiescence exit. RSC depletion caused severe quiescence exit defects: a global decrease in RNA polymerase II (Pol II) loading, Pol II accumulation at transcription start sites, initiation from ectopic upstream loci, and aberrant antisense transcription. These phenomena were due to a combination of highly robust Pol II transcription and severe chromatin defects in the promoter regions and gene bodies. Together, these results uncovered multiple mechanisms by which RSC facilitates initiation and maintenance of large-scale, rapid gene expression despite a globally repressive chromatin state.
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Reviewer #3 (Public Review):
Cellular quiescence, the reversible exit from the cell cycle, is essential for long-term cell survival. One feature of quiescent cells is transcription inactivation and this paper examines gene reactivation during quiescence exit and the accompanying changes to chromatin structure. Using a variety of genome-wide analyses, including 4tU-seq, ChIP-seq, and MNase-seq, the authors show that transcription occurs within minutes of quiescence exit, and for most genes, the initial rate of transcription exceeds that of normal cycling cells. Moreover, this work shows that gene repression during quiescence, and activation upon quiescence exit, are associated with distinct chromatin organization, particularly over promoters. Finally, the authors uncover a role for the RSC chromatin-remodeling complex in establishing a …
Reviewer #3 (Public Review):
Cellular quiescence, the reversible exit from the cell cycle, is essential for long-term cell survival. One feature of quiescent cells is transcription inactivation and this paper examines gene reactivation during quiescence exit and the accompanying changes to chromatin structure. Using a variety of genome-wide analyses, including 4tU-seq, ChIP-seq, and MNase-seq, the authors show that transcription occurs within minutes of quiescence exit, and for most genes, the initial rate of transcription exceeds that of normal cycling cells. Moreover, this work shows that gene repression during quiescence, and activation upon quiescence exit, are associated with distinct chromatin organization, particularly over promoters. Finally, the authors uncover a role for the RSC chromatin-remodeling complex in establishing a chromatin organization that facilitates normal gene expression during quiescence exit. To support the above findings, the authors generated an impressive amount of sequencing datasets that robustly support their findings and will undoubtedly be of great use to many yeast transcription researchers. Although more transparent and consistent bioinformatic analyses of these data would better communicate the findings, this work enhances our understanding of gene expression changes during the transition between key cell states and thus will be of interest to a broad spectrum of readers ranging from molecular to developmental biologists.
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Reviewer #2 (Public Review):
In this manuscript, Cucinotta et al investigate the role of the conserved RSC chromatin remodeler in preparing cells for hypertranscription during exit from quiescence using cellular perturbations and a range of genomic techniques. They find that upon exit from quiescence there is a large and rapid increase in transcription (within 5 minutes) and this hypertranscription cannot be explained solely by alterations to histone acetylation. Therefore, the authors investigated what is driving this process and identified that RSC, a well describe chromatin remodeler with activities in altering chromatin structure to promote transcription, has altered binding profiles within quiescent cells relative to log cells, and loss of RSC results in altered nucleosome positioning within gene bodies and increased histone …
Reviewer #2 (Public Review):
In this manuscript, Cucinotta et al investigate the role of the conserved RSC chromatin remodeler in preparing cells for hypertranscription during exit from quiescence using cellular perturbations and a range of genomic techniques. They find that upon exit from quiescence there is a large and rapid increase in transcription (within 5 minutes) and this hypertranscription cannot be explained solely by alterations to histone acetylation. Therefore, the authors investigated what is driving this process and identified that RSC, a well describe chromatin remodeler with activities in altering chromatin structure to promote transcription, has altered binding profiles within quiescent cells relative to log cells, and loss of RSC results in altered nucleosome positioning within gene bodies and increased histone occupancy within nucleosome depleted regions (NDRs). They find that RSCs biochemical activity is important for promoting transcription and is required for appropriate RNAPII occupancy during exit. Finally, they find that RSC is required for appropriate transcription as depletion of RSC results in increase aberrant transcription, leading to the model that RSC is important for regulating chromatin structure for appropriate binding of RNAPII throughout the genome during exit from quiescence. The conclusions of this paper are well supported by data, but some aspects of data analysis need to be extended.
