Investigation of epigenome changes in quiescent fission yeast cells

Cellular quiescence is a reversible, non-proliferative state in which cells temporarily exit the cell cycle while maintaining the capacity to re-enter it upon receiving appropriate stimuli. This state enables cells to survive under unfavorable environmental conditions and protects them from stress-induced damage.

In unicellular organisms such as bacteria and yeast, quiescence serves as a vital survival strategy in response to nutrient depletion, extreme temperatures, or pH fluctuations. Similarly, in multicellular organisms, quiescence is a hallmark of adult stem cells and other stress-responsive cell types, contributing to long-term tissue homeostasis and regeneration. Given its critical role in cellular stress resistance and regenerative capacity, elucidating the molecular mechanisms that govern quiescence has significant implications for regenerative medicine, particularly in enhancing stem cell activation and tissue repair strategies.

In this study, the fission yeast Schizosaccharomyces pombe was employed as a model system to dissect the molecular basis of cellular quiescence. We integrated multiple high-throughput and functional genomics approaches- including fluorescence-activated cell sorting (FACS), fluorescence microscopy, RNA sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP- seq), and chromosome conformation capture (Hi-C)-to investigate quiescence entry potential, chromosome condensation, genome-wide transcriptional changes, histone modification dynamics, and three-dimensional genome architecture.

Our overarching goal was to gain mechanistic insight into how transcriptional programs and epigenomic landscapes are dynamically restructured during the transition into, and maintenance of, both early and long-term quiescence.

In Study I, we identified 149 genes that were significantly upregulated during early quiescence, in contrast to a global downregulation of transcriptional activity across the genome. Among these, 16 genes sustained high expression levels from early to late quiescence and were predominantly located in subtelomeric regions. Genetic analyses using mutants of INO80 complex subunits and the H2A.Z-encoding gene revealed that INO80 plays a critical role in evicting H2A.Z prior to transcriptional elongation, thereby facilitating proper activation of quiescence-specific genes.

In Study II, ChIP-seq profiling demonstrated that the active histone mark H3K4me3 is strongly associated with quiescence gene activation. H3K4me3 was found to regulate RNAPII pausing at promoter-proximal regions, and its deposition was dependent on set1, the catalytic component of the COMPASS complex. These results underscore the importance of H3K4me3 as a central epigenetic mark in the regulation of quiescence transcriptional programs.

In Study III, live-cell fluorescence microscopy and flow cytometry (FACS) revealed that after 16 hours of nitrogen starvation, cells entering quiescence exhibited a halt in further chromosomal condensation. Complementary Hi-C analysis uncovered the emergence of highly interactive topologically associating domains (TADs) in subtelomeric regions, along with telomere hyper-clustering-a structural feature reminiscent of quiescent genome organization observed in other eukaryotic systems.

Collectively, these three studies establish a core set of quiescence-specific genes and define an integrated epigenetic and 3D structural framework that orchestrates the establishment and maintenance of cellular quiescence in Schizosaccharomyces pombe.

List of scientific papers

I. Yasaman Zahedi, Shengyuan Zeng, Karl Ekwall. An essential role for the Ino80 chromatin remodeling complex in regulation of gene expression during cellular quiescence. Chromosome Res. 2023 Apr 12;31(2):14. https://doi.org/10.1007/s10577-023-09723-x

II. Shengyuan Zeng, Karl Ekwall. Epigenome Mapping in Quiescent Cells Reveals a Key Role for H3K4me3 in Regulation of RNA Polymerase II Activity. Epigenomes. 2024 Oct 22;8(4):39. https://doi.org/10.3390/epigenomes8040039

III. Shengyuan Zeng, Mickael Durand-Dubief, Job Dekker, Jean-Paul Javerzat, Karl Ekwall. Extensive chromosome reorganization in quiescent fission yeast cells. [Manuscript]