Strengths:
To my knowledge, this is the first mechanistic description of quiescent exit, adding to the many roles of the important RSC chromatin remodeling complex. The data are extensive to support the claims made by the authors. Data are also clearly described within the text and put into great context within the field.
Weaknesses:
Correlations are not directly drawn across the datasets, and aspects of data presentation could be clarified. For example, there is little comparison between the expression data (4tU-seq) and the localization (ChIP-seq) or nucleosome positioning (MNase-seq) datasets. Direct comparisons of where locations have altered factor occupancy and/or nucleosome changes with the expression changes or aberrant transcription increases would help facilitate a mechanistic description.
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Reviewer #1 (Public Review):
Cucinotta et al. examine the widespread, transient transcription of genes that occurs within minutes of refeeding quiescent Saccharomyces cerevisiae cells, focusing on the role of the RSC remodeler complex in this process. A range of appropriate genomic approaches are used to characterize the initial burst of transcription, changes in localization of RSC and RNA Pol II, and changes in the occupancy and positioning of nucleosomes during the first minutes after nutrient repletion. Several new insights are reported including the role of RSC in maintaining promoters in state that is ready to respond rapidly to nutrient repletion, the relocalization of RSC into genes following initiation of transcription, a role for TFIIS in exiting quiescence that was not apparent in log phase, the timing of histone acetylation …
Reviewer #1 (Public Review):
Cucinotta et al. examine the widespread, transient transcription of genes that occurs within minutes of refeeding quiescent Saccharomyces cerevisiae cells, focusing on the role of the RSC remodeler complex in this process. A range of appropriate genomic approaches are used to characterize the initial burst of transcription, changes in localization of RSC and RNA Pol II, and changes in the occupancy and positioning of nucleosomes during the first minutes after nutrient repletion. Several new insights are reported including the role of RSC in maintaining promoters in state that is ready to respond rapidly to nutrient repletion, the relocalization of RSC into genes following initiation of transcription, a role for TFIIS in exiting quiescence that was not apparent in log phase, the timing of histone acetylation in response to transcription, changes in chromatin architecture during the exit from quiescence, and the effects of chromatin changes on transcription start site selection and repression of antisense transcription from downstream nucleosome depleted regions. Given how little is known about the emergences of cells from quiescence and how common and important this transition is in long-term viability, development, and carcinogenesis, these insights are certain to have broad impact. The data are of high quality and the manuscript is very clearly written, with good correlation between the level of support provided by the data and the strength of the conclusions drawn. Only minor issues remain to be addressed.
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Evaluation Summary:
Cucinotta et al. describe mechanisms that support an intense burst of transcription from many genes within minutes of nutrient repletion as Saccharomyces cerevisiae cells emerge from quiescence. They focus primarily on the role of the nucleosome remodeler RSC in managing chromatin architecture over promoters during quiescence and as cells re-enter the cell cycle using a broad range of genome-wide measurements that strongly support the conclusions. This important process of cell cycle re-entry from quiescence is understudied but impacts areas as diverse as development and carcinogenesis in multicellular organisms to long-term survival and adaptation of microorganisms to environmental cues, so the results will be of interest to a broad audience.
(This preprint has been reviewed by eLife. We include the public reviews …
Evaluation Summary:
Cucinotta et al. describe mechanisms that support an intense burst of transcription from many genes within minutes of nutrient repletion as Saccharomyces cerevisiae cells emerge from quiescence. They focus primarily on the role of the nucleosome remodeler RSC in managing chromatin architecture over promoters during quiescence and as cells re-enter the cell cycle using a broad range of genome-wide measurements that strongly support the conclusions. This important process of cell cycle re-entry from quiescence is understudied but impacts areas as diverse as development and carcinogenesis in multicellular organisms to long-term survival and adaptation of microorganisms to environmental cues, so the results will be of interest to a broad audience.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